Publications

Global genomic diversity of Pseudomonas aeruginosa in bronchiectasis

Harrington et al., 2024 | Bacteria and Bacterial Diseases | Link

Background Pseudomonas aeruginosa is the dominant pathogen causing lung infections in people with both cystic fibrosis CF and bronchiectasis associated with poorer outcomes Unlike CF bronchiectasis has been a neglected disease More extensive genomic studies of larger bronchiectasis patient cohorts and within patient sampling are needed to improve understanding of the evolutionary mechanisms underpinning P aeruginosa infections to guide novel and improved treatments Methods We have performed genome sequencing of P aeruginosa isolates from patients attending clinics worldwide to analyse the genomic diversity between and within patient infections Results We observed high genetic diversity between infections with low incidence of ... More |

Background Pseudomonas aeruginosa is the dominant pathogen causing lung infections in people with both cystic fibrosis (CF) and bronchiectasis, associated with poorer outcomes. Unlike CF, bronchiectasis has been a neglected disease. More extensive genomic studies of larger bronchiectasis patient cohorts and within patient sampling are needed to improve understanding of the evolutionary mechanisms underpinning P. aeruginosa infections to guide novel and improved treatments.

Methods We have performed genome sequencing of 2,854 P. aeruginosa isolates from 180 patients attending clinics worldwide to analyse the genomic diversity between and within patient infections.

Results We observed high genetic diversity between infections with low incidence of highly transmissible strains. Our genomic data provide evidence for the mutational targets driving P. aeruginosa evolution in bronchiectasis. Some functions found to gain mutations were comparable to CF, including biofilm and iron acquisition, whilst others highlighted distinct evolutionary paths in bronchiectasis such as pyocin production and resistance, and a novel efflux pump gene (PA1874). We also show a high incidence of antimicrobial resistance-associated mutations and acquired resistance genes, in particular multidrug efflux and fluoroquinolone resistance mechanisms.

Conclusions Our findings highlight important differences between P. aeruginosa infections in bronchiectasis and CF and provide evidence of the relatively minor role transmissible strains play in bronchiectasis. Our study provides a 10-fold increase in the available genomic data for these infections and is a global resource to improve our knowledge and understanding, to facilitate better patient outcomes.

Summary The largest genomic study of Pseudomonas aeruginosa bronchiectasis isolates to-date, providing an unprecedented global genomic resource. We highlight important differences between bronchiectasis and cystic fibrosis, including key genes under selection. Less |

Elucidating signal transduction in multi-domain BLUF photoreceptors by studying the Photoactivated Adenylate Cyclase OaPAC

Chretien et al., 2024 | Thesis/ Dessertation | Link

Photosensory receptors essential molecular entities across all domains of life enable organisms to detect and respond to light stimuli underpinning their critical involvement in regulating biological processes such as phototropism circadian rhythms photomorphogenesis and photosynthesis Among the myriad types of photosensory receptors blue light sensing proteins such as Blue Light Using Flavin BLUF photoreceptors distinguish themselves through their ability to utilize blue light for signalling Characterized by the conserved structure of their sensor domain BLUF photoreceptors are found in a wide array of organisms from bacteria and algae to plants and certain fungi Known for their capacity to bind flavin ... More |

Photosensory receptors, essential molecular entities across all domains of life, enable organisms to
detect and respond to light stimuli, underpinning their critical involvement in regulating biological
processes such as phototropism, circadian rhythms, photomorphogenesis, and photosynthesis.
Among the myriad types of photosensory receptors, blue light sensing proteins such as Blue Light
Using Flavin (BLUF) photoreceptors distinguish themselves through their ability to utilize blue
light for signalling. Characterized by the conserved structure of their sensor domain, BLUF
photoreceptors are found in a wide array of organisms, from bacteria and algae to plants and certain
fungi. Known for their capacity to bind flavin chromophores, typically flavin adenine dinucleotide
(FAD), they undergo conformational changes upon blue photon absorption, leading to downstream
signalling events, highlighting their pivotal role in the adaptive responses of various organisms to
light. This dissertation provides a comprehensive exploration of the BLUF photoreceptors,
particularly focusing on the Photoactivated Adenylate Cyclase protein from Oscillatoria acuminata
(OaPAC), which comprises a BLUF sensor domain linked to an Adenylate Cyclase (AC) effector
domain, catalysing the conversion of ATP into cAMP. This study aims to elucidate the
photoactivation mechanism of OaPAC and the ensuing signal transduction pathway, employing an
integrative approach that leverages time-resolved crystallography, small angle X-ray scattering,
spectroscopy, and biochemical characterization techniques. Special emphasis is placed on the TyrGln-Met triad in the BLUF domain, which plays a crucial role in the initial light-induced
rearrangements. Additionally, significant attention is given to the less understood aspects of BLUF
photoreceptors, particularly the transduction of the initial light signal to more distal parts of the
protein, which ultimately leads to biological activity. This research identifies a Metout/Trpin
transition as a crucial element in conveying the signal to the α-helix linker region. Finally, structural
models of OaPAC with ATP bound in the active site, along with complementary FTIR
investigations, provide a thorough understanding of ATP binding and allosteric communication. As
a result, the research presented in this dissertation not only expands the fundamental understanding
of BLUF photoreceptor biology, but also provides a framework for future studies aimed at
deciphering complete signal transduction pathways in multi-domain BLUF photoreceptors and
towards the development of optogenetic tools Less |

The toxicity of alpha-syn condensates in the presence and absence of metal ions

Pinto et al., 2024 | Thesis/ Dessertation | Link

Neurodegenerative diseases NDDs characterized by progressive neuronal death and misfolded protein aggregation pose significant clinical social and personal challenges Parkinson's Disease PD the second most common neurological disorder is notably associated with the aggregation of alpha-synuclein aSyn Despite its prominence the transition of monomeric aSyn to aggregates remains inadequately understood Recent studies suggest that Liquid-Liquid Phase Separation LLPS and disease related metal ions involve the transition in the molecular pathogenesis of PD LLPS involves the separation of biomolecules into distinct phases without a membrane potentially facilitating aSyn aggregation through dynamic condensates that eventually form solid deposits I aim to investigate ... More |

Neurodegenerative diseases (NDDs), characterized by progressive neuronal death and misfolded protein aggregation, pose significant clinical, social, and personal challenges. Parkinson's Disease (PD), the second most common neurological disorder, is notably associated with the aggregation of alpha-synuclein (aSyn). Despite its prominence, the transition of monomeric aSyn to aggregates remains inadequately understood. Recent studies suggest that Liquid-Liquid Phase Separation (LLPS) and disease related metal ions involve the transition in the molecular pathogenesis of PD. LLPS involves the separation of biomolecules into distinct phases without a membrane, potentially facilitating aSyn aggregation through dynamic condensates that eventually form solid deposits. I aim to investigate LLPS of aSyn and macroscopic dynamics of its formed droplets over time, and examine how PD related metal ions, affect the dynamic process of LLPS and modulate its toxicity to neuroblastoma cells. These metal ions, prevalent in the brain and specifically interacting with aSyn are presumably modulating LLPS, toxicity and aggregation of aSyn, making it crucial to understand their roles in the molecular pathogenesis of PD I expressed α-synuclein (aSyn) proteins in E. coli and studied the biophysical properties and toxicities of aSyn-metal ion coacervates using various techniques, including a protein crystallization robotic dispenser and confocal microscopy. In the presence of metal ions such as CuCl₂, MnCl₂, ZnCl₂, and FeCl₃, the number of droplets significantly decreased. I found that CuCl₂ ions immobilize aSyn condensates and increase their toxicity. In contrast, MnCl₂, ZnCl₂, and FeCl₃ help maintain a longer metastable state of the condensates, reducing their toxicity. This project highlights the crucial role of metal ions in modulating aSyn phase behavior, condensate toxicity and their potential involvement in the progression of PD. Less |

Short CDRL1 in intermediate VRC01-like mAbs is not sufficient to overcome key glycan barriers on HIV-1 Env

Agrawal et al., 2024 | Journal of Virology | Link

VRC -class broadly neutralizing antibodies bnAbs have been isolated from people with HIV- but they have not yet been elicited by vaccination They are extensively somatically mutated and sometimes accumulate CDRL deletions Such indels may allow VRC -class antibodies to accommodate the glycans expressed on a conserved N N-linked glycosylation site in loop D of the gp subunit These glycans constitute a major obstacle in the development of VRC -class antibodies as unmutated antibody forms are unable to accommodate them Although immunizations of knock-in mice expressing human VRC -class B-cell receptors BCRs with specifically designed Env-derived immunogens lead to the ... More |

VRC01-class broadly neutralizing antibodies (bnAbs) have been isolated from people with HIV-1, but they have not yet been elicited by vaccination. They are extensively somatically mutated and sometimes accumulate CDRL1 deletions. Such indels may allow VRC01-class antibodies to accommodate the glycans expressed on a conserved N276 N-linked glycosylation site in loop D of the gp120 subunit. These glycans constitute a major obstacle in the development of VRC01-class antibodies, as unmutated antibody forms are unable to accommodate them. Although immunizations of knock-in mice expressing human VRC01-class B-cell receptors (BCRs) with specifically designed Env-derived immunogens lead to the accumulation of somatic mutations in VRC01-class BCRs, CDRL1 deletions are rarely observed, and the elicited antibodies display narrow neutralizing activities. The lack of broad neutralizing potential could be due to the absence of deletions, the lack of appropriate somatic mutations, or both. To address this point, we modified our previously determined prime-boost immunization with a germline-targeting immunogen nanoparticle (426c.Mod.Core), followed by a heterologous core nanoparticle (HxB2.WT.Core), by adding a final boost with a cocktail of various stabilized soluble Env trimers. We isolated VRC01-like antibodies with extensive somatic mutations and, in one case, a seven-amino acid CDRL1 deletion. We generated chimeric antibodies that combine the vaccine-elicited somatic mutations with CDRL1 deletions present in human mature VRC01 bnAbs. We observed that CDRL1 indels did not improve the neutralizing antibody activities. Our study indicates that CDRL1 length by itself is not sufficient for the broadly neutralizing phenotype of this class of antibodies. Less |

Development of a sensitive high-throughput enzymatic assay capable of measuring sub-nanomolar inhibitors of SARS-CoV2 Mpro

Kovar et al., 2024 | SLAS Discovery | Link

The SARS-CoV- main protease Mpro is essential for viral replication because it is responsible for the processing of most of the non-structural proteins encoded by the virus Inhibition of Mpro prevents viral replication and therefore constitutes an attractive antiviral strategy We set out to develop a high-throughput Mpro enzymatic activity assay using fluorescently labeled peptide substrates A library of fluorogenic substrates of various lengths sequences and dye quencher positions was prepared and tested against full length SARS-CoV- Mpro enzyme for optimal activity The addition of buffers containing strongly hydrated kosmotropic anion salts such as citrate from the Hofmeister series significantly ... More |

The SARS-CoV-2 main protease (Mpro) is essential for viral replication because it is responsible for the processing of most of the non-structural proteins encoded by the virus. Inhibition of Mpro prevents viral replication and therefore constitutes an attractive antiviral strategy. We set out to develop a high-throughput Mpro enzymatic activity assay using fluorescently labeled peptide substrates. A library of fluorogenic substrates of various lengths, sequences and dye/quencher positions was prepared and tested against full length SARS-CoV-2 Mpro enzyme for optimal activity. The addition of buffers containing strongly hydrated kosmotropic anion salts, such as citrate, from the Hofmeister series significantly boosted the enzyme activity and enhanced the assay detection limit, enabling the ranking of sub-nanomolar inhibitors without relying on the low-throughput Morrison equation method. By comparing cooperativity in citrate or non-citrate buffer while titrating the Mpro enzyme concentration, we found full positive cooperativity of Mpro with citrate buffer at less than one nanomolar (nM), but at a much higher enzyme concentration (∼320 nM) with non-citrate buffer. In addition, using a tight binding Mpro inhibitor, we confirmed there was only one active catalytical site in each Mpro monomer. Since cooperativity requires at least two binding sites, we hypothesized that citrate facilitates dimerization of Mpro at sub-nanomolar concentration as one of the mechanisms enhances Mpro catalytic efficiency. This assay has been used in high-throughput screening and structure activity relationship (SAR) studies to support medicinal chemistry efforts. IC50 values determined in this assay correlates well with EC50 values generated by a SARS-CoV-2 antiviral assay after adjusted for cell penetration. Less |

Therapeutic Potential of Wharton's Jelly Mesenchymal Stem Cells-derived Secretome (S-MSCs) in Psoriasis Vulgaris: A Case Study

Azzahara et al., 2024 | International Journal of Cell and Biomedical Science | Link

Background Psoriasis is a chronic immune-mediated skin disease that also has systemic manifestations Case In this report we discuss our findings about a -years old psoriasis suffering male patient with a Psoriasis Area Severity Index PASI score of treated with Wharton s Jelly Mesenchymal Stem Cells-derived Secretome S-MSCs Remarkably complete regression was recorded within a treatment period of a week only Result The patient demonstrated a decrease in PASI from to after infusion and followed by intramuscular injections of S-MSCs Bioactive factors secreted by MSCs cytokines and growth factors are very likely to be the principal molecules which play a ... More |

Background: Psoriasis is a chronic, immune-mediated skin disease that also has systemic manifestations. Case: In this report, we discuss our findings about a 47-years old psoriasis suffering male patient with a Psoriasis Area Severity Index (PASI) score of 10.8, treated with Wharton’s Jelly Mesenchymal Stem Cells-derived Secretome (S-MSCs). Remarkably, complete regression was recorded within a treatment period of a week only. Result: The patient demonstrated a decrease in PASI, from 10.8 to 3.2 after 1 infusion and followed by 4 intramuscular injections of S-MSCs. Bioactive factors secreted by MSCs, cytokines and growth factors, are very likely to be the principal molecules which play a vital role in inflammatory modulation and skin tissue regeneration. No serious adverse events were noted for the patient as a result of secretome infusion and intramuscular injection. Conclusion: This report demonstrates safety and promises to be an effective strategy using S-MSCs treatment for managing the psoriatic issue and, thus, may offer as an alternative approach to overcome the limitations of the cell-based therapy. Less |

Bioprospecting of inhibitors of EPEC virulence from metabolites of marine actinobacteria from the Arctic Sea

Pylkko et al., 2024 | Frontiers in Microbiology | Link

A considerable number of antibacterial agents are derived from bacterial metabolites Similarly numerous known compounds that impede bacterial virulence stem from bacterial metabolites Enteropathogenic Escherichia coli EPEC is a notable human pathogen causing intestinal infections particularly affecting infant mortality in developing regions These infections are characterized by microvilli effacement and intestinal epithelial lesions linked with aberrant actin polymerization This study aimed to identify potential antivirulence compounds for EPEC infections among bacterial metabolites harvested from marine actinobacteria Kocuria sp and Rhodococcus spp from the Arctic Sea by the application of virulence-based screening assays Moreover we demonstrate the suitability of these antivirulence ... More |

A considerable number of antibacterial agents are derived from bacterial metabolites. Similarly, numerous known compounds that impede bacterial virulence stem from bacterial metabolites. Enteropathogenic Escherichia coli (EPEC) is a notable human pathogen causing intestinal infections, particularly affecting infant mortality in developing regions. These infections are characterized by microvilli effacement and intestinal epithelial lesions linked with aberrant actin polymerization. This study aimed to identify potential antivirulence compounds for EPEC infections among bacterial metabolites harvested from marine actinobacteria (Kocuria sp. and Rhodococcus spp.) from the Arctic Sea by the application of virulence-based screening assays. Moreover, we demonstrate the suitability of these antivirulence assays to screen actinobacteria extract fractions for the bioassay-guided identification of metabolites. We discovered a compound in the fifth fraction of a Kocuria strain that interferes with EPEC-induced actin polymerization without affecting growth. Furthermore, a growth-inhibiting compound was identified in the fifth fraction of a Rhodococcus strain. Our findings include the bioassay-guided identification, HPLC-MS-based dereplication, and isolation of a large phospholipid and a likely antimicrobial peptide, demonstrating the usefulness of this approach in screening for compounds capable of inhibiting EPEC virulence. Less |

The C2 domain augments Ras GTPase Activating Protein catalytic activity

Paul et al., 2024 | Preprint | Link

Regulation of Ras GTPases by GTPase activating proteins GAP is essential for their normal signaling Nine of the ten GAPs for Ras contain a C domain immediately proximal to their canonical GAP domain and in RasGAP p GAP p RasGAP RASA mutation of this domain is associated with vascular malformations in humans Here we show that the C domain of RasGAP is required for full catalytic activity towards Ras Analysis of the RasGAP C -GAP crystal structure AlphaFold models and sequence conservation reveal direct C domain interaction with the Ras allosteric lobe This is achieved by an evolutionarily conserved surface ... More |

Regulation of Ras GTPases by GTPase activating proteins (GAP) is essential for their normal signaling. Nine of the ten GAPs for Ras contain a C2 domain immediately proximal to their canonical GAP domain, and in RasGAP (p120GAP, p120RasGAP; RASA1) mutation of this domain is associated with vascular malformations in humans. Here, we show that the C2 domain of RasGAP is required for full catalytic activity towards Ras. Analysis of the RasGAP C2-GAP crystal structure, AlphaFold models, and sequence conservation reveal direct C2 domain interaction with the Ras allosteric lobe. This is achieved by an evolutionarily conserved surface centered around RasGAP residue R707, point mutation of which impairs the catalytic advantage conferred by the C2 domain in vitro. In mice, R707C mutation phenocopies the vascular and signaling defects resulting from constitutive disruption of the RASA1 gene. In SynGAP, mutation of the equivalent conserved C2 domain surface impairs catalytic activity. Our results indicate that the C2 domain is required to achieve full catalytic activity of Ras GTPase activating proteins. Less |

Bimodal substrate binding in the active site of the glycosidase BcX

Saberi et al., 2024 | The FEBS journal | Link

Bacillus circulans xylanase BcX from the glycoside hydrolase family degrades xylan through a retaining double-displacement mechanism The enzyme is thought to hydrolyze glycosidic bonds in a processive manner and has a large active site cleft with six subsites allowing the binding of six xylose units Such an active site architecture suggests that oligomeric xylose substrates can bind in multiple ways In the crystal structure of the catalytically inactive variant BcX E Q the substrate xylotriose is observed in the active site as well as bound to the known secondary binding site and a third site on the protein surface Nuclear ... More |

Bacillus circulans xylanase (BcX) from the glycoside hydrolase family 11 degrades xylan through a retaining, double-displacement mechanism. The enzyme is thought to hydrolyze glycosidic bonds in a processive manner and has a large, active site cleft, with six subsites allowing the binding of six xylose units. Such an active site architecture suggests that oligomeric xylose substrates can bind in multiple ways. In the crystal structure of the catalytically inactive variant BcX E78Q, the substrate xylotriose is observed in the active site, as well as bound to the known secondary binding site and a third site on the protein surface. Nuclear magnetic resonance (NMR) titrations with xylose oligomers of different lengths yield nonlinear chemical shift trajectories for active site nuclei resonances, indicative of multiple binding orientations for these substrates for which binding and dissociation are in fast exchange on the NMR timescale, exchanging on the micro- to millisecond timescale. Active site binding can be modeled with a 2 : 1 model with dissociation constants in the low and high millimolar range. Extensive mutagenesis of active site residues indicates that tight binding occurs in the glycon binding site and is stabilized by Trp9 and the thumb region. Mutations F125A and W71A lead to large structural rearrangements. Binding at the glycon site is sensed throughout the active site, whereas the weak binding mostly affects the aglycon site. The interactions with the two active site locations are largely independent of each other and of binding at the secondary binding site. Less |

Prevotella are major contributors of sialidases in the human vaginal microbiome

Pelayo et al., 2024 | PNAS Microbiology | Link

Elevated bacterial sialidase activity in the female genital tract is strongly associated with poor health outcomes including preterm birth and bacterial vaginosis These negative effects may arise from sialidase-mediated degradation of the protective mucus layer in the cervicovaginal environment Prior biochemical studies of vaginal bacterial sialidases have focused solely on the bacterial vaginosis-associated organism Gardnerella vaginalis Despite their implications for sexual and reproductive health sialidases from other vaginal bacteria have not been characterized Here we show that vaginal Prevotella species produce active sialidases that possess variable activity toward mucin These sialidases are highly conserved across clades of Prevotella from different ... More |

Elevated bacterial sialidase activity in the female genital tract is strongly associated with poor health outcomes including preterm birth and bacterial vaginosis. These negative effects may arise from sialidase-mediated degradation of the protective mucus layer in the cervicovaginal environment. Prior biochemical studies of vaginal bacterial sialidases have focused solely on the bacterial vaginosis-associated organism Gardnerella vaginalis. Despite their implications for sexual and reproductive health, sialidases from other vaginal bacteria have not been characterized. Here, we show that vaginal Prevotella species produce active sialidases that possess variable activity toward mucin. These sialidases are highly conserved across clades of Prevotella from different geographies, hinting at their importance globally. Finally, we find that Prevotella sialidases, including mucin-degrading enzymes from Prevotella timonensis, are highly prevalent and abundant in human vaginal metagenomes and metatranscriptomes, Together, our results identify Prevotella as a critical source of sialidases in the vaginal microbiome, improving our understanding of this detrimental bacterial activity. Less |

Discovery of potent SARS-CoV-2 nsp3 macrodomain inhibitors uncovers lack of translation to cellular antiviral response

Lee et al., 2024 | Preprint | Link

A strategy for pandemic preparedness is the development of antivirals against a wide set of viral targets with complementary mechanisms of action SARS-CoV- nsp -mac is a viral macrodomain with ADP-ribosylhydrolase activity which counteracts host immune response Targeting the virus' immunomodulatory functionality offers a differentiated strategy to inhibit SARS-CoV- compared to approved therapeutics which target viral replication directly Here we report a fragment-based lead generation campaign guided by computational approaches We discover tool compounds which inhibit nsp -mac activity at low nanomolar concentrations and with responsive structure-activity relationships high selectivity and drug-like properties Using our inhibitors we show that inhibition ... More |

A strategy for pandemic preparedness is the development of antivirals against a wide set of viral targets with complementary mechanisms of action. SARS-CoV-2 nsp3-mac1 is a viral macrodomain with ADP-ribosylhydrolase activity, which counteracts host immune response. Targeting the virus' immunomodulatory functionality offers a differentiated strategy to inhibit SARS-CoV-2 compared to approved therapeutics, which target viral replication directly. Here we report a fragment-based lead generation campaign guided by computational approaches. We discover tool compounds which inhibit nsp3-mac1 activity at low nanomolar concentrations, and with responsive structure-activity relationships, high selectivity, and drug-like properties. Using our inhibitors, we show that inhibition of nsp3-mac1 increases ADP-ribosylation, but surprisingly does not translate to demonstrable antiviral activity in cell culture and iPSC-derived pneumocyte models. Further, no synergistic activity is observed in combination with interferon gamma, a main protease inhibitor, nor a papain-like protease inhibitor. Our results question the extent to which targeting modulation of innate immunitydriven ADP-ribosylation can influence SARS-CoV-2 replication. Moreover, these findings suggest that nsp3-mac1 might not be a suitable target for antiviral therapeutics development. Less |

Substrate‐Based Ligand Design for Phenazine Biosynthesis Enzyme PhzF

Baumgarten et al., 2024 | Chemistry Europe | Link

The phenazine pyocyanin is an important virulence factor of the pathogen Pseudomonas aeruginosa which is on the WHO list of antibiotic resistant priority pathogens In this study the isomerase PhzF a key bacterial enzyme of the pyocyanin biosynthetic pathway was investigated as a pathoblocker target The aim of the pathoblocker strategy is to reduce the virulence of the pathogen without killing it thus preventing the rapid development of resistance Based on crystal structures of PhzF derivatives of the inhibitor hydroxyanthranilic acid were designed Co-crystal structures of the synthesized derivatives with PhzF revealed spacial limitations of the binding pocket of PhzF ... More |

The phenazine pyocyanin is an important virulence factor of the pathogen Pseudomonas aeruginosa, which is on the WHO list of antibiotic resistant “priority pathogens”. In this study the isomerase PhzF, a key bacterial enzyme of the pyocyanin biosynthetic pathway, was investigated as a pathoblocker target. The aim of the pathoblocker strategy is to reduce the virulence of the pathogen without killing it, thus preventing the rapid development of resistance. Based on crystal structures of PhzF, derivatives of the inhibitor 3–hydroxyanthranilic acid were designed. Co-crystal structures of the synthesized derivatives with PhzF revealed spacial limitations of the binding pocket of PhzF in the closed conformation. In contrast, ligands aligned to the open conformation of PhzF provided more room for structural modifications. The intrinsic fluorescence of small 3–hydroxyanthranilic acid derivatives enabled direct affinity determinations using FRET assays. The analysis of structure-activity relationships showed that the carboxylic acid moiety is essential for binding to the target enzyme. The results of this study provide fundamental structural insights that will be useful for the design of PhzF-inhibitors. Less |

Crystallisation protocol for SARS-CoV-2 nsp3 macrodomain in P1 21 1

Aschenbrenner et al., 2024 | Protocols part of Springer nature | Link

The COVID- pandemic has demonstrated the need for novel therapeutic interventions and improved pandemic preparedness strategies against severe acute respiratory syndrome coronavirus SARS-CoV- This protocol details an optimized crystallization method for the SARS-CoV- nsp macrodomain a potential drug target Using sitting drop vapor diffusion with seeding we describe specific buffer conditions and procedures to consistently produce high-quality crystals suitable for XChem fragment screening The method yields crystals that diffract to an average resolution of enabling high-resolution structural studies and can also be used for compound development through co-crystallization experiments All structures solved during the development of tool compounds for the ... More |

The COVID-19 pandemic has demonstrated the need for novel therapeutic interventions and improved pandemic preparedness strategies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This protocol details an optimized crystallization method for the SARS-CoV-2 nsp3 macrodomain, a potential drug target. Using sitting drop vapor diffusion with seeding, we describe specific buffer conditions and procedures to consistently produce high-quality crystals suitable for XChem fragment screening. The method yields crystals that diffract to an average resolution of 1.5 Å, enabling high-resolution structural studies and can also be used for compound development through co-crystallization experiments.
All structures solved during the development of tool compounds for the SARS-CoV-2 nsp3 macrodomain are deposited on the PDB (Group deposition: G_1002283). Less |

Crystallisation protocol for SARS-CoV-2 nsp3 macrodomain in P43

Aschenbrenner et al., 2024 | Protocols part of Springer nature | Link

The COVID- pandemic has demonstrated the need for novel therapeutic interventions and improved pandemic preparedness strategies against severe acute respiratory syndrome coronavirus SARS-CoV- This protocol details an optimized crystallization method for the SARS-CoV- nsp macrodomain a potential drug target Using sitting drop vapor diffusion we describe specific buffer conditions and procedures to consistently produce high-quality crystals suitable for XChem fragment screening The method yields crystals that diffract to an average resolution of enabling high-resolution structural studies All structures solved during the development of tool compounds for the SARS-CoV- nsp macrodomain are deposited on the PDB Group deposition G |

Charge Detection Mass Spectrometry Enables Molecular Characterization of Nucleic Acid Nanoparticles

Ofoegbu et al., 2024 | ACS NANO | Link

Nucleic acid nanoparticles NANPs are increasingly used in preclinical investigations as delivery vectors Tools that can characterize assembly and assess quality will accelerate their development and clinical translation Standard techniques used to characterize NANPs like gel electrophoresis lack the resolution for precise characterization Here we introduce the use of charge detection mass spectrometry CD-MS to characterize these materials Using this technique we determined the mass of NANPs varying in size shape and molecular mass NANPs varying in production quality due to formulations lacking component oligonucleotides and NANPs functionalized with protein and nucleic acid-based secondary molecules Based on these demonstrations CD-MS ... More |

Nucleic acid nanoparticles (NANPs) are increasingly used in preclinical investigations as delivery vectors. Tools that can characterize assembly and assess quality will accelerate their development and clinical translation. Standard techniques used to characterize NANPs, like gel electrophoresis, lack the resolution for precise characterization. Here, we introduce the use of charge detection mass spectrometry (CD-MS) to characterize these materials. Using this technique, we determined the mass of NANPs varying in size, shape, and molecular mass, NANPs varying in production quality due to formulations lacking component oligonucleotides, and NANPs functionalized with protein and nucleic acid-based secondary molecules. Based on these demonstrations, CD-MS is a promising tool to precisely characterize NANPs, enabling more precise assessments of the manufacturing and processing of these materials. Less |

Therapeutic Potential of Secretome Hypoxia Mesenchymal Stem Cells: Downregulation of TNF-α and HIF-2α in Metabolic Syndrome-Induced Inflammation in Wistar Rats

Dewi et al., 2024 | Biosaintifika | Link

Metabolic syndrome MetS has become a global health challenge with several associated issues such as obesity insulin resistance dyslipidemia and hypertension Important proteins such as Tumor Necrosis Factor-alpha TNF- and Hypoxia Inducible Factor- alpha HIF- regulate the inflammatory process by inducing the expression of pro-inflammatory proteins This study aims to determine the effect of administering SH-MSCSs on the expression of the TNF- and Hypoxia Inducible Factor HIF - genes in the male Wistar rat model with Metabolic Syndrome This research is an experimental study with a Post-test Only Control Group Design using a total of male Wistar rats divided into ... More |

Metabolic syndrome (MetS) has become a global health challenge with several associated issues, such as obesity, insulin resistance, dyslipidemia, and hypertension. Important proteins such as Tumor Necrosis Factor-alpha (TNF-α) and Hypoxia Inducible Factor-2 alpha (HIF-2α) regulate the inflammatory process by inducing the expression of pro-inflammatory proteins. This study aims to determine the effect of administering SH-MSCSs on the expression of the TNF-α and Hypoxia Inducible Factor (HIF)-2α genes in the male Wistar rat model with Metabolic Syndrome. This research is an experimental study with a Post-test Only Control Group Design, using a total of 24 male Wistar rats divided into four groups: T1 (Healthy control), T2 (MetS + NaCl), T3 (MetS + administration of SH-MSCs dose 150 uL), and T4 (MetS + administration of SH-MSCs dose 300 uL). SH-MSCSs were administered intraperitoneally four times over 14 days. Adipose tissue TNF-α and HIF-2α gene expression were measured on day 15 using qRT-PCR. TNF-α and HIF-2α gene expression was significantly lower in T3 and T4, compared with the MetS control group (T2). Administration of SH-MSCs was able to reduce the expression of the Tumor Necrosis Factor (TNF-α) and Hypoxia Inducible Factor (HIF)-2α genes in fatty tissue in the male Wistar rat model with Metabolic Syndrome.This study presents a novel approach to treating MetS by demonstrating that the administration of SH-MSCs significantly reduces the expression of pro-inflammatory genes TNF-α and HIF-2α. This finding is beneficial for society as it suggests a potential newtherapeutic strategy that could mitigate inflammation and improve health outcomes for individuals suffering from MetS, thereby addressing a critical global health challenge. Less |

Overcome the challenge for intratumoral injection of STING agonist for pancreatic cancer by systemic administration

Li et al., 2024 | Journal of Hematology & Oncology | Link

Objective Due to the challenge for intratumoral administration innate agonists have not made it beyond preclinical studies for efficacy testing in most of tumor types Pancreatic ductal adenocarcinoma PDAC has a T-cell excluded or deserted tumor microenvironment Innate agonist treatments may serve as a T cell priming mechanism to sensitize PDACs to anti-PD- antibody a-PD- treatment Design Using a transplant murine model with spontaneously formed liver metastasis and also the genetically engineered KPC mouse model that spontaneously develops PDAC we compared the antitumor efficacy between intrahepatic intratumoral and intramuscular systemic administration of BMS- a next-generation STING agonist Flow cytometry Nanostring ... More |

Objective: Due to the challenge for intratumoral administration, innate agonists have not made it beyond preclinical studies for efficacy testing in most of tumor types. Pancreatic ductal adenocarcinoma (PDAC) has a T-cell excluded or deserted tumor microenvironment. Innate agonist treatments may serve as a T cell priming mechanism to sensitize PDACs to anti-PD-1 antibody (a-PD-1) treatment. Design: Using a transplant murine model with spontaneously formed liver metastasis and also the genetically engineered KPC mouse model that spontaneously develops PDAC, we compared the antitumor efficacy between intrahepatic/intratumoral and intramuscular systemic administration of BMS-986301, a next-generation STING agonist. Flow cytometry, Nanostring, and cytokine assays were used to evaluate local and systemic immune responses. Results: The study demonstrated that administration of STING agonist systemically via intramuscular injection is equivalent or potentially superior to its intratumoral injection in inducing both effector T cell response and antitumor efficacy. Compared to intratumoral administration, T cell exhaustion and immunosuppressive signals induced by systemic administration were attenuated. Nonetheless, either local or systemic treatment of STING agonist was associated with increased expression of CTLA-4 in the tumors. However, the combination of a-PD-1 and anti-CTLA-4 antibody with systemic STING agonist demonstrated the antitumor efficacy in the KPC mouse spontaneous PDAC model. Our study also demonstrated the feasibility and antitumor efficacy of systemic administration of BMS-986299, a new NLRP3 agonist. Conclusion: For the first time, our study supports the clinical development of innate agonists via systemic administration, instead of local administration, for treating PDAC. Less |

Borrelia burgdorferi BB0346 is an Essential, Structurally Variant LolA Homolog that is Primarily Required for Homeostatic Localization of Periplasmic Lipoproteins

Murphy et al., 2024 | Preprint | Link

In diderm bacteria the Lol pathway canonically mediates the periplasmic transport of lipoproteins from the inner membrane IM to the outer membrane OM and therefore plays an essential role in bacterial envelope homeostasis After extrusion of modified lipoproteins from the IM via the LolCDE complex the periplasmic chaperone LolA carries lipoproteins through the periplasm and transfers them to the OM lipoprotein insertase LolB itself a lipoprotein with a LolA-like fold Yet LolB homologs appear restricted to -proteobacteria and are missing from spirochetes like the tick-borne Lyme disease pathogen Borrelia burgdorferi suggesting a different hand-off mechanism at the OM Here we ... More |

In diderm bacteria, the Lol pathway canonically mediates the periplasmic transport of lipoproteins from the inner membrane (IM) to the outer membrane (OM) and therefore plays an essential role in bacterial envelope homeostasis. After extrusion of modified lipoproteins from the IM via the LolCDE complex, the periplasmic chaperone LolA carries lipoproteins through the periplasm and transfers them to the OM lipoprotein insertase LolB, itself a lipoprotein with a LolA-like fold. Yet, LolB homologs appear restricted to ψ-proteobacteria and are missing from spirochetes like the tick-borne Lyme disease pathogen Borrelia burgdorferi, suggesting a different hand-off mechanism at the OM. Here, we solved the crystal structure of the B. burgdorferi LolA homolog BB0346 (LolABb) at 1.9 Å resolution. We identified multiple structural deviations in comparative analyses to other solved LolA structures, particularly a unique LolB-like protruding loop domain. LolABb failed to complement an Escherichia coli lolA knockout, even after codon optimization, signal I peptide adaptation, and a C-terminal chimerization which had allowed for complementation with an α-proteobacterial LolA. Analysis of a conditional B. burgdorferi lolA knockout strain indicated that LolABb was essential for growth. Intriguingly, protein localization assays indicated that initial depletion of LolABb led to an emerging mislocalization of both IM and periplasmic OM lipoproteins, but not surface lipoproteins. Together, these findings further support the presence of two separate primary secretion pathways for periplasmic and surface OM lipoproteins in B. burgdorferi and suggest that the distinct structural features of LolABb allow it to function in a unique LolB-deficient lipoprotein sorting system. Less |

The crystal structure of Acinetobacter baumannii bacterioferritin reveals a heteropolymer of bacterioferritin and ferritin subunits

Yao et al., 2024 | Nature Scientific Reports | Link

Iron storage proteins e g vertebrate ferritin and the ferritin-like bacterioferritin Bfr and bacterial ferritin Ftn are spherical hollow proteins that catalyze the oxidation of Fe at binuclear iron ferroxidase centers FOC and store the Fe in their interior thus protecting cells from unwanted Fe Fe redox cycling and storing iron at concentrations far above the solubility of Fe Vertebrate ferritins are heteropolymers of H and L subunits with only the H subunits having FOC Bfr and Ftn were thought to coexist in bacteria as homopolymers but recent evidence indicates these molecules are heteropolymers assembled from Bfr and Ftn subunits ... More |

Iron storage proteins, e.g., vertebrate ferritin, and the ferritin-like bacterioferritin (Bfr) and bacterial ferritin (Ftn), are spherical, hollow proteins that catalyze the oxidation of Fe2+ at binuclear iron ferroxidase centers (FOC) and store the Fe3+ in their interior, thus protecting cells from unwanted Fe3+/Fe2+ redox cycling and storing iron at concentrations far above the solubility of Fe3+. Vertebrate ferritins are heteropolymers of H and L subunits with only the H subunits having FOC. Bfr and Ftn were thought to coexist in bacteria as homopolymers, but recent evidence indicates these molecules are heteropolymers assembled from Bfr and Ftn subunits. Despite the heteropolymeric nature of vertebrate and bacterial ferritins, structures have been determined only for recombinant proteins constituted by a single subunit type. Herein we report the structure of Acinetobacter baumannii bacterioferritin, the first structural example of a heteropolymeric ferritin or ferritin-like molecule, assembled from completely overlapping Ftn homodimers harboring FOC and Bfr homodimers devoid of FOC but binding heme. The Ftn homodimers function by catalyzing the oxidation of Fe2+ to Fe3+, while the Bfr homodimers bind a cognate ferredoxin (Bfd) which reduces the stored Fe3+ by transferring electrons via the heme, enabling Fe2+ mobilization to the cytosol for incorporation in metabolism. Less |

Use of protease substrate specificity screening in the rational design of selective protease inhibitors with unnatural amino acids: Application to HGFA, matriptase, and hepsin

Mahoney et al., 2024 | Protein Science | Link

Inhibition of the proteolytic processing of hepatocyte growth factor HGF and macrophage stimulating protein MSP is an attractive approach for the drug discovery of novel anticancer therapeutics which prevent tumor progression and metastasis Here we utilized an improved and expanded version of positional scanning of substrate combinatorial libraries PS-SCL technique called HyCoSuL to optimize peptidomimetic inhibitors of the HGF MSP activating serine proteases HGFA matriptase and hepsin These inhibitors have an electrophilic ketone serine trapping warhead and thus form a reversible covalent bond to the protease We demonstrate that by varying the P P and P positions of the inhibitor ... More |

Inhibition of the proteolytic processing of hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP) is an attractive approach for the drug discovery of novel anticancer therapeutics which prevent tumor progression and metastasis. Here, we utilized an improved and expanded version of positional scanning of substrate combinatorial libraries (PS-SCL) technique called HyCoSuL to optimize peptidomimetic inhibitors of the HGF/MSP activating serine proteases, HGFA, matriptase, and hepsin. These inhibitors have an electrophilic ketone serine trapping warhead and thus form a reversible covalent bond to the protease. We demonstrate that by varying the P2, P3, and P4 positions of the inhibitor with unnatural amino acids based on the protease substrate preferences learned from HyCoSuL, we can predictably modify the potency and selectivity of the inhibitor. We identified the tetrapeptide JH-1144 (8) as a single digit nM inhibitor of HGFA, matriptase and hepsin with excellent selectivity over Factor Xa and thrombin. These unnatural peptides have increased metabolic stability relative to natural peptides of similar structure. The tripeptide inhibitor PK-1-89 (2) has excellent pharmacokinetics in mice with good compound exposure out to 24 h. In addition, we obtained an X-ray structure of the inhibitor MM1132 (15) bound to matriptase revealing an interesting binding conformation useful for future inhibitor design. Less |

Rational exploration of 2,4-diaminopyrimidines as DHFR inhibitors active against Mycobacterium abscessus and Mycobacterium avium, two emerging human pathogens

Meirelles et al., 2024 | Biological and Medicinal Chemistry | Link

Nontuberculous mycobacteria NTM are emerging human pathogens linked to severe pulmonary diseases Current treatments involve the prolonged use of multiple drugs and are often ineffective Bacterial dihydrofolate reductase DHFR is a key enzyme targeted by antibiotics in Gram-negative bacterial infections However existing DHFR inhibitors designed for Gram-negative bacteria often fail against mycobacterial DHFRs Here we detail the rational design of NTM DHFR inhibitors based on P a malarial DHFR inhibitor We identified a -diaminopyrimidine exhibiting improved pharmacological properties and activity against purified DHFR and whole cell cultures of two predominant NTM species Mycobacterium avium and Mycobacterium abscessus This study underscores ... More |

Nontuberculous mycobacteria (NTM) are emerging human pathogens linked to severe pulmonary diseases. Current treatments involve the prolonged use of multiple drugs and are often ineffective. Bacterial dihydrofolate reductase (DHFR) is a key enzyme targeted by antibiotics in Gram-negative bacterial infections. However, existing DHFR inhibitors designed for Gram-negative bacteria often fail against mycobacterial DHFRs. Here, we detail the rational design of NTM DHFR inhibitors based on P218, a malarial DHFR inhibitor. We identified 8, a 2,4-diaminopyrimidine exhibiting improved pharmacological properties and activity against purified DHFR and whole cell cultures of two predominant NTM species: Mycobacterium avium and Mycobacterium abscessus. This study underscores the potential of 8 as a promising candidate for the in vivo validation of DHFR as an effective treatment against NTM infections. Less |

Structural insight into the functional regulation of Elongation factor Tu by reactive oxygen species in Synechococcus elongatus PCC 7942

Cheng et al., 2024 | International Journal of Biological Macromolecules | Link

In cyanobacteria Elongation factor Tu EF-Tu plays a crucial role in the repair of photosystem II PSII which is highly susceptible to oxidative stress induced by light exposure and regulated by reactive oxygen species ROS However the specific molecular mechanism governing the functional regulation of EF-Tu by ROS remains unclear Previous research has shown that a mutated EF-Tu where C is substituted with a Ser residue can alleviate photoinhibition highlighting the important role of C in EF-Tu photosensitivity In this study we elucidated how ROS deactivate EF-Tu by examining the crystal structures of EF-Tu in both wild-type and mutated form ... More |

In cyanobacteria, Elongation factor Tu (EF-Tu) plays a crucial role in the repair of photosystem II (PSII), which is highly susceptible to oxidative stress induced by light exposure and regulated by reactive oxygen species (ROS). However, the specific molecular mechanism governing the functional regulation of EF-Tu by ROS remains unclear. Previous research has shown that a mutated EF-Tu, where C82 is substituted with a Ser residue, can alleviate photoinhibition, highlighting the important role of C82 in EF-Tu photosensitivity. In this study, we elucidated how ROS deactivate EF-Tu by examining the crystal structures of EF-Tu in both wild-type and mutated form (C82S) individually at resolutions of 1.7 Å and 2.0 Å in Synechococcus elongatus PCC 7942 complexed with GDP. Specifically, the GDP-bound form of EF-Tu adopts an open conformation with C82 located internally, making it resistant to oxidation. Coordinated conformational changes in switches I and II create a tunnel that positions C82 for ROS interaction, revealing the vulnerability of the closed conformation of EF-Tu to oxidation. An analysis of these two structures reveals that the precise spatial arrangement of C82 plays a crucial role in modulating EF-Tu's response to ROS, serving as a regulatory element that governs photosynthetic biosynthesis. Less |

Structural and biochemical characterization of USP28 inhibition by small molecule inhibitors

Nair et al., 2024 | Thesis/ Dessertation | Link

Ubiquitination is an important post-translational modification that maintains cellular homeostasis by regulating various biological processes Deubiquitinases DUBs are enzymes that reverse the ubiquitination process by catalyzing the removal of ubiquitin from a substrate Abnormal expression or function of DUBs is often associated with the onset and progression of various diseases including cancer Ubiquitin specific proteases USPs which constitute the largest family of DUBs in humans have become the center of interest as potential targets in cancer therapy as many of them display increased activity or are overexpressed in a range of malignant tumors or the tumor microenvironment Two related members ... More |

Ubiquitination is an important post-translational modification that maintains cellular homeostasis by regulating various biological processes. Deubiquitinases (DUBs) are enzymes that reverse the ubiquitination process by catalyzing the removal of ubiquitin from a substrate. Abnormal expression or function of DUBs is often associated with the onset and progression of various diseases, including cancer. Ubiquitin specific proteases (USPs), which constitute the largest family of DUBs in humans, have become the center of interest as potential targets in cancer therapy as many of them display increased activity or are overexpressed in a range of malignant tumors or the tumor microenvironment. Two related members of the USP family, USP28 and USP25, share high sequence identities but play diverse biological roles. USP28 regulates cell proliferation, oncogenesis, DNA damage repair and apoptosis, whereas USP25 is involved in the anti-viral response, innate immunity and ER-associated degradation in addition to carcinogenesis. USP28 and USP25 also exhibit different oligomeric states – while USP28 is a constitutively active dimer, USP25 assumes an auto-inhibited tetrameric structure. The catalytic domains of both USP28 and USP25 comprise the canonical, globular USP-domain but contain an additional, extended insertion site called USP25/28 catalytic domain inserted domain (UCID) that mediates oligomerization of the proteins. Disruption of the USP25 tetramer leads to the formation of an activated dimeric protein. However, it is still not clear what triggers its activation. Due to their role in maintaining and stabilizing numerous oncoproteins, USP28 and USP25 have emerged as interesting candidates for anti-cancer therapy. Recent advances in smallmolecular inhibitor development have led to the discovery of relatively potent inhibitors of USP28 and USP25. This thesis focuses on the structural elucidation of USP28 and the biochemical characterization of USP28/USP25, both in complex with representatives of three out of the eight compound classes reported as USP28/USP25-specific inhibitors. The crystal structures of USP28 in complex with the AZ compounds, Vismodegib and FT206 reveal that all three inhibitor classes bind into the same allosteric pocket distant from the catalytic center, located between the palm and the thumb subdomains (the S1-site). Intriguingly, this binding pocket is identical to the UCID-tip binding interface in the USP25 tetramer, rendering the protein in a locked, inactive conformation. Formation of the binding pocket in USP28 requires a shift in the helix α5, which induces conformational changes and local distortion of the binding channel that typically accommodates the C-terminal tail of I Ubiquitin, thus preventing catalysis and abrogating USP28 activity. The key residues of the USP28-inhibitor binding pocket are highly conserved in USP25. Mutagenesis studies of these residues accompanied by biochemical and biophysical assays confirm the proposed mechanism of inhibition and similar binding to USP25. This work provides valuable insights into the inhibition mechanism of the small molecule compounds specifically for the DUBs USP28 and USP25. The USP28-inhibitor complex structures offer a framework to develop more specific and potent inhibitors. Less |

Small-Angle X‑ray Scattering as a Powerful Tool for Phase and Crystallinity Assessment of Monoclonal Antibody Crystallites in Support of Batch Crystallization

Larpent et al., 2024 | Molecular Pharmaceutics | Link

Crystalline suspensions of monoclonal antibodies mAbs have great potential to improve drug substance isolation and purification on a large scale and to be used for drug delivery via high-concentration formulations Crystalline mAb suspensions are expected to have enhanced chemical and physical properties relative to mAb solutions delivered intravenously making them attractive candidates for subcutaneous delivery In contrast to small molecules the development of protein crystalline suspensions is not a widely used approach in the pharmaceutical industry This is mainly due to the challenges in finding crystalline hits and the suboptimal physical properties of the resulting crystallites when hits are found ... More |

Crystalline suspensions of monoclonal antibodies (mAbs) have great potential to improve drug substance isolation
and purification on a large scale and to be used for drug delivery via high-concentration formulations. Crystalline mAb suspensions
are expected to have enhanced chemical and physical properties relative to mAb solutions delivered intravenously, making them
attractive candidates for subcutaneous delivery. In contrast to small molecules, the development of protein crystalline suspensions is
not a widely used approach in the pharmaceutical industry. This is mainly due to the challenges in finding crystalline hits and the
suboptimal physical properties of the resulting crystallites when hits are found. Modern advances in instrumentation and increased
knowledge of mAb crystallization have, however, resulted in higher probabilities of discovering crystal forms and improving their
particle properties and characterization. In this regard, physical, analytical characterization plays a central role in the initial steps of
understanding and later optimizing the crystallization of mAbs and requires careful selection of the appropriate tools. This
contribution describes a novel crystal structure of the antibody pembrolizumab and demonstrates the usefulness of small-angle X-ray
scattering (SAXS) for characterizing its crystalline suspensions. It illustrates the advantages of SAXS when used to (i) confirm
crystallinity and crystal phase of crystallites produced in batch mode; (ii) confirm crystallinity under various conditions and detect
variations in crystal phases, enabling fine-tuning of the crystallizations for phase control across multiple batches; (iii) monitor the
physical response and stability of the crystallites in suspension with regard to filtration and washing; and (iv) monitor the physical
stability of the crystallites upon drying. Overall, this work highlights how SAXS is an essential tool for mAb crystallization
characterization. Less |

Structure-Guided Design of Potent Coronavirus Inhibitors with a 2‑Pyrrolidone Scaffold: Biochemical, Crystallographic, and Virological Studies

Dampalla et al., 2024 | Journal of Medicinal Chemistry | Link

Zoonotic coronaviruses are known to produce severe infections in humans and have been the cause of significant morbidity and mortality worldwide SARS-CoV- was the largest and latest contributor of fatal cases even though MERS-CoV has the highest case-fatality ratio among zoonotic coronaviruses These infections pose a high risk to public health worldwide warranting efforts for the expeditious discovery of antivirals Hence we hereby describe a novel series of inhibitors of coronavirus CLpro embodying an N-substituted -pyrrolidone scaffold envisaged to exploit favorable interactions with the S S subsites and connected to an invariant Leu-Gln P P recognition element Several inhibitors showed ... More |

Zoonotic coronaviruses are known to produce
severe infections in humans and have been the cause of significant
morbidity and mortality worldwide. SARS-CoV-2 was the largest
and latest contributor of fatal cases, even though MERS-CoV has
the highest case-fatality ratio among zoonotic coronaviruses. These
infections pose a high risk to public health worldwide warranting
efforts for the expeditious discovery of antivirals. Hence, we hereby
describe a novel series of inhibitors of coronavirus 3CLpro
embodying an N-substituted 2-pyrrolidone scaffold envisaged to
exploit favorable interactions with the S3−S4 subsites and
connected to an invariant Leu-Gln P2−P1 recognition element.
Several inhibitors showed nanomolar antiviral activity in enzyme and cell-based assays, with no significant cytotoxicity. Highresolution
crystal structures of inhibitors bound to the 3CLpro were determined to probe and identify the molecular determinants
associated with binding, to inform the structure-guided optimization of the inhibitors, and to confirm the mechanism of action of the
inhibitors. Less |

READDI protocol: Crystallisation of CHIKV nsP3 macrodomain

Aschenbrenner et al., 2024 | Protocols part of Springer nature | Link

Chikungunya virus CHIKV causes severe fever rash and debilitating joint pain that can last for months or even years Millions of people have been infected with CHIKV mostly in low- and middle-income countries and the virus continues to spread into new areas due to the geographical expansion of its mosquito hosts The crystallization protocol and buffer conditions used to obtain reproducible Chikungunya Virus nsP macrodomain crystals suitable for XChem fragment screening |

Discovery of an antivirulence compound that targets the Staphylococcus aureus SaeRS two-component system to inhibit toxic shock syndrome toxin-1 production

Dufresne et al., 2024 | Journal of Biological Chemistry | Link

Menstrual toxic shock syndrome mTSS is a rare but severe disorder associated with the use of menstrual products such as high-absorbency tampons and is caused by Staphylococcus aureus strains that produce the toxic shock syndrome toxin- TSST- superantigen Herein we screened a library of small bioactive molecules for the ability to inhibit transcription of the TSST- gene without inhibiting the growth of S aureus The dominant positive regulator of TSST- is the SaeRS two-component system TCS and we identified phenazopyridine hydrochloride PP-HCl that repressed the production of TSST- by inhibiting the kinase function of SaeS PP-HCl competed with ATP for ... More |

Menstrual toxic shock syndrome (mTSS) is a rare but severe disorder associated with the use of menstrual products such as high-absorbency tampons and is caused by Staphylococcus aureus strains that produce the toxic shock syndrome toxin-1 (TSST-1) superantigen. Herein, we screened a library of 3920 small bioactive molecules for the ability to inhibit transcription of the TSST-1 gene without inhibiting the growth of S. aureus. The dominant positive regulator of TSST-1 is the SaeRS two-component system (TCS), and we identified phenazopyridine hydrochloride (PP-HCl) that repressed the production of TSST-1 by inhibiting the kinase function of SaeS. PP-HCl competed with ATP for binding of the kinase SaeS leading to decreased phosphorylation of SaeR and reduced expression of TSST-1 as well as several other secreted virulence factors known to be regulated by SaeRS. PP-HCl targets the virulence of S. aureus, and it also decreases the impact of TSST-1 on human lymphocytes without affecting the healthy vaginal microbiota. Our findings demonstrate the promising potential of PP-HCl as a therapeutic strategy against mTSS. Less |

Synthesis and biological characterization of an orally bioavailable lactate dehydrogenase-A inhibitor against pancreatic cancer

Sharma et al., 2024 | European Journal of Medicinal Chemistry | Link

Lactate dehydrogenase-A LDHA is the major isoform of lactate dehydrogenases LDH that is overexpressed and linked to poor survival in pancreatic ductal adenocarcinoma PDAC Despite some progress current LDH inhibitors have poor structural and physicochemical properties or exhibit unfavorable pharmacokinetics that have hampered their development The present study reports the synthesis and biological evaluation of a novel class of LDHA inhibitors comprising a succinic acid monoamide motif Compounds and are structurally related analogs that demonstrated potent inhibition of LDHA with IC s of nM and nM respectively We solved cocrystal structures of compound -bound to LDHA that showed that the ... More |

Lactate dehydrogenase-A (LDHA) is the major isoform of lactate dehydrogenases (LDH) that is overexpressed and linked to poor survival in pancreatic ductal adenocarcinoma (PDAC). Despite some progress, current LDH inhibitors have poor structural and physicochemical properties or exhibit unfavorable pharmacokinetics that have hampered their development. The present study reports the synthesis and biological evaluation of a novel class of LDHA inhibitors comprising a succinic acid monoamide motif. Compounds 6 and 21 are structurally related analogs that demonstrated potent inhibition of LDHA with IC50s of 46 nM and 72 nM, respectively. We solved cocrystal structures of compound 21-bound to LDHA that showed that the compound binds to a distinct allosteric site between the two subunits of the LDHA tetramer. Inhibition of LDHA correlated with reduced lactate production and reduction of glycolysis in MIA PaCa-2 pancreatic cancer cells. The lead compounds inhibit the proliferation of human pancreatic cancer cell lines and patient-derived 3D organoids and exhibit a synergistic cytotoxic effect with the OXPHOS inhibitor phenformin. Unlike current LDHA inhibitors, 6 and 21 have appropriate pharmacokinetics and ligand efficiency metrics, exhibit up to 73% oral bioavailability, and a cumulative half-life greater than 4 h in mice. Less |

Structure and Stability of Ago2 MID-Nucleotide Complexes: All-in-One (Drop) His6-SUMO Tag Removal, Nucleotide Binding, and Crystal Growth

Lei et al., 2024 | Current Protocols | Link

The middle MID domain of eukaryotic Argonaute Ago proteins and archaeal and bacterial homologues mediates the interaction with the -terminal nucleotide of miRNA and siRNA guide strands The MID domain of human Ago hAgo is comprised of amino acids with a molecular weight of kDa MID adopts a Rossman-like beta -alpha -beta -alpha -beta -alpha -beta -alpha fold with a nucleotide specificity loop between beta and alpha Multiple crystal structures of nucleotides bound to hAgo MID have been reported whereby complexes were obtained by soaking ligands into crystals of MID domain alone This protocol describes a simplified one-step approach to ... More |

The middle (MID) domain of eukaryotic Argonaute (Ago) proteins and archaeal and bacterial homologues mediates the interaction with the 5′-terminal nucleotide of miRNA and siRNA guide strands. The MID domain of human Ago2 (hAgo2) is comprised of 139 amino acids with a molecular weight of 15.56 kDa. MID adopts a Rossman-like beta1-alpha1-beta2-alpha2-beta3-alpha3-beta4-alpha4 fold with a nucleotide specificity loop between beta3 and alpha3. Multiple crystal structures of nucleotides bound to hAgo2 MID have been reported, whereby complexes were obtained by soaking ligands into crystals of MID domain alone. This protocol describes a simplified one-step approach to grow well-diffracting crystals of hAgo2 MID-nucleotide complexes by mixing purified His6-SUMO-MID fusion protein, Ulp1 protease, and excess nucleotide in the presence of buffer and precipitant. The crystal structures of MID complexes with UMP, UTP and 2′-3′ linked α-L-threofuranosyl thymidine-3′-triphosphate (tTTP) are presented. This article also describes fluorescence-based assays to measure dissociation constants (Kd) of MID-nucleotide interactions for nucleoside 5′-monophosphates and nucleoside 3′,5′-bisphosphates. Less |

The impact of exchanging the light and heavy chains on the structures of bovine ultralong antibodies

Clarke et al., 2024 | Structural Biology Communications | Link

The third complementary-determining regions of the heavy-chain CDR H variable regions VH of some cattle antibodies are highly extended consisting of or more residues These ultralong CDR Hs form -ribbon stalks that protrude from the surface of the antibody with a disulfide cross-linked knob region at their apex that dominates antigen interactions over the other CDR loops The structure of the Fab fragment of a naturally paired bovine ultralong antibody D identified by single B-cell sequencing has been determined to A resolution By swapping the D native light chain with that of an unrelated antigen-unknown ultralong antibody it is shown ... More |

The third complementary-determining regions of the heavy-chain (CDR3H) variable regions (VH) of some cattle antibodies are highly extended, consisting of 48 or more residues. These ‘ultralong’ CDR3Hs form �-ribbon stalks that protrude from the surface of the antibody with a disulfide cross-linked knob region at their apex that dominates antigen interactions over the other CDR loops. The structure of the Fab fragment of a naturally paired bovine ultralong antibody (D08), identified by single B-cell sequencing, has been determined to 1.6 A ˚ resolution. By swapping the D08 native light chain with that of an unrelated antigen-unknown ultralong antibody, it is shown that interactions between the CDR3s of the variable domains potentially affect the fine positioning of the ultralong CDR3H; however, comparison with other crystallographic structures shows that crystalline packing is also a major contributor. It is concluded that, on balance, the exact positioning of ultralong CDR3H loops is most likely to be due to the constraints of crystal packing. Less |

Discovery of JNJ-74856665: A Novel Isoquinolinone DHODH Inhibitor for the Treatment of AML

DeRatt et al., 2024 | Journal of Medicinal Chemistry | Link

Acute myelogenous leukemia AML a heterogeneous disease of the blood and bone marrow is characterized by the inability of myeloblasts to differentiate into mature cell types Dihydroorotate dehydrogenase DHODH is an enzyme well-known in the pyrimidine biosynthesis pathway and preclinical findings demonstrated that DHODH is a metabolic vulnerability in AML as inhibitors can induce differentiation across multiple AML subtypes As a result of virtual screening and structure-based drug design approaches a novel series of isoquinolinone DHODH inhibitors was identified Further lead optimization afforded JNJ- as an orally bioavailable potent and selective DHODH inhibitor with favorable physicochemical properties selected for clinical ... More |

Acute myelogenous leukemia (AML), a heterogeneous disease of the blood and bone marrow, is characterized by the inability of myeloblasts to differentiate into mature cell types. Dihydroorotate dehydrogenase (DHODH) is an enzyme well-known in the pyrimidine biosynthesis pathway and preclinical findings demonstrated that DHODH is a metabolic vulnerability in AML as inhibitors can induce differentiation across multiple AML subtypes. As a result of virtual screening and structure-based drug design approaches, a novel series of isoquinolinone DHODH inhibitors was identified. Further lead optimization afforded JNJ-74856665 as an orally bioavailable, potent, and selective DHODH inhibitor with favorable physicochemical properties selected for clinical development in patients with AML and myelodysplastic syndromes (MDS). Less |

A ‘rich-get-richer’ mechanism drives patchy dynamics and resistance evolution in antibiotic-treated bacteria

Emrah et al., 2024 | Molecular Systems Biology | Link

Bacteria in nature often form surface-attached communities that initially comprise distinct subpopulations or patches For pathogens these patches can form at infection sites persist during antibiotic treatment and develop into mature biofilms Evidence suggests that patches can emerge due to heterogeneity in the growth environment and bacterial seeding as well as cell-cell signaling However it is unclear how these factors contribute to patch formation and how patch formation might affect bacterial survival and evolution Here we demonstrate that a 'rich-get-richer' mechanism drives patch formation in bacteria exhibiting collective survival CS during antibiotic treatment Modeling predicts that the seeding heterogeneity of ... More |

Bacteria in nature often form surface-attached communities that initially comprise distinct subpopulations, or patches. For pathogens, these patches can form at infection sites, persist during antibiotic treatment, and develop into mature biofilms. Evidence suggests that patches can emerge due to heterogeneity in the growth environment and bacterial seeding, as well as cell-cell signaling. However, it is unclear how these factors contribute to patch formation and how patch formation might affect bacterial survival and evolution. Here, we demonstrate that a 'rich-get-richer' mechanism drives patch formation in bacteria exhibiting collective survival (CS) during antibiotic treatment. Modeling predicts that the seeding heterogeneity of these bacteria is amplified by local CS and global resource competition, leading to patch formation. Increasing the dose of a non-eradicating antibiotic treatment increases the degree of patchiness. Experimentally, we first demonstrated the mechanism using engineered Escherichia coli and then demonstrated its applicability to a pathogen, Pseudomonas aeruginosa. We further showed that the formation of P. aeruginosa patches promoted the evolution of antibiotic resistance. Our work provides new insights into population dynamics and resistance evolution during surface-attached bacterial growth. Less |

Managing macromolecular crystallographic data with a laboratory information management system

Daniel et al., 2024 | Structural Biology | Link

Protein crystallography is an established method to study the atomic structures of macromolecules and their complexes A prerequisite for successful structure determination is diffraction-quality crystals which may require extensive optimization of both the protein and the conditions and hence projects can stretch over an extended period with multiple users being involved The workflow from crystallization and crystal treatment to deposition and publication is well defined and therefore an electronic laboratory information management system LIMS is well suited to management of the data Completion of the project requires key information on all the steps being available and this information should also ... More |

Protein crystallography is an established method to study the atomic structures of macromolecules and their complexes. A prerequisite for successful structure determination is diffraction-quality crystals, which may require extensive optimization of both the protein and the conditions, and hence projects can stretch over an extended period, with multiple users being involved. The workflow from crystallization and crystal treatment to deposition and publication is well defined, and therefore an electronic laboratory information management system (LIMS) is well suited to management of the data. Completion of the project requires key information on all the steps being available and this information should also be made available according to the FAIR principles. As crystallized samples are typically shipped between facilities, a key feature to be captured in the LIMS is the exchange of metadata between the crystallization facility of the home laboratory and, for example, synchrotron facilities. On completion, structures are deposited in the Protein Data Bank (PDB) and the LIMS can include the PDB code in its database, completing the chain of custody from crystallization to structure deposition and publication. A LIMS designed for macromolecular crystallography, IceBear, is available as a standalone installation and as a hosted service, and the implementation of key features for the capture of metadata in IceBear is discussed as an example. Less |

Rapid high-throughput method for investigating physiological regulation of neutrophil extracellular trap formation

Zukas et al., 2024 | Journal of Thrombosis and Haemostasis | Link

Background Neutrophils the most abundant white blood cells in humans play pivotal roles in innate immunity rapidly migrating to sites of infection and inflammation to phagocytose neutralize and eliminate invading pathogens Neutrophil extracellular trap NET formation is increasingly recognized as an essential rapid innate immune response but when dysregulated it contributes to pathogenesis of sepsis and immunothrombotic disease Objectives Current NETosis models are limited routinely employing nonphysiological triggers that can bypass natural NET regulatory pathways Models utilizing isolated neutrophils and immortalized cell lines do not reflect the complex biology underlying neutrophil activation and NETosis that occurs in whole blood To ... More |

Background
Neutrophils, the most abundant white blood cells in humans, play pivotal roles in innate immunity, rapidly migrating to sites of infection and inflammation to phagocytose, neutralize, and eliminate invading pathogens. Neutrophil extracellular trap (NET) formation is increasingly recognized as an essential rapid innate immune response, but when dysregulated, it contributes to pathogenesis of sepsis and immunothrombotic disease.

Objectives
Current NETosis models are limited, routinely employing nonphysiological triggers that can bypass natural NET regulatory pathways. Models utilizing isolated neutrophils and immortalized cell lines do not reflect the complex biology underlying neutrophil activation and NETosis that occurs in whole blood. To our knowledge, we report the first human ex vivo model utilizing naturally occurring molecules to induce NETosis in whole blood. This approach could be used for drug screening and, importantly, inadvertent activators of NETosis.

Methods
Here we describe a novel, high-throughput ex vivo whole blood–induced NETosis model using combinatorial pooling of native NETosis-inducing factors in a more biologically relevant Synthetic-Sepsis model.

Results
We found different combinations of factors evoked distinct neutrophil responses in the rate of NET generation and/or magnitude of NETosis. Despite interdonor variability, similar sets of proinflammatory molecules induced consistent responses across donors. We found that at least 3 biological triggers were necessary to induce NETosis in our system including either tumor necrosis factor-α or lymphotoxin-α.

Conclusion
These findings emphasize the importance of investigating neutrophil physiology in a biologically relevant context to enable a better understanding of disease pathology, risk factors, and therapeutic targets, potentially providing novel strategies for disease intervention and treatment. Less |

Protocol for generation of and high-throughput drug testing with patient-derived colorectal cancer organoids

Tan et al., 2024 | STAR Protocols | Link

Drug sensitivity testing of patient-derived tumor organoids PDTOs is a promising tool for personalizing cancer treatment Here we present a protocol for generation of and high-throughput drug testing with PDTOs We describe detailed steps for PDTO establishment from colorectal cancer tissues preparation of PDTOs for high-throughput drug testing and quantification of drug testing results using image analysis This protocol provides a standardized workflow for PDTO testing of standard-of-care therapies along with exploring the activity of new agents for translational research |

Pseudomonas aeruginosa gene PA4880 encodes a Dps-like protein with a Dps fold, bacterioferritin-type ferroxidase centers, and endonuclease activity

Rajapaksha et al., 2024 | Frontiers in Molecular Biosciences | Link

We report the biochemical structural and functional characterization of the protein coded by gene PA in the P aeruginosa PAO genome The PA gene had been annotated as coding a probable bacterioferritin Our structural work shows that the product of gene PA is a protein that adopts the Dps subunit fold which oligomerizes into a -mer quaternary structure Unlike Dps however the ferroxidase di-iron centers and iron coordinating ligands are buried within each subunit in a manner identical to that observed in the ferroxidase center of P aeruginosa bacterioferritin Since these structural characteristics correspond to Dps-like proteins we term the ... More |

We report the biochemical, structural, and functional characterization of the protein coded by gene PA4880 in the P. aeruginosa PAO1 genome. The PA4880 gene had been annotated as coding a probable bacterioferritin. Our structural work shows that the product of gene PA4880 is a protein that adopts the Dps subunit fold, which oligomerizes into a 12-mer quaternary structure. Unlike Dps, however, the ferroxidase di-iron centers and iron coordinating ligands are buried within each subunit, in a manner identical to that observed in the ferroxidase center of P. aeruginosa bacterioferritin. Since these structural characteristics correspond to Dps-like proteins, we term the protein as P. aeruginosa Dps-like, or Pa DpsL. The ferroxidase centers in Pa DpsL catalyze the oxidation of Fe2+ utilizing O2 or H2O2 as oxidant, and the resultant Fe3+ is compartmentalized in the interior cavity. Interestingly, incubating Pa DpsL with plasmid DNA results in efficient nicking of the DNA and at higher concentrations of Pa DpsL the DNA is linearized and eventually degraded. The nickase and endonuclease activities suggest that Pa DpsL, in addition to participating in the defense of P. aeruginosa cells against iron-induced toxicity, may also participate in the innate immune mechanisms consisting of restriction endonucleases and cognate methyl transferases. Less |

Macrocyclic Azapeptide Nitriles: Structure-Based Discovery of Potent SARS-CoV‑2 Main Protease Inhibitors as Antiviral Drugs

Breidenbach et al., 2024 | Journal of Medicinal Chemistry | Link

Given the crucial role of the main protease Mpro in the replication cycle of SARS-CoV- this viral cysteine protease constitutes a high-profile drug target We investigated peptidomimetic azapeptide nitriles as auspicious irreversibly acting inhibitors of Mpro Our systematic approach combined an Mpro active-site scanning by combinatorially assembled azanitriles with structure-based design Encouraged by the bioactive conformation of open-chain inhibitors we conceptualized the novel chemotype of macrocyclic azanitriles whose binding mode was elucidated by cocrystallization This strategy provided a favorable entropic contribution to target binding and resulted in the development of the extraordinarily potent Mpro inhibitor with an IC value of ... More |

Given the crucial role of the main protease (Mpro) in the replication cycle of SARS-CoV-2, this viral cysteine protease constitutes a high-profile drug target. We investigated peptidomimetic azapeptide nitriles as auspicious, irreversibly acting inhibitors of Mpro. Our systematic approach combined an Mpro active-site scanning by combinatorially assembled azanitriles with structure-based design. Encouraged by the bioactive conformation of open-chain inhibitors, we conceptualized the novel chemotype of macrocyclic azanitriles whose binding mode was elucidated by cocrystallization. This strategy provided a favorable entropic contribution to target binding and resulted in the development of the extraordinarily potent Mpro inhibitor 84 with an IC50 value of 3.23 nM and a second-order rate constant of inactivation, kinac/Ki, of 448,000 M–1s–1. The open-chain Mpro inhibitor 58, along with the macrocyclic compounds 83 and 84, a broad-spectrum anticoronaviral agent, demonstrated the highest antiviral activity with EC50 values in the single-digit micromolar range. Our findings are expected to promote the future development of peptidomimetic Mpro inhibitors as anti-SARS-CoV-2 agents. Less |

Caskin2 is a novel talin- and Abi1-binding protein that promotes cell motility

Wang et al., 2024 | Journal of Cell Science | Link

Talin herein referring collectively to talin and couples the actomyosin cytoskeleton to integrins and transmits tension to the extracellular matrix Talin also interacts with numerous additional proteins capable of modulating the actin-integrin linkage and thus downstream mechanosignaling cascades Here we demonstrate that the scaffold protein Caskin interacts directly with the R domain of talin through its C-terminal LD motif Caskin also associates with the WAVE regulatory complex to promote cell migration in an Abi -dependent manner Furthermore we demonstrate that the Caskin Abi interaction is regulated by growth factor-induced phosphorylation of Caskin on serine In MCF and UACC cells which ... More |

Talin (herein referring collectively to talin 1 and 2) couples the actomyosin cytoskeleton to integrins and transmits tension to the extracellular matrix. Talin also interacts with numerous additional proteins capable of modulating the actin-integrin linkage and thus downstream mechanosignaling cascades. Here, we demonstrate that the scaffold protein Caskin2 interacts directly with the R8 domain of talin through its C-terminal LD motif. Caskin2 also associates with the WAVE regulatory complex to promote cell migration in an Abi1-dependent manner. Furthermore, we demonstrate that the Caskin2–Abi1 interaction is regulated by growth factor-induced phosphorylation of Caskin2 on serine 878. In MCF7 and UACC893 cells, which contain an amplification of CASKIN2, Caskin2 localizes in plasma membrane-associated plaques and around focal adhesions in cortical microtubule stabilization complexes. Taken together, our results identify Caskin2 as a novel talin-binding protein that might not only connect integrin-mediated adhesion to actin polymerization but could also play a role in crosstalk between integrins and microtubules. Less |

Spectroscopic insights into multi-phase protein crystallization in complex lysate using Raman spectroscopy and a particle-free bypass

Wegner et al., 2024 | Frontiers in Bioengineering and Biotechnology | Link

Protein crystallization as opposed to well-established chromatography processes has the benefits to reduce production costs while reaching a comparable high purity However monitoring crystallization processes remains a challenge as the produced crystals may interfere with analytical measurements Especially for capturing proteins from complex feedstock containing various impurities establishing reliable process analytical technology PAT to monitor protein crystallization processes can be complicated In heterogeneous mixtures important product characteristics can be found by multivariate analysis and chemometrics thus contributing to the development of a thorough process understanding In this project an analytical set-up is established combining offline analytics on-line ultraviolet visible light ... More |

Protein crystallization as opposed to well-established chromatography processes has the benefits to reduce production costs while reaching a comparable high purity. However, monitoring crystallization processes remains a challenge as the produced crystals may interfere with analytical measurements. Especially for capturing proteins from complex feedstock containing various impurities, establishing reliable process analytical technology (PAT) to monitor protein crystallization processes can be complicated. In heterogeneous mixtures, important product characteristics can be found by multivariate analysis and chemometrics, thus contributing to the development of a thorough process understanding. In this project, an analytical set-up is established combining offline analytics, on-line ultraviolet visible light (UV/Vis) spectroscopy, and in-line Raman spectroscopy to monitor a stirred-batch crystallization process with multiple phases and species being present. As an example process, the enzyme Lactobacillus kefir alcohol dehydrogenase (LkADH) was crystallized from clarified Escherichia coli (E. coli) lysate on a 300 mL scale in five distinct experiments, with the experimental conditions changing in terms of the initial lysate solution preparation method and precipitant concentration. Since UV/Vis spectroscopy is sensitive to particles, a cross-flow filtration (cross-flow filtration)-based bypass enabled the on-line analysis of the liquid phase providing information on the lysate composition regarding the nucleic acid to protein ratio. A principal component analysis (PCA) of in situ Raman spectra supported the identification of spectra and wavenumber ranges associated with productspecific information and revealed that the experiments followed a comparable, spectral trend when crystals were present. Based on preprocessed Raman spectra, a partial least squares (PLS) regression model was optimized to monitor the target molecule concentration in real-time. The off-line sample analysis provided information on the crystal number and crystal geometry by automated image analysis as well as the concentration of LkADH and host cell proteins (HCPs) In spite of a complex lysate suspension containing scattering crystals and various impurities, it was possible to monitor the target molecule concentration in a heterogeneous, multi-phase process using spectroscopic methods. With the presented analytical set-up of off-line, particle-sensitive on-line, and in-line analyzers, a crystallization capture process can be characterized better in terms of the geometry, yield, and purity of the crystals. Less |

Cyanobacterial α-carboxysome carbonic anhydrase is allosterically regulated by the Rubisco substrate RuBP

Pulsford et al., 2024 | Science Advances | Link

Cyanobacterial CO concentrating mechanisms CCMs sequester a globally consequential proportion of carbon into the biosphere Proteinaceous microcompartments called carboxysomes play a critical role in CCM function housing two enzymes to enhance CO fixation carbonic anhydrase CA and Rubisco Despite its importance our current understanding of the carboxysomal CAs found in -cyanobacteria CsoSCA remains limited particularly regarding the regulation of its activity Here we present a structural and biochemical study of CsoSCA from the cyanobacterium Cyanobium sp PCC Our results show that the Cyanobium CsoSCA is allosterically activated by the Rubisco substrate ribulose- -bisphosphate and forms a hexameric trimer of dimers ... More |

Cyanobacterial CO2 concentrating mechanisms (CCMs) sequester a globally consequential proportion of carbon into the biosphere. Proteinaceous microcompartments, called carboxysomes, play a critical role in CCM function, housing two enzymes to enhance CO2 fixation: carbonic anhydrase (CA) and Rubisco. Despite its importance, our current understanding of the carboxysomal CAs found in α-cyanobacteria, CsoSCA, remains limited, particularly regarding the regulation of its activity. Here, we present a structural and biochemical study of CsoSCA from the cyanobacterium Cyanobium sp. PCC7001. Our results show that the Cyanobium CsoSCA is allosterically activated by the Rubisco substrate ribulose-1,5-bisphosphate and forms a hexameric trimer of dimers. Comprehensive phylogenetic and mutational analyses are consistent with this regulation appearing exclusively in cyanobacterial α-carboxysome CAs. These findings clarify the biologically relevant oligomeric state of α-carboxysomal CAs and advance our understanding of the regulation of photosynthesis in this globally dominant lineage. Less |

AvrSr27 is a zinc-bound effector with a modular structure important for immune recognition

Outram et al., 2024 | New Phytologist | Link

Effector proteins are central to the success of plant pathogens while immunity in hostplants is driven by receptor-mediated recognition of these effectors Understanding the mole-cular details of effector receptor interactions is key for the engineering of novel immunereceptors Here we experimentally determined the crystal structure of the Puccinia graminis f sp tri-tici Pgt effector AvrSr which was not accurately predicted using AlphaFold We charac-terised the role of the conserved cysteine residues in AvrSr using in vitro biochemical assaysand examined Sr -mediated recognition using transient expression in Nicotiana spp andwheat protoplasts The AvrSr structure contains a novel b-strand rich modular fold ... More |

Effector proteins are central to the success of plant pathogens, while immunity in hostplants is driven by receptor-mediated recognition of these effectors. Understanding the mole-cular details of effector–receptor interactions is key for the engineering of novel immunereceptors. Here, we experimentally determined the crystal structure of the Puccinia graminis f. sp. tri-tici (Pgt) effector AvrSr27, which was not accurately predicted using AlphaFold2. We charac-terised the role of the conserved cysteine residues in AvrSr27 using in vitro biochemical assaysand examined Sr27-mediated recognition using transient expression in Nicotiana spp. andwheat protoplasts. The AvrSr27 structure contains a novel b-strand rich modular fold consisting of two structu-rally similar domains that bind to Zn2+ ions. The N-terminal domain of AvrSr27 is sufﬁcient forinteraction with Sr27 and triggering cell death. We identiﬁed two Pgt proteins structurallyrelated to AvrSr27 but with low sequence identity that can also associate with Sr27, albeitmore weakly. Though only the full-length proteins, trigger Sr27-dependent cell death in tran-sient expression systems. Collectively, our ﬁndings have important implications for utilising protein prediction plat-forms for effector proteins, and those embarking on bespoke engineering of immunity recep-tors as solutions to plant disease Less |

A versatile bacterial innate immunity protein directly senses two disparate phage proteins

Zhang et al., 2024 | Preprint | Link

Eukaryotic innate immune systems use pattern recognition receptors PRRs to sense infection by detecting pathogen-associated molecular patterns which then triggers an immune response Bacteria have similarly evolved immunity proteins that sense certain components of their viral predators known as bacteriophages Although different immunity proteins can recognize different phage-encoded triggers individual bacterial immunity proteins have only been found to sense a single trigger during infection suggesting a one-to-one relationship between bacterial PRRs and their ligands Here we demonstrate that the anti-phage defense protein CapRelSJ in Escherichia coli can directly bind and sense two completely unrelated and structurally different proteins using the ... More |

Eukaryotic innate immune systems use pattern recognition receptors (PRRs) to sense infection by detecting pathogen-associated molecular patterns, which then triggers an immune response. Bacteria have similarly evolved immunity proteins that sense certain components of their viral predators known as bacteriophages1–6. Although different immunity proteins can recognize different phage-encoded triggers, individual bacterial immunity proteins have only been found to sense a single trigger during infection, suggesting a one-to-one relationship between bacterial PRRs and their ligands7–11. Here, we demonstrate that the anti-phage defense protein CapRelSJ46 in Escherichia coli can directly bind and sense two completely unrelated and structurally different proteins using the same sensory domain, with overlapping but distinct interfaces. Our results highlight the remarkable versatility of an immune sensory domain, which may be a common property of anti-phage defense systems and enable them to keep pace with their rapidly evolving viral predators. We found that Bas11 phages harbor both trigger proteins that are sensed by CapRelSJ46 during infection, and we demonstrate that such phage can only fully evade CapRelSJ46 defense when both triggers are mutated. Our work reveals how a bacterial immune system that senses more than one trigger can help prevent phages from easily escaping detection, and it may allow detection of a broader range of phages. More generally, our findings illustrate unexpected multifactorial sensing by bacterial defense systems and complex coevolutionary relationships between them and their phage-encoded triggers. Less |

High Resolution Crystal Structure of the Pyruvate Kinase Tetramer in Complex with the Allosteric Activator Mitapivat/AG-348

Xiao et al., 2024 | Crystals | Link

Pyruvate kinase PK deficiency is a rare genetic disorder that affects this critical enzyme within the glycolysis pathway In recent years Mitapivat MTPV AG- has emerged as a notable allosteric activator for treating PK deficiency However the allosteric regulatory effects exerted on PK by MTPV are yet to be comprehensively elucidated To shed light on the molecular mechanisms of the allosteric effects we employed crystallography and biophysical methods Our efforts yielded a high-resolution crystal structure of the PK tetramer complexed with MTPV at resolution Isothermal titration calorimetry measurements revealed that MTPV binds to human PK with an affinity of M ... More |

Pyruvate kinase (PK) deficiency is a rare genetic disorder that affects this critical enzyme within the glycolysis pathway. In recent years, Mitapivat (MTPV, AG-348) has emerged as a notable allosteric activator for treating PK deficiency. However, the allosteric regulatory effects exerted on PK by MTPV are yet to be comprehensively elucidated. To shed light on the molecular mechanisms of the allosteric effects, we employed crystallography and biophysical methods. Our efforts yielded a high-resolution crystal structure of the PK tetramer complexed with MTPV at 2.1 Å resolution. Isothermal titration calorimetry measurements revealed that MTPV binds to human PK with an affinity of 1 μM. The enhanced structural details now allow for unambiguous analysis of the MTPV-filled cavity intricately embedded within the enzyme. Finally, the structure suggests that MTPV binding induces an allosteric effect on the B-domain situated proximal to the active site. In summary, our study provides valuable insights into the allosteric regulation of PK by MTPV and paves the way for further structure-based drug optimization for therapeutic interventions in PK deficiency. Less |

Dynamic Clinical Assay Pipeline for Detecting a Virus

Williams et al., 2024 | Google Patents | Link

Disclosed herein are methods and systems comprising obtaining nucleic acid from a sample that was obtained from a subject capturing and amplifying a target molecule in the nucleic acid using a molecular inversion probe under hybridization conditions ligating an adapter to create a circular molecule sequencing the circular molecule to obtain sequence reads generating a sequencing file comprising the sequence reads of each molecule and a position of each sequence read in a reference genome of a virus and generating a reporting file for the subject comprising a predicted lineage of the virus in the sample |

Crystallographic Fragment Screen of Coxsackievirus A16 2A Protease identifies new opportunities for the development of broad-spectrum anti-enterovirals

Lithgo et al., 2024 | Preprint | Link

Enteroviruses are the causative agents of paediatric hand-foot-and-mouth disease and a target for pandemic preparedness due to the risk of higher order complications in a large-scale outbreak The A protease of these viruses is responsible for the self-cleavage of the poly protein allowing for correct folding and assembly of capsid proteins in the final stages of viral replication These A proteases are highly conserved between Enterovirus species such as Enterovirus A and Coxsackievirus A Inhibition of the A protease deranges capsid folding and assembly preventing formation of mature virions in host cells and making the protease a valuable target for ... More |

Enteroviruses are the causative agents of paediatric hand-foot-and-mouth disease, and a target for pandemic preparedness due to the risk of higher order complications in a large-scale outbreak. The 2A protease of these viruses is responsible for the self-cleavage of the poly protein, allowing for correct folding and assembly of capsid proteins in the final stages of viral replication. These 2A proteases are highly conserved between Enterovirus species, such as Enterovirus A71 and Coxsackievirus A16. Inhibition of the 2A protease deranges capsid folding and assembly, preventing formation of mature virions in host cells and making the protease a valuable target for antiviral activity. Herein, we describe a crystallographic fragment screening campaign that identified 75 fragments which bind to the 2A protease including 38 unique compounds shown to bind within the active site. These fragments reveal a path for the development of non-peptidomimetic inhibitors of the 2A protease with broad-spectrum anti-enteroviral activity. Less |

Structural Insights into Partial Activation of the Prototypic G Protein-Coupled Adenosine A2A Receptor

Claff et al., 2024 | ACS Pharmacology & Translational Science | Link

The adenosine A A receptor A AAR belongs to the rhodopsin-like G protein-coupled receptor GPCR family which constitutes the largest class of GPCRs Partial agonists show reduced efficacy as compared to physiological agonists and can even act as antagonists in the presence of a full agonist Here we determined an X-ray crystal structure of the partial A AAR agonist -amino- - H-imidazol- -ylmethyl sulfanyl - -p-hydroxyphenyl- -pyridinedicarbonitrile LUF in complex with the A AAR construct A A-PSB -bRIL stabilized in its inactive conformation and being devoid of any mutations in the ligand binding pocket The determined high-resolution structure resolved water ... More |

The adenosine A2A receptor (A2AAR) belongs to the rhodopsin-like G protein-coupled receptor (GPCR) family, which constitutes the largest class of GPCRs. Partial agonists show reduced efficacy as compared to physiological agonists and can even act as antagonists in the presence of a full agonist. Here, we determined an X-ray crystal structure of the partial A2AAR agonist 2-amino-6-[(1H-imidazol-2-ylmethyl)sulfanyl]-4-p-hydroxyphenyl-3,5-pyridinedicarbonitrile (LUF5834) in complex with the A2AAR construct A2A-PSB2-bRIL, stabilized in its inactive conformation and being devoid of any mutations in the ligand binding pocket. The determined high-resolution structure (2.43 Å) resolved water networks and crucial binding pocket interactions. A direct hydrogen bond of the p-hydroxy group of LUF5834 with T883.36 was observed, an amino acid that was mutated to alanine in the most frequently used A2AAR crystallization constructs thus preventing the discovery of its interactions in most of the previous A2AAR co-crystal structures. G protein dissociation studies confirmed partial agonistic activity of LUF5834 as compared to that of the full agonist N-ethylcarboxamidoadenosine (NECA). In contrast to NECA, the partial agonist was still able to bind to the receptor construct locked in its inactive conformation by an S913.39K mutation, although with an affinity lower than that at the native receptor. This could explain the compound’s partial agonistic activity: while full A2AAR agonists bind exclusively to the active conformation, likely following conformational selection, partial agonists bind to active as well as inactive conformations, showing higher affinity for the active conformation. This might be a general mechanism of partial agonism also applicable to other GPCRs. Less |

Crystallization of SARS-CoV-2 N Protein

Marples et al., 2024 | Protocols part of Springer nature | Link

The crystallization protocol and buffer conditions used to obtain reproducible SARS C V- Nucelocapsid crystals suitable for XChem fragment screening |

Crystallization of Zika virus NS3 helicase

Godoy et al., 2024 | Protocols part of Springer nature | Link

The crystallization protocol and buffer conditions used to obtain Zika NS helicase crystals suitable for XChem fragment screening The Zika virus ZIKV discovered in Africa in swiftly spread across continents causing significant concern due to its recent association with microcephaly in newborns and Guillain-Barr syndrome in adults Despite a decrease in prevalence the potential for a resurgence remains necessitating urgent therapeutic interventions Like other flaviviruses ZIKV presents promising drug targets within its replication machinery notably the NS helicase NS Hel protein which plays critical roles in viral replication However a lack of structural information impedes the development of specific inhibitors ... More |

The crystallization protocol and buffer conditions used to obtain Zika NS3 helicase crystals suitable for XChem fragment screening. The Zika virus (ZIKV), discovered in Africa in 1947, swiftly spread across continents, causing significant concern due to its recent association with microcephaly in newborns and Guillain-Barré syndrome in adults. Despite a decrease in prevalence, the potential for a resurgence remains, necessitating urgent therapeutic interventions. Like other flaviviruses, ZIKV presents promising drug targets within its replication machinery, notably the NS3 helicase (NS3Hel) protein, which plays critical roles in viral replication. However, a lack of structural information impedes the development of specific inhibitors targeting NS3Hel. This protocol was used to grow Zika NS3 crystals that were applied high-throughput crystallographic fragment screening on ZIKV NS3 Helicase. Less |

Crystallization of Enterovirus D68 3C protease

Lithgo et al., 2024 | Protocols part of Springer nature | Link

The development of effective broad-spectrum antivirals forms an important part of preparing for future pandemics A cause for concern is the currently emerging pathogen Enterovirus D EV-D which primarily spreads through respiratory routes causing mostly mild to severe respiratory illness but in severe cases acute flaccid myelitis The C protease of EV-D is a potential target for the development of antiviral drugs due to its essential role in the viral life cycle and high sequence conservation This protocol was used to grow D C ProB crystals that were applied high-throughput crystallographic follow up compound screening on D C |

Insights into Early Phases of Phycocyanin Crystal Formation via SONICC Spectroscopy

Pechkova et al., 2024 | Crystals | Link

This research delves into the early nucleation stages of phycocyanin a protein pivotal for its fluorescent properties and crystalline stability and holding considerable potential for biotechnological applications The paper contrasts traditional crystallization methods with the innovative Langmuir Blodgett nanotemplate approach aiming to enhance molecular assembly and nucleation processes The study employs Langmuir Blodgett nanotemplates alongside second-order nonlinear imaging of chiral crystal SONICC spectroscopy This combination is designed to orderly organize phycocyanin molecules and provide a sensitive visualization of early-stage crystal formation capturing the intricate dynamics of protein crystallization The experiments were conducted under controlled conditions where surface pressure was maintained ... More |

This research delves into the early nucleation stages of phycocyanin, a protein pivotal for its fluorescent properties and crystalline stability and holding considerable potential for biotechnological applications. The paper contrasts traditional crystallization methods with the innovative Langmuir–Blodgett nanotemplate approach, aiming to enhance molecular assembly and nucleation processes. The study employs Langmuir–Blodgett nanotemplates alongside second-order nonlinear imaging of chiral crystal (SONICC) spectroscopy. This combination is designed to orderly organize phycocyanin molecules and provide a sensitive visualization of early-stage crystal formation, capturing the intricate dynamics of protein crystallization. The experiments were conducted under controlled conditions, where surface pressure was maintained at 26 mN/m and barrier speed at 70 cm/min to optimize the monolayer formation at the air–water interface. The Langmuir–Blodgett method, compared to traditional vapor diffusion techniques, shows improvements in the uniformity and efficiency of nucleation. The sensitivity of SONICC spectroscopy significantly enhances the visualization of the nucleation process, revealing a more structured and uniform crystalline assembly in the early stages of formation. This method demonstrates a substantial improvement in nucleation dynamics, leading to a more orderly growth process and potentially larger, well-ordered crystals. Integrating Langmuir–Blodgett nanotemplates with SONICC spectroscopy offers a significant step in understanding protein crystallization processes with insights into the nucleation and growth of protein crystals and broad implications for refining crystallography methodologies of protein-based biomaterials, contributing to the advancement of structural biology and materials science. Less |

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