1181 Citations
Ketol-acid reductoisomerase KARI the second enzyme in the branched-chain amino acid biosynthesis pathway is a potential drug target for bacterial infections including Mycobacterium tuberculosis Here we have screened the Medicines for Malaria Venture Pathogen Box against purified M tuberculosis Mt KARI and identified two compounds that have Ki values below nm In Mt cell susceptibility assays one of these compounds exhibited an IC value of m Co-crystallization of this compound - methylsulfonyl methyl - H-benzo b oxazin- -one MMV in complex with Staphylococcus aureus KARI which has identity with Mt KARI NADPH and Mg yielded a structure to resolution However ... More
Ketol-acid reductoisomerase (KARI), the second enzyme in the branched-chain amino acid biosynthesis pathway, is a potential drug target for bacterial infections including Mycobacterium tuberculosis. Here, we have screened the Medicines for Malaria Venture Pathogen Box against purified M. tuberculosis (Mt) KARI and identified two compounds that have Ki values below 200 nm. In Mt cell susceptibility assays one of these compounds exhibited an IC50 value of 0.8 μm. Co-crystallization of this compound, 3-((methylsulfonyl)methyl)-2H-benzo[b][1,4]oxazin-2-one (MMV553002), in complex with Staphylococcus aureus KARI, which has 56 % identity with Mt KARI, NADPH and Mg2+ yielded a structure to 1.72 Å resolution. However, only a hydrolyzed product of the inhibitor (i.e. 3-(methylsulfonyl)-2-oxopropanic acid, missing the 2-aminophenol attachment) is observed in the active site. Surprisingly, Mt cell susceptibility assays showed that the 2-aminophenol product is largely responsible for the anti-TB activity of the parent compound. Thus, 3-(methylsulfonyl)-2-oxopropanic acid was identified as a potent KARI inhibitor that could be further explored as a potential biocidal agent and we have shown 2-aminophenol, as an anti-TB drug lead, especially given it has low toxicity against human cells. The study highlights that careful analysis of broad screening assays is required to correctly interpret cell-based activity data. Less
Outbreaks of human epidemic nonbacterial gastroenteritis are mainly caused by noroviruses Viral replication requires a C-like cysteine protease CLpro which processes the kDa viral polyprotein into six functional proteins The CLpro has attracted much interest due to its potential as a target for antiviral drugs A system for growing high-quality crystals of native Southampton norovirus CLpro SV CP has been established allowing the ligand-free crystal structure to be determined to in a tetrameric state This also allowed crystal-based fragment screening to be performed with various compound libraries ultimately to guide drug discovery for SV CP A total of fragments were ... More
Outbreaks of human epidemic nonbacterial gastroenteritis are mainly caused by noroviruses. Viral replication requires a 3C-like cysteine protease (3CLpro) which processes the 200 kDa viral polyprotein into six functional proteins. The 3CLpro has attracted much interest due to its potential as a target for antiviral drugs. A system for growing high-quality crystals of native Southampton norovirus 3CLpro (SV3CP) has been established, allowing the ligand-free crystal structure to be determined to 1.3 Å in a tetrameric state. This also allowed crystal-based fragment screening to be performed with various compound libraries, ultimately to guide drug discovery for SV3CP. A total of 19 fragments were found to bind to the protease out of the 844 which were screened. Two of the hits were located at the active site of SV3CP and showed good inhibitory activity in kinetic assays. Another 5 were found at the enzyme’s putative RNA-binding site and a further 11 were located in the symmetric central cavity of the tetramer. Less
The ability to interrogate total RNA content of single cells would enable better mapping of the transcriptional logic behind emerging cell types and states However current RNA-seq methods are unable to simultaneously monitor both short and long poly A and poly A -transcripts at the single-cell level and thus deliver only a partial snapshot of the cellular RNAome Here we describe Smart-seq-total a method capable of assaying a broad spectrum of coding and non-coding RNA from a single cell Built upon the template-switch mechanism Smart-seq-total bears the key feature of its predecessor Smart-seq namely the ability to capture full-length transcripts ... More
The ability to interrogate total RNA content of single cells would enable better mapping of the transcriptional logic behind emerging cell types and states. However, current RNA-seq methods are unable to simultaneously monitor both short and long, poly(A)+ and poly(A)-transcripts at the single-cell level, and thus deliver only a partial snapshot of the cellular RNAome. Here, we describe Smart-seq-total, a method capable of assaying a broad spectrum of coding and non-coding RNA from a single cell. Built upon the template-switch mechanism, Smart-seq-total bears the key feature of its predecessor, Smart-seq2, namely, the ability to capture full-length transcripts with high yield and quality. It also outperforms current poly(A)–independent total RNA-seq protocols by capturing transcripts of a broad size range, thus, allowing us to simultaneously analyze protein-coding, long non-coding, microRNA and other non-coding RNA transcripts from single cells. We used Smart-seq-total to analyze the total RNAome of human primary fibroblasts, HEK293T and MCF7 cells as well as that of induced murine embryonic stem cells differentiated into embryoid bodies. We show that simultaneous measurement of non-coding RNA and mRNA from the same cell enables elucidation of new roles of non-coding RNA throughout essential processes such as cell cycle or lineage commitment. Moreover, we show that cell types can be distinguished based on the abundance of non-coding transcripts alone. Less
N -Methyladenosine m A is the most widespread internal messenger RNA modification in humans Despite recent progress in understanding the biological roles of m A the inability to install m A site specifically in individual transcripts has hampered efforts to elucidate causal relationships between the presence of a specific m A and phenotypic outcomes In the present study we demonstrate that nucleus-localized dCas fusions with a truncated METTL methyltransferase domain and cytoplasm-localized fusions with a modified METTL METTL methyltransferase complex can direct site-specific m A incorporation in distinct cellular compartments with the former fusion protein having particularly low off-target activity ... More
N6-Methyladenosine (m6A) is the most widespread internal messenger RNA modification in humans. Despite recent progress in understanding the biological roles of m6A, the inability to install m6A site specifically in individual transcripts has hampered efforts to elucidate causal relationships between the presence of a specific m6A and phenotypic outcomes. In the present study, we demonstrate that nucleus-localized dCas13 fusions with a truncated METTL3 methyltransferase domain and cytoplasm-localized fusions with a modified METTL3:METTL14 methyltransferase complex can direct site-specific m6A incorporation in distinct cellular compartments, with the former fusion protein having particularly low off-target activity. Independent cellular assays across multiple sites confirm that this targeted RNA methylation (TRM) system mediates efficient m6A installation in endogenous RNA transcripts with high specificity. Finally, we show that TRM can induce m6A-mediated changes to transcript abundance and alternative splicing. These findings establish TRM as a tool for targeted epitranscriptome engineering that can reveal the effect of individual m6A modifications and dissect their functional roles. Less
ADP-ribosylation is a post-translational modification involved in the regulation of many vital cellular processes This posttranslational modification is carried out by ADP-ribosyltransferases converting -NAD into nicotinamide and a protein-linked ADP-ribosyl group or a chain of PAR The reverse reaction release of ADP-ribose from the acceptor molecule is catalyzed by ADP-ribosylhydrolases Several hydrolases contain a macrodomain fold and activities of human macrodomain protein modules vary from reading or erasing mono- and poly-ADP-ribosylation Macrodomains have been linked to diseases such as cancer making them potential drug targets Discovery of inhibitors requires robust biochemical tools mostly lacking for hydrolases and here we describe ... More
ADP-ribosylation is a post-translational modification involved in the regulation of many vital cellular processes. This posttranslational modification is carried out by ADP-ribosyltransferases converting β-NAD+ into nicotinamide and a protein-linked ADP-ribosyl group or a chain of PAR. The reverse reaction, release of ADP-ribose from the acceptor molecule, is catalyzed by ADP-ribosylhydrolases. Several hydrolases contain a macrodomain fold, and activities of human macrodomain protein modules vary from reading or erasing mono- and poly-ADP-ribosylation. Macrodomains have been linked to diseases such as cancer, making them potential drug targets. Discovery of inhibitors requires robust biochemical tools mostly lacking for hydrolases, and here we describe an inhibitor screening assay against mono-ADP-ribosylhydrolyzing enzymes. The activity-based assay uses an α-NAD+, anomer of β-NAD+, which is accepted as a substrate by MacroD1, MacroD2, and ARH3 due to its resemblance to the protein-linked ADP-ribose. The amount of α-NAD+ present after hydrolysis is measured by chemically converting it on a microtiter plate to a fluorescent compound. We optimized the assay for MacroD2 and performed a proof-of-concept compound screening. Three compounds were identified as screening hits with micromolar potency. However, further characterization of the compounds identified them as protein destabilizers, excluding further follow-up studies. Validation and screening demonstrated the usability of the in vitro assay for MacroD2, and we also demonstrate the applicability of the assay as a tool for other human ADP-ribosylhydrolases. Less
The intracellular parasite Toxoplasma gondii employs a vast array of effector proteins from the rhoptry and dense granule organelles to modulate host cell biology these effectors are known as ROPs and GRAs respectively To examine the individual impacts of ROPs and GRAs on host gene expression we developed a robust novel protocol to enrich for ultrapure populations of a naturally occurring and reproducible population of host cells called uninfected-injected U-I cells which Toxoplasma injects with ROPs but subsequently fails to invade We then performed single-cell transcriptomic analysis at to h postinfection on U-I cells as well as on uninfected and ... More
The intracellular parasite Toxoplasma gondii employs a vast array of effector proteins from the rhoptry and dense granule organelles to modulate host cell biology; these effectors are known as ROPs and GRAs, respectively. To examine the individual impacts of ROPs and GRAs on host gene expression, we developed a robust, novel protocol to enrich for ultrapure populations of a naturally occurring and reproducible population of host cells called uninfected-injected (U-I) cells, which Toxoplasma injects with ROPs but subsequently fails to invade. We then performed single-cell transcriptomic analysis at 1 to 3 h postinfection on U-I cells (as well as on uninfected and infected controls) arising from infection with either wild-type parasites or parasites lacking the MYR1 protein, which is required for soluble GRAs to cross the parasitophorous vacuole membrane (PVM) and reach the host cell cytosol. Based on comparisons of infected and U-I cells, the host’s earliest response to infection appears to be driven primarily by the injected ROPs, which appear to induce immune and cellular stress pathways. These ROP-dependent proinflammatory signatures appear to be counteracted by at least some of the MYR1-dependent GRAs and may be enhanced by the MYR-independent GRAs (which are found embedded within the PVM). Finally, signatures detected in uninfected bystander cells from the infected monolayers suggest that MYR1-dependent paracrine effects also counteract inflammatory ROP-dependent processes. Less
The pharmaceutical industry is continuing to face high research and development R D costs and low overall success rates of clinical compounds during drug development There is an increasing demand for development and validation of healthy or disease-relevant and physiological human cellular models that can be implemented in early-stage discovery thereby shifting attrition of future therapeutics to a point in discovery at which the costs are significantly lower There needs to be a paradigm shift in the early drug discovery phase which is lengthy and costly away from simplistic cellular models that show an inability to effectively and efficiently reproduce ... More
The pharmaceutical industry is continuing to face high research and development (R&D) costs and low overall success rates of clinical compounds during drug development. There is an increasing demand for development and validation of healthy or disease-relevant and physiological human cellular models that can be implemented in early-stage discovery, thereby shifting attrition of future therapeutics to a point in discovery at which the costs are significantly lower. There needs to be a paradigm shift in the early drug discovery phase (which is lengthy and costly), away from simplistic cellular models that show an inability to effectively and efficiently reproduce healthy or human disease-relevant states to steer target and compound selection for safety, pharmacology, and efficacy questions. This perspective article covers the various stages of early drug discovery from target identification (ID) and validation to the hit/lead discovery phase, lead optimization, and preclinical safety. We outline key aspects that should be considered when developing, qualifying, and implementing complex in vitro models (CIVMs) during these phases, because criteria such as cell types (e.g., cell lines, primary cells, stem cells, and tissue), platform (e.g., spheroids, scaffolds or hydrogels, organoids, microphysiological systems, and bioprinting), throughput, automation, and single and multiplexing endpoints will vary. The article emphasizes the need to adequately qualify these CIVMs such that they are suitable for various applications (e.g., context of use) of drug discovery and translational research. The article ends looking to the future, in which there is an increase in combining computational modeling, artificial intelligence and machine learning (AI/ML), and CIVMs. Less
Interleukin- IL- is a key cytokine implicated in the pathogenesis of autoimmune disorders including psoriasis and ulcerative colitis Although targeted IL- antibody therapeutics are used clinically there are no small-molecule therapeutics that selectively inhibit IL- signaling To address this gap we developed a high-throughput screening strategy employing an IL- -responsive cell-based luciferase reporter gene assay as the primary screen with cellular cytotoxicity and off-target counter screening assays to identify IL- pathway-specific inhibitors The primary screening assay utilized avian DT cells genetically engineered to overexpress IL- R IL- R STAT and firefly luciferase in a -well format Treatment of these cells ... More
Interleukin-23 (IL-23) is a key cytokine implicated in the pathogenesis of autoimmune disorders, including psoriasis and ulcerative colitis. Although targeted IL-23 antibody therapeutics are used clinically, there are no small-molecule therapeutics that selectively inhibit IL-23 signaling. To address this gap, we developed a high-throughput screening strategy employing an IL-23-responsive cell-based luciferase reporter gene assay as the primary screen, with cellular cytotoxicity and off-target counter screening assays to identify IL-23 pathway-specific inhibitors. The primary screening assay utilized avian DT40 cells, genetically engineered to overexpress IL-23R, IL-12Rβ1, STAT5, and firefly luciferase, in a 1536-well format. Treatment of these cells with IL-23 resulted in the phosphorylation and activation of STAT5, which was completely inhibited by the pan-JAK inhibitor tofacitinib. Assay performance was robust, with signal-to-background >7-fold and Z′ > 0.5 over 40 screening plates (approximately 24,000 compounds), with a hit rate of 5% (>66.9% activity cutoff). Of these 1288 hits, 66% were identified as cytotoxic by incubating the IL-23 reporter cells with compound overnight and measuring cell viability. Further assessment of specificity via examination of impact on off-target IFN-γ signaling eliminated an additional 230 compounds, leaving 209 that were evaluated for dose–response activity. Of these compounds, 24 exhibited IC50 values of <7 µM and ≥80% inhibition of IL-23 activity, with >3-fold selectivity over IFN-γ inhibition, thus representing promising starting points for prospective IL-23 pathway small-molecule inhibitors. Less
The enzyme pyridoxal kinase PdxK catalyzes the conversion of pyridoxal to pyridoxal- -phosphate PLP using ATP as the co-factor The product pyridoxal- -phosphate plays a key role in several biological processes such as transamination decarboxylation and deamination In the present study full-length ORF of PdxK from Leishmania donovani LdPdxK was cloned and then purified using affinity chromatography LdPdxK exists as a homo-dimer in solution and shows more activity at near to physiological pH Biochemical analysis of LdPdxK with pyridoxal pyridoxamine pyridoxine and ginkgotoxin revealed its affinity preference towards different substrates The secondary structure analysis using circular dichroism spectroscopy showed LdPdxK ... More
The enzyme pyridoxal kinase (PdxK) catalyzes the conversion of pyridoxal to pyridoxal-5′-phosphate (PLP) using ATP as the co-factor. The product pyridoxal-5′-phosphate plays a key role in several biological processes such as transamination, decarboxylation and deamination. In the present study, full-length ORF of PdxK from Leishmania donovani (LdPdxK) was cloned and then purified using affinity chromatography. LdPdxK exists as a homo-dimer in solution and shows more activity at near to physiological pH. Biochemical analysis of LdPdxK with pyridoxal, pyridoxamine, pyridoxine and ginkgotoxin revealed its affinity preference towards different substrates. The secondary structure analysis using circular dichroism spectroscopy showed LdPdxK to be predominantly α-helical in organization which tends to decline at lower and higher pH. Simultaneously, LdPdxK was crystallized and its three-dimensional structure in complex with ADP and different substrates were determined. Crystal structure of LdPdxK delineated that it has a central core of β-sheets surrounded by α-helices with a conserved GTGD ribokinase motif. The structures of LdPdxK disclosed no major structural changes between ADP and ADP- substrate bound structures. In addition, comparative structural analysis highlighted the key differences between the active site pockets of leishmanial and human PdxK, rendering LdPdxK an attractive candidate for the designing of novel and specific inhibitors. Less
Of the eighteen hemagglutinin HA subtypes H H that have been identified in bats and aquatic birds many HA subtypes have been structurally characterized However several subtypes H H and H still require characterization To better understand all of these HA subtypes at the molecular level HA structures from an A H N A swine Missouri A an A H N A turkey Ontario an A H N A duck Memphis an A H N A mallard Gurjev and an A H N A wedge-tailed shearwater Western Australia were determined by X-ray crystallography at and resolution respectively The interactions between ... More
Of the eighteen hemagglutinin (HA) subtypes (H1–H18) that have been identified in bats and aquatic birds, many HA subtypes have been structurally characterized. However, several subtypes (H8, H11 and H12) still require characterization. To better understand all of these HA subtypes at the molecular level, HA structures from an A(H4N6) (A/swine/Missouri/A01727926/2015), an A(H8N4) (A/turkey/Ontario/6118/1968), an A(H11N9) (A/duck/Memphis/546/1974), an A(H14N5) A/mallard/Gurjev/263/1982, and an A(H15N9) (A/wedge-tailed shearwater/Western Australia/2576/1979 were determined by X-ray crystallography at 2.2Å, 2.3Å, 2.8Å, 3.0Å and 2.5Å resolution, respectively. The interactions between these viruses and host receptors were studied utilizing glycan-binding analyses with their recombinant HA. The data show that all avian HAs retain their strict binding preference to avian receptors, whereas swine H4 has a weak human receptor binding. The molecular characterization and structural analyses of the HA from these zoonotic influenza viruses not only provide a deeper appreciation and understanding of the structure of all HA subtypes, but also re-iterate why continuous global surveillance is needed. Less
NS is a kDa major nonstructural protein of avian reoviruses which cause significant economic losses in the poultry industry They replicate inside viral factories in host cells and the NS protein has been suggested to be the minimal viral factor required for factory formation Thus determining the structure of NS is of great importance for understanding its role in viral infection In the study presented here a fragment consisting of residues - of NS was expressed as an EGFP fusion protein in Sf insect cells EGFP- NS - crystallization in Sf cells was monitored and verified by several imaging techniques ... More
μNS is a 70 kDa major nonstructural protein of avian reoviruses, which cause significant economic losses in the poultry industry. They replicate inside viral factories in host cells, and the �NS protein has been suggested to be the minimal viral factor required for factory formation. Thus, determining the structure of �NS is of great importance for understanding its role in viral infection. In the study presented here, a fragment consisting of residues 448-605 of �NS was expressed as an EGFP fusion protein in Sf9 insect cells. EGFP-�NS(448-605) crystallization in Sf9 cells was monitored and verified by several imaging techniques. Cells infected with the EGFP-�NS(448-605) baculovirus formed rod-shaped microcrystals (5-15 �m in length) which were reconstituted in high-viscosity media (LCP and agarose) and investigated by serial femtosecond X-ray diffraction using viscous jets at an X-ray free-electron laser (XFEL). The crystals diffracted to 4.5 � resolution. A total of 4227 diffraction snapshots were successfully indexed into a hexagonal lattice with unit-cell parameters a = 109.29, b = 110.29, c = 324.97 �. The final data set was merged and refined to 7.0 � resolution. Preliminary electron-density maps were obtained. While more diffraction data are required to solve the structure of �NS(448-605), the current experimental strategy, which couples high-viscosity crystal delivery at an XFEL with in cellulo crystallization, paves the way towards structure determination of the �NS protein. Less
Synthetic lethal screens have the potential to identify new vulnerabilities incurred by specific cancer mutations but have been hindered by lack of agreement between studies In the case of KRAS we identify that published synthetic lethal screen hits significantly overlap at the pathway rather than gene level Analysis of pathways encoded as protein networks could identify synthetic lethal candidates that are more reproducible than those previously reported Lack of overlap likely stems from biological rather than technical limitations as most synthetic lethal phenotypes are strongly modulated by changes in cellular conditions or genetic context the latter determined using a pairwise ... More
Synthetic lethal screens have the potential to identify new vulnerabilities incurred by specific cancer mutations but have been hindered by lack of agreement between studies. In the case of KRAS, we identify that published synthetic lethal screen hits significantly overlap at the pathway rather than gene level. Analysis of pathways encoded as protein networks could identify synthetic lethal candidates that are more reproducible than those previously reported. Lack of overlap likely stems from biological rather than technical limitations as most synthetic lethal phenotypes are strongly modulated by changes in cellular conditions or genetic context, the latter determined using a pairwise genetic interaction map that identifies numerous interactions that suppress synthetic lethal effects. Accounting for pathway, cellular and genetic context nominates a DNA repair dependency in KRAS-mutant cells, mediated by a network containing BRCA1. We provide evidence for why most reported synthetic lethals are not reproducible which is addressable using a multi-faceted testing framework. Less
We engineered a concatenated fluorescent biosensor and dual-wavelength fluorescence lifetime FLT detection to perform high-throughput screening HTS in living cells for discovery of potential heart-failure drugs Heart failure is correlated with insufficient activity of the sarcoplasmic reticulum Ca-pump SERCA a often due to excessive inhibition by phospholamban PLB a small transmembrane protein We sought to discover small molecules that restore SERCA a activity by disrupting this inhibitory interaction between PLB and SERCA a Our approach was to fluorescently tag the two proteins and measure fluorescence resonance energy transfer FRET to detect changes in binding or structure of the complex To ... More
We engineered a concatenated fluorescent biosensor and dual-wavelength fluorescence lifetime (FLT) detection, to perform high-throughput screening (HTS) in living cells for discovery of potential heart-failure drugs. Heart failure is correlated with insufficient activity of the sarcoplasmic reticulum Ca-pump (SERCA2a), often due to excessive inhibition by phospholamban (PLB), a small transmembrane protein. We sought to discover small molecules that restore SERCA2a activity by disrupting this inhibitory interaction between PLB and SERCA2a. Our approach was to fluorescently tag the two proteins and measure fluorescence resonance energy transfer (FRET) to detect changes in binding or structure of the complex. To optimize sensitivity to these changes, we engineered a biosensor that concatenates the two fluorescently labeled proteins on a single polypeptide chain. This SERCA2a-PLB FRET biosensor construct is functionally active and effective for HTS. By implementing 2-wavelength FLT detection at extremely high speed during primary HTS, we culled fluorescent compounds as false-positive Hits. In pilot screens, we identified Hits that alter the SERCA2a-PLB interaction, and a newly developed secondary calcium uptake assay revealed both activators and inhibitors of Ca-transport. We are implementing this approach for large-scale screens to discover new drug-like modulators of SERCA2a-PLB interactions for heart failure therapeutic development. Less
We present a deep learning-based framework to design and quantify point-of-care sensors As a use-case we demonstrated a low-cost and rapid paper-based vertical flow assay VFA for high sensitivity C-Reactive Protein hsCRP testing commonly used for assessing risk of cardio-vascular disease CVD A machine learning-based framework was developed to determine an optimal configuration of immunoreaction spots and conditions spatially-multiplexed on a sensing membrane and to accurately infer target analyte concentration Using a custom-designed handheld VFA reader a clinical study with human samples showed a competitive coefficient-of-variation of and linearity of R among blindly-tested VFAs in the hsCRP range i e ... More
We present a deep learning-based framework to design and quantify point-of-care sensors. As a use-case, we demonstrated a low-cost and rapid paper-based vertical flow assay (VFA) for high sensitivity C-Reactive Protein (hsCRP) testing, commonly used for assessing risk of cardio-vascular disease (CVD). A machine learning-based framework was developed to (1) determine an optimal configuration of immunoreaction spots and conditions, spatially-multiplexed on a sensing membrane, and (2) to accurately infer target analyte concentration. Using a custom-designed handheld VFA reader, a clinical study with 85 human samples showed a competitive coefficient-of-variation of 11.2% and linearity of R2 = 0.95 among blindly-tested VFAs in the hsCRP range (i.e., 0–10 mg/L). We also demonstrated a mitigation of the hook-effect due to the multiplexed immunoreactions on the sensing membrane. This paper-based computational VFA could expand access to CVD testing, and the presented framework can be broadly used to design cost-effective and mobile point-of-care sensors. Less
Treatments for cognitive deficits associated with central nervous system CNS disorders such as Alzheimer disease and schizophrenia remain significant unmet medical needs that incur substantial pressure on the health care system The nicotinic acetylcholine receptor nAChR has garnered substantial attention as a target for cognitive deficits based on receptor localization robust preclinical effects genetics implicating its involvement in cognitive disorders and encouraging albeit mixed clinical data with nAChR orthosteric agonists Importantly previous orthosteric agonists at this receptor suffered from off-target activity receptor desensitization and an inverted U-shaped dose-effect curve in preclinical assays that limit their clinical utility To overcome the ... More
Treatments for cognitive deficits associated with central nervous system (CNS) disorders such as Alzheimer disease and schizophrenia remain significant unmet medical needs that incur substantial pressure on the health care system. The α7 nicotinic acetylcholine receptor (nAChR) has garnered substantial attention as a target for cognitive deficits based on receptor localization, robust preclinical effects, genetics implicating its involvement in cognitive disorders, and encouraging, albeit mixed, clinical data with α7 nAChR orthosteric agonists. Importantly, previous orthosteric agonists at this receptor suffered from off-target activity, receptor desensitization, and an inverted U-shaped dose-effect curve in preclinical assays that limit their clinical utility. To overcome the challenges with orthosteric agonists, we have identified a novel selective α7 positive allosteric modulator (PAM), BNC375. This compound is selective over related receptors and potentiates acetylcholine-evoked α7 currents with only marginal effect on the receptor desensitization kinetics. In addition, BNC375 enhances long-term potentiation of electrically evoked synaptic responses in rat hippocampal slices and in vivo. Systemic administration of BNC375 reverses scopolamine-induced cognitive deficits in rat novel object recognition and rhesus monkey object retrieval detour (ORD) task over a wide range of exposures, showing no evidence of an inverted U-shaped dose-effect curve. The compound also improves performance in the ORD task in aged African green monkeys. Moreover, ex vivo 13C-NMR analysis indicates that BNC375 treatment can enhance neurotransmitter release in rat medial prefrontal cortex. These findings suggest that α7 nAChR PAMs have multiple advantages over orthosteric α7 nAChR agonists for the treatment of cognitive dysfunction associated with CNS diseases. Less
Microbial rhodopsins appeared to be the most abundant light-harvesting proteins on the Earth and are the major contributes to the solar energy captured in the sea They possess highly diverse biological functions Explosion of research on microbial rhodopsins led to breakthroughs in their applications in particular in neuroscience An unexpected new discovery was a Na -pumping KR rhodopsin from Krokinobacter eikastus the first light-driven non-proton cation pump A fundamental difference between proton and other cation pumps is that non-proton pumps cannot use tunneling or Grotthuss mechanism for the ion translocation and therefore Na pumping cannot be understood in the framework ... More
Microbial rhodopsins appeared to be the most abundant light-harvesting proteins on the Earth and are the major contributes to the solar energy captured in the sea. They possess highly diverse biological functions. Explosion of research on microbial rhodopsins led to breakthroughs in their applications, in particular, in neuroscience. An unexpected new discovery was a Na+-pumping KR2 rhodopsin from Krokinobacter eikastus, the first light-driven non-proton cation pump. A fundamental difference between proton and other cation pumps is that non-proton pumps cannot use tunneling or Grotthuss mechanism for the ion translocation and, therefore, Na+ pumping cannot be understood in the framework of classical proton pump, like bacteriorhodopsin. Extensive studies on the molecular mechanism of KR2 failed to reveal mechanism of pumping. The existing high-resolution structures relate only to the ground state of the protein and revealed no Na+ inside the protein, which is unusual for active ion transporters. KR2 is only known non proton cation transporter with demonstrated remarkable potential for optogenetic applications and, therefore, elucidation of the mechanism of cation transport is important. To understand conception of cation pumping we solved crystal structures of the functionally key O-intermediate state of physiologically relevant pentameric form of KR2 and its D116N and H30A key mutants at high resolution and performed additional functional studies. The structure of the O-state reveals a sodium ion near the retinal Schiff base coordinated by N112 and D116 residues of the characteristic (for the whole family) NDQ triad. The structural and functional data show that cation uptake and release are driven by a switching mechanism. Surprisely, Na+ pathway in KR2 is lined with the chain of polar pores/cavities, similarly to the channelrhodopsin-2. Using Parinello fast molecular dynamics approach we obtained a molecular movie of a probable ion release. Our data provides insight into the mechanism of a non-proton cation light-driven pumping, strongly suggest close relation of sodium pumps to channel rhodopsins and, we believe, expand the present knowledge of rhodopsin world. Certainly they might be used for engineering of cation pumps and ion channels for optogenetics. Less
The major bottlenecks in structure elucidation of nucleic acids are crystallization and phasing Co-crystallization with proteins is a straight forward approach to overcome these challenges The human RNA-binding protein U A has previously been established as crystallization module however the absence of UV-active residues and the predetermined architecture in the asymmetric unit constitute clear limitations of the U A system Here we report three crystal structures of tryptophan-containing U A variants which expand the crystallization toolbox for nucleic acids Analysis of the structures complemented by SAXS NMR spectroscopy and optical spectroscopy allow for insights into the potential of the U ... More
The major bottlenecks in structure elucidation of nucleic acids are crystallization and phasing. Co-crystallization with proteins is a straight forward approach to overcome these challenges. The human RNA-binding protein U1A has previously been established as crystallization module, however, the absence of UV-active residues and the predetermined architecture in the asymmetric unit constitute clear limitations of the U1A system. Here, we report three crystal structures of tryptophan-containing U1A variants, which expand the crystallization toolbox for nucleic acids. Analysis of the structures complemented by SAXS, NMR spectroscopy, and optical spectroscopy allow for insights into the potential of the U1A variants to serve as crystallization modules for nucleic acids. In addition, we report a fast and efficient protocol for crystallization of RNA by soaking and present a fluorescence-based approach for detecting RNA-binding in crystallo. Our results provide a new tool set for the crystallization of RNA and RNA:DNA complexes. Less
Membrane-embedded sensor histidine kinases HKs and chemoreceptors are used ubiquitously by bacteria and archaea to percept the environment and are often crucial for their survival and pathogenicity The proteins can transmit the signal from the sensor domain to the catalytic kinase domain reliably over the span of several hundreds of angstroms and regulate the activity of the cognate response regulator proteins with which they form two-component signaling systems TCSs Several mechanisms of transmembrane signal transduction in TCS receptors have been proposed dubbed swinging piston helical rotation and diagonal scissoring Yet despite decades of studies there is no consensus on whether ... More
Membrane-embedded sensor histidine kinases (HKs) and chemoreceptors are used ubiquitously by bacteria and archaea to percept the environment, and are often crucial for their survival and pathogenicity. The proteins can transmit the signal from the sensor domain to the catalytic kinase domain reliably over the span of several hundreds of angstroms, and regulate the activity of the cognate response regulator proteins, with which they form two-component signaling systems (TCSs). Several mechanisms of transmembrane signal transduction in TCS receptors have been proposed, dubbed (swinging) piston, helical rotation, and diagonal scissoring. Yet, despite decades of studies, there is no consensus on whether these mechanisms are common for all TCS receptors. Here, we extend our previous work on Escherichia coli nitrate/nitrite sensor kinase NarQ. We determined a crystallographic structure of the sensor-TM-HAMP fragment of the R50S mutant, which, unexpectedly, was found in a ligand-bound-like conformation, despite an inability to bind nitrate. Subsequently, we reanalyzed the structures of the ligand-free and ligand-bound NarQ and NarX sensor domains, and conducted extensive molecular dynamics simulations of ligand-free and ligand-bound wild type and mutated NarQ. Based on the data, we show that binding of nitrate to NarQ causes, first and foremost, helical rotation and diagonal scissoring of the α-helices at the core of the sensor domain. These conformational changes are accompanied by a subtle piston-like motion, which is amplified by a switch in the secondary structure of the linker between the sensor and TM domains. We conclude that helical rotation, diagonal scissoring, and piston are simply different degrees of freedom in coiled-coil proteins and are not mutually exclusive in NarQ, and likely in other nitrate sensors and TCS proteins as well. Less
Tumor-associated peptide human leukocyte antigen complexes pHLAs represent the largest pool of cell surface expressed cancer-specific epitopes making them attractive targets for cancer therapies Soluble bispecific molecules that incorporate an anti-CD effector function are being developed to redirect T cells against these targets using different approaches The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs e g immune-mobilizing monoclonal T cell receptors against cancer ImmTAC molecules whereas the second harnesses an antibody-based format TCR-mimic antibodies For both classes of reagent target specificity is vital considering the vast universe of potential pHLA molecules that can be presented on healthy ... More
Tumor-associated peptide–human leukocyte antigen complexes (pHLAs) represent the largest pool of cell surface–expressed cancer-specific epitopes, making them attractive targets for cancer therapies. Soluble bispecific molecules that incorporate an anti-CD3 effector function are being developed to redirect T cells against these targets using 2 different approaches. The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs (e.g., immune-mobilizing monoclonal T cell receptors against cancer [ImmTAC] molecules), whereas the second harnesses an antibody-based format (TCR-mimic antibodies). For both classes of reagent, target specificity is vital, considering the vast universe of potential pHLA molecules that can be presented on healthy cells. Here, we made use of structural, biochemical, and computational approaches to investigate the molecular rules underpinning the reactivity patterns of pHLA-targeting bispecifics. We demonstrate that affinity-enhanced TCRs engage pHLA using a comparatively broad and balanced energetic footprint, with interactions distributed over several HLA and peptide side chains. As ImmTAC molecules, these TCRs also retained a greater degree of pHLA selectivity, with less off-target activity in cellular assays. Conversely, TCR-mimic antibodies tended to exhibit binding modes focused more toward hot spots on the HLA surface and exhibited a greater degree of crossreactivity. Our findings extend our understanding of the basic principles that underpin pHLA selectivity and exemplify a number of molecular approaches that can be used to probe the specificity of pHLA-targeting molecules, aiding the development of future reagents. Less
Ruthenium-catalysed azide alkyne cycloaddition RuAAC provides access to -disubstituted -triazole motifs in peptide engineering applications However investigation of this motif as a disulfide mimetic in cyclic peptides has been limited and the structural consequences remain to be studied We report synthetic strategies to install various triazole linkages into cyclic peptides through backbone cyclisation and RuAAC cross-linking reactions These linkages were evaluated in four serine protease inhibitors based on sunflower trypsin inhibitor- NMR and X-ray crystallography revealed exceptional consensus of bridging distance and backbone conformations RMSD of the triazole linkages compared to the parent disulfide molecules The triazole-bridged peptides also displayed ... More
Ruthenium-catalysed azide–alkyne cycloaddition (RuAAC) provides access to 1,5-disubstituted 1,2,3-triazole motifs in peptide engineering applications. However, investigation of this motif as a disulfide mimetic in cyclic peptides has been limited, and the structural consequences remain to be studied. We report synthetic strategies to install various triazole linkages into cyclic peptides through backbone cyclisation and RuAAC cross-linking reactions. These linkages were evaluated in four serine protease inhibitors based on sunflower trypsin inhibitor-1. NMR and X-ray crystallography revealed exceptional consensus of bridging distance and backbone conformations (RMSD<0.5 Å) of the triazole linkages compared to the parent disulfide molecules. The triazole-bridged peptides also displayed superior half-lives in liver S9 stability assays compared to disulfide-bridged peptides. This work establishes a foundation for the application of 1,5-disubstituted 1,2,3-triazoles as disulfide mimetics. Less
Single-cell RNA sequencing scRNA-seq is the leading technique for characterizing the transcriptomes of individual cells in a sample The latest protocols are scalable to thousands of cells and are being used to compile cell atlases of tissues organs and organisms However the protocols differ substantially with respect to their RNA capture efficiency bias scale and costs and their relative advantages for different applications are unclear In the present study we generated benchmark datasets to systematically evaluate protocols in terms of their power to comprehensively describe cell types and states We performed a multicenter study comparing commonly used scRNA-seq and single-nucleus ... More
Single-cell RNA sequencing (scRNA-seq) is the leading technique for characterizing the transcriptomes of individual cells in a sample. The latest protocols are scalable to thousands of cells and are being used to compile cell atlases of tissues, organs and organisms. However, the protocols differ substantially with respect to their RNA capture efficiency, bias, scale and costs, and their relative advantages for different applications are unclear. In the present study, we generated benchmark datasets to systematically evaluate protocols in terms of their power to comprehensively describe cell types and states. We performed a multicenter study comparing 13 commonly used scRNA-seq and single-nucleus RNA-seq protocols applied to a heterogeneous reference sample resource. Comparative analysis revealed marked differences in protocol performance. The protocols differed in library complexity and their ability to detect cell-type markers, impacting their predictive value and suitability for integration into reference cell atlases. These results provide guidance both for individual researchers and for consortium projects such as the Human Cell Atlas. Less
Cellular signaling via binding of the cytokines IL- and along with binding of the accessory protein IL- RAcP to their cognate receptor IL- R is believed to play a major role in epithelial and immune cell-mediated inflammation responses Antagonizing the signaling cascade that results from these binding events via a directed monoclonal antibody provides an opportunity to suppress such immune responses We report here the molecular structure of a complex between an extracellular portion of human IL- R and a Fab derived from a high affinity anti-IL- R neutralizing monoclonal antibody at resolution This structure the first of IL- R ... More
Cellular signaling via binding of the cytokines IL-36α, β, and γ along with binding of the accessory protein IL-36RAcP, to their cognate receptor IL-36R is believed to play a major role in epithelial and immune cell-mediated inflammation responses. Antagonizing the signaling cascade that results from these binding events via a directed monoclonal antibody provides an opportunity to suppress such immune responses. We report here the molecular structure of a complex between an extracellular portion of human IL-36R and a Fab derived from a high affinity anti-IL-36R neutralizing monoclonal antibody at 2.3 Å resolution. This structure, the first of IL-36R, reveals similarities with other structurally characterized IL-1R family members and elucidates the molecular determinants leading to the high affinity binding of the monoclonal antibody. The structure of the complex reveals that the epitope recognized by the Fab is remote from both the putative ligand and accessory protein binding interfaces on IL-36R, suggesting that the functional activity of the antibody is noncompetitive for these binding events. Less
The molecular rules driving TCR cross-reactivity are poorly understood and consequently it is unclear the extent to which TCRs targeting the same Ag recognize the same off-target peptides We determined TCR peptide HLA crystal structures and using a single-chain peptide HLA phage library we generated peptide specificity profiles for three newly identified human TCRs specific for the cancer testis Ag NY-ESO- HLA-A Two TCRs engaged the same central peptide feature although were more permissive at peripheral peptide positions and accordingly possessed partially overlapping peptide specificity profiles The third TCR engaged a flipped peptide conformation leading to the recognition of off-target ... More
The molecular rules driving TCR cross-reactivity are poorly understood and, consequently, it is unclear the extent to which TCRs targeting the same Ag recognize the same off-target peptides. We determined TCR–peptide–HLA crystal structures and, using a single-chain peptide–HLA phage library, we generated peptide specificity profiles for three newly identified human TCRs specific for the cancer testis Ag NY-ESO-1157–165–HLA-A2. Two TCRs engaged the same central peptide feature, although were more permissive at peripheral peptide positions and, accordingly, possessed partially overlapping peptide specificity profiles. The third TCR engaged a flipped peptide conformation, leading to the recognition of off-target peptides sharing little similarity with the cognate peptide. These data show that TCRs specific for a cognate peptide recognize discrete peptide repertoires and reconciles how an individual’s limited TCR repertoire following negative selection in the thymus is able to recognize a vastly larger antigenic pool. Less
Monoheme c-type cytochromes are important electron transporters in all domains of life They possess a common fold hallmarked by three -helices that surround a covalently attached heme An intriguing feature of many monoheme c-type cytochromes is their capacity to form oligomers by exchanging at least one of their -helices which is often referred to as D domain swapping Here the crystal structure of NirC a c-type cytochrome co-encoded with other proteins involved in nitrite reduction by the opportunistic pathogen Pseudomonas aeruginosa has been determined The crystals diffracted anisotropically to a maximum resolution of spherical resolution of and initial phases were ... More
Monoheme c-type cytochromes are important electron transporters in all domains of life. They possess a common fold hallmarked by three α-helices that surround a covalently attached heme. An intriguing feature of many monoheme c-type cytochromes is their capacity to form oligomers by exchanging at least one of their α-helices, which is often referred to as 3D domain swapping. Here, the crystal structure of NirC, a c-type cytochrome co-encoded with other proteins involved in nitrite reduction by the opportunistic pathogen Pseudomonas aeruginosa, has been determined. The crystals diffracted anisotropically to a maximum resolution of 2.12 Å (spherical resolution of 2.83 Å) and initial phases were obtained by Fe-SAD phasing, revealing the presence of 11 NirC chains in the asymmetric unit. Surprisingly, these protomers arrange into one monomer and two different types of 3D domain-swapped dimers, one of which shows pronounced asymmetry. While the simultaneous observation of monomers and dimers probably reflects the interplay between the high protein concentration required for crystallization and the structural plasticity of monoheme c-type cytochromes, the identification of conserved structural motifs in the monomer together with a comparison with similar proteins may offer new leads to unravel the unknown function of NirC. Less
Brain endothelial cells BECs are key constituents of the blood-brain barrier BBB protecting the brain from pathogens and restricting access of circulatory factors Yet because circulatory proteins have prominent age-related effects on adult neurogenesis neuroinflammation and cognitive function in mice we wondered whether BECs receive and potentially relay signals between the blood and brain Using single-cell RNA sequencing of hippocampal BECs we discover that capillary BECs compared with arterial and venous BECs undergo the greatest transcriptional changes in normal aging upregulating innate immunity and oxidative stress response pathways Short-term infusions of aged plasma into young mice recapitulate key aspects of ... More
Brain endothelial cells (BECs) are key constituents of the blood-brain barrier (BBB), protecting the brain from pathogens and restricting access of circulatory factors. Yet, because circulatory proteins have prominent age-related effects on adult neurogenesis, neuroinflammation, and cognitive function in mice, we wondered whether BECs receive and potentially relay signals between the blood and brain. Using single-cell RNA sequencing of hippocampal BECs, we discover that capillary BECs—compared with arterial and venous BECs—undergo the greatest transcriptional changes in normal aging, upregulating innate immunity and oxidative stress response pathways. Short-term infusions of aged plasma into young mice recapitulate key aspects of this aging transcriptome, and remarkably, infusions of young plasma into aged mice exert rejuvenation effects on the capillary transcriptome. Together, these findings suggest that the transcriptional age of BECs is exquisitely sensitive to age-related circulatory cues and pinpoint the BBB itself as a promising therapeutic target to treat brain disease. Less
Light-oxygen-voltage LOV domains are ubiquitous photosensory modules found in proteins from bacteria archaea and eukaryotes Engineered versions of LOV domains have found widespread use in fluorescence microscopy and optogenetics with improved versions being continuously developed Many of the engineering efforts focused on the thermal stabilization of LOV domains Recently we described a naturally thermostable LOV domain from Chloroflexus aggregans Here we show that the discovered protein can be further stabilized using proline substitution We tested the effects of three mutations and found that the melting temperature of the A P mutant is raised by approximately C whereas mutations A P ... More
Light-oxygen-voltage (LOV) domains are ubiquitous photosensory modules found in proteins from bacteria, archaea and eukaryotes. Engineered versions of LOV domains have found widespread use in fluorescence microscopy and optogenetics, with improved versions being continuously developed. Many of the engineering efforts focused on the thermal stabilization of LOV domains. Recently, we described a naturally thermostable LOV domain from Chloroflexus aggregans. Here we show that the discovered protein can be further stabilized using proline substitution. We tested the effects of three mutations, and found that the melting temperature of the A95P mutant is raised by approximately 2 °C, whereas mutations A56P and A58P are neutral. To further evaluate the effects of mutations, we crystallized the variants A56P and A95P, while the variant A58P did not crystallize. The obtained crystal structures do not reveal any alterations in the proteins other than the introduced mutations. Molecular dynamics simulations showed that mutation A58P alters the structure of the respective loop (Aβ-Bβ), but does not change the general structure of the protein. We conclude that proline substitution is a viable strategy for the stabilization of the Chloroflexus aggregans LOV domain. Since the sequences and structures of the LOV domains are overall well-conserved, the effects of the reported mutations may be transferable to other proteins belonging to this family. Less
Formulation conditions have a significant influence on the degree of freeze thaw FT stress-induced protein instabilities Adding cryoprotectants might stabilize the induced FT stress instabilities However a simple preservation of protein stability might be insufficient and further methods are necessary This study aims to evaluate the addition of a heat cycle following FT application as a function of different cryoprotectants with lysozyme as exemplary protein Sucrose and glycerol were shown to be the most effective cryoprotectants when compared to PEG and Tween In terms of heat-induced reversibility of aggregates glycerol showed the best performance followed by sucrose NaCl and Tween ... More
Formulation conditions have a significant influence on the degree of freeze/thaw (FT) stress-induced protein instabilities. Adding cryoprotectants might stabilize the induced FT stress instabilities. However, a simple preservation of protein stability might be insufficient and further methods are necessary. This study aims to evaluate the addition of a heat cycle following FT application as a function of different cryoprotectants with lysozyme as exemplary protein. Sucrose and glycerol were shown to be the most effective cryoprotectants when compared to PEG200 and Tween20. In terms of heat-induced reversibility of aggregates, glycerol showed the best performance followed by sucrose, NaCl and Tween20 systems. The analysis was performed using a novel approach to visualize complex interplays by a clustering and data reduction scheme. In addition, solubility and structural integrity were measured and confirmed the obtained results. Less
Next generation sequencing is in the process of evolving from a technology used for research purposes to one which is applied in clinical diagnostics Recently introduced high throughput and benchtop instruments offer fully automated sequencing runs at a lower cost per base and faster assay times In turn the complex and cumbersome library preparation starting with isolated nucleic acids and resulting in amplified and barcoded DNA with sequencing adapters has been identified as a significant bottleneck Library preparation protocols usually consist of a multistep process and require costly reagents and substantial hands-on-time Considerable emphasis will need to be placed on ... More
Next generation sequencing is in the process of evolving from a technology used for research purposes to one which is applied in clinical diagnostics. Recently introduced high throughput and benchtop instruments offer fully automated sequencing runs at a lower cost per base and faster assay times. In turn, the complex and cumbersome library preparation, starting with isolated nucleic acids and resulting in amplified and barcoded DNA with sequencing adapters, has been identified as a significant bottleneck. Library preparation protocols usually consist of a multistep process and require costly reagents and substantial hands-on-time. Considerable emphasis will need to be placed on standardisation to ensure robustness and reproducibility. This review presents an overview of the current state of automation of library preparation for next generation sequencing. Major challenges associated with library preparation are outlined and different automation strategies are classified according to their functional principle. Pipetting workstations allow high-throughput processing yet offer limited flexibility, whereas microfluidic solutions offer great potential due to miniaturisation and decreased investment costs. For the emerging field of single cell transcriptomics for example, microfluidics enable singularisation of tens of thousands of cells in nanolitre droplets and barcoding of the RNA to assign each nucleic acid sequence to its cell of origin. Finally, two applications, the characterisation of bacterial pathogens and the sequencing within human immunogenetics, are outlined and benefits of automation are discussed. Less
The glycyl radical enzyme GRE superfamily utilizes a glycyl radical cofactor to catalyze difficult chemical reactions in a variety of anaerobic microbial metabolic pathways Recently a GRE trans- -hydroxy-L-proline Hyp dehydratase HypD was discovered that catalyzes the dehydration of Hyp to S - -pyrroline- -carboxylic acid P C This enzyme is abundant in the human gut microbiome and also present in prominent bacterial pathogens However we lack an understanding of how HypD performs its unusual chemistry Here we have solved the crystal structure of HypD from the pathogen Clostridioides difficile with Hyp bound in the active site Biochemical studies have ... More
The glycyl radical enzyme (GRE) superfamily utilizes a glycyl radical cofactor to catalyze difficult chemical reactions in a variety of anaerobic microbial metabolic pathways. Recently, a GRE, trans-4-hydroxy-L-proline (Hyp) dehydratase (HypD), was discovered that catalyzes the dehydration of Hyp to (S)-Δ1-pyrroline-5-carboxylic acid (P5C). This enzyme is abundant in the human gut microbiome and also present in prominent bacterial pathogens. However, we lack an understanding of how HypD performs its unusual chemistry. Here, we have solved the crystal structure of HypD from the pathogen Clostridioides difficile with Hyp bound in the active site. Biochemical studies have led to the identification of key catalytic residues and have provided insight into the radical mechanism of Hyp dehydration. Less
Perovskites have seen significant research interest in the last decade As ternary and quaternary compounds their chemical space is exceptionally large yet perovskite development has been limited to a restricted set of chemical constituents often discovered through trial and error Here we report a high-throughput experimental framework for the discovery of new perovskite single crystals We use machine learning ML to guide the sequence of ever-improved robotic synthetic trials We perform high-throughput syntheses of perovskite single crystals with a protein crystallization robot and characterize the outcomes with the aid of convolutional neural network-based image recognition We then use an ML ... More
Perovskites have seen significant research interest in the last decade. As ternary and quaternary compounds, their chemical space is exceptionally large, yet perovskite development has been limited to a restricted set of chemical constituents often discovered through trial and error. Here, we report a high-throughput experimental framework for the discovery of new perovskite single crystals. We use machine learning (ML) to guide the sequence of ever-improved robotic synthetic trials. We perform high-throughput syntheses of perovskite single crystals with a protein crystallization robot and characterize the outcomes with the aid of convolutional neural network-based image recognition. We then use an ML model to predict the optimal conditions for the synthesis of a new perovskite single crystal, enabling us to report the first synthesis of (3-PLA)2PbCl4.This material exhibits strong blue emission, illustrating the applicability of the method in identifying new optoelectronic materials. Less
Glucagon-like peptide- receptor GLP- R is a class B G protein-coupled receptor that plays an important role in glucose homeostasis and treatment of type diabetes Structures of full-length class B receptors were determined in complex with their orthosteric agonist peptides however little is known about their extracellular domain ECD conformations in the absence of orthosteric ligands which has limited our understanding of their activation mechanism Here we report the resolution peptide-free crystal structure of the full-length human GLP- R in an inactive state which reveals a unique closed conformation of the ECD Disulfide cross-linking validates the physiological relevance of the ... More
Glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor that plays an important role in glucose homeostasis and treatment of type 2 diabetes. Structures of full-length class B receptors were determined in complex with their orthosteric agonist peptides, however, little is known about their extracellular domain (ECD) conformations in the absence of orthosteric ligands, which has limited our understanding of their activation mechanism. Here, we report the 3.2 Å resolution, peptide-free crystal structure of the full-length human GLP-1R in an inactive state, which reveals a unique closed conformation of the ECD. Disulfide cross-linking validates the physiological relevance of the closed conformation, while electron microscopy (EM) and molecular dynamic (MD) simulations suggest a large degree of conformational dynamics of ECD that is necessary for binding GLP-1. Our inactive structure represents a snapshot of the peptide-free GLP-1R and provides insights into the activation pathway of this receptor family. Less
Small-molecule drug discovery can be hindered by the formation of aggregates that act as non-selective inhibitors of drug targets Such aggregates appear as false positives in high-throughput screening campaigns and can bedevil structure-activity relationships during compound optimization Protocols are described for resonant waveguide grating RWG and dynamic light scattering DLS as microplate-based high-throughput approaches to identify compound aggregation Resonant waveguide grating and dynamic light scattering give equivalent results for the compound test set as assessed with Bland-Altman analysis
The human formyl peptide receptor FPR plays a crucial role in host defense and inflammation and has been considered as a drug target for chronic inflammatory diseases A variety of peptides with different structures and origins have been characterized as FPR ligands However the ligand-binding modes of FPR remain elusive thereby limiting the development of potential drugs Here we report the crystal structure of FPR bound to the potent peptide agonist WKYMVm at resolution The structure adopts an active conformation and exhibits a deep ligand-binding pocket Combined with mutagenesis ligand binding and signaling studies key interactions between the agonist and ... More
The human formyl peptide receptor 2 (FPR2) plays a crucial role in host defense and inflammation, and has been considered as a drug target for chronic inflammatory diseases. A variety of peptides with different structures and origins have been characterized as FPR2 ligands. However, the ligand-binding modes of FPR2 remain elusive, thereby limiting the development of potential drugs. Here we report the crystal structure of FPR2 bound to the potent peptide agonist WKYMVm at 2.8 Å resolution. The structure adopts an active conformation and exhibits a deep ligand-binding pocket. Combined with mutagenesis, ligand binding and signaling studies, key interactions between the agonist and FPR2 that govern ligand recognition and receptor activation are identified. Furthermore, molecular docking and functional assays reveal key factors that may define binding affinity and agonist potency of formyl peptides. These findings deepen our understanding about ligand recognition and selectivity mechanisms of the formyl peptide receptor family. Less
One central question surrounding the biosynthesis of fatty acids and polyketide-derived natural products is how the -phosphopantetheinyl transferase PPTase interrogates the essential acyl carrier protein ACP domain to fulfill the initial activation step The triggering factor of this study was the lack of structural information on PPTases at physiological pH which could bias our comprehension of the mechanism of action of these important enzymes Structural and functional studies on the family II PPTase PptAb of Mycobacterium abscessus show that pH has a profound effect on the coordination of metal ions and on the conformation of endogenously bound coenzyme A CoA ... More
One central question surrounding the biosynthesis of fatty acids and polyketide-derived natural products is how the 4′-phosphopantetheinyl transferase (PPTase) interrogates the essential acyl carrier protein (ACP) domain to fulfill the initial activation step. The triggering factor of this study was the lack of structural information on PPTases at physiological pH, which could bias our comprehension of the mechanism of action of these important enzymes. Structural and functional studies on the family II PPTase PptAb of Mycobacterium abscessus show that pH has a profound effect on the coordination of metal ions and on the conformation of endogenously bound coenzyme A (CoA). The observed conformational flexibility of CoA at physiological pH is accompanied by a disordered 4′-phosphopantetheine (Ppant) moiety. Finally, structural and dynamical information on an isolated mycobacterial ACP domain, in its apo form and in complex with the activator PptAb, suggests an alternate mechanism for the post-translational modification of modular megasynthases. Less
Membrane proteins are highly interesting targets due to their pivotal role in cell function and disease They are inserted in cell membranes are often intrinsically flexible and can adopt several conformational states to carry out their function Although most overall folds of membrane proteins are known many questions remain about specific functionally relevant intramolecular rearrangements that require experimental structure determination Here using the example of rhodopsin we describe how to prepare and analyze membrane protein crystals for serial crystallography at room temperature a new technique allowing to merge diffraction data from thousands of injector-delivered crystals that are too tiny for ... More
Membrane proteins are highly interesting targets due to their pivotal role in cell function and disease. They are inserted in cell membranes, are often intrinsically flexible, and can adopt several conformational states to carry out their function. Although most overall folds of membrane proteins are known, many questions remain about specific functionally relevant intramolecular rearrangements that require experimental structure determination. Here, using the example of rhodopsin, we describe how to prepare and analyze membrane protein crystals for serial crystallography at room temperature, a new technique allowing to merge diffraction data from thousands of injector-delivered crystals that are too tiny for classical single-crystal analysis even in cryogenic conditions. The application of serial crystallography for studying protein dynamics is mentioned. Less
The lipid cubic phases LCP have enabled the determination of many important high-resolution structures of membrane proteins such as G-protein-coupled receptors photosensitive proteins enzymes channels and transporters However harvesting the crystals from the glass or plastic plates in which crystals grow is challenging The in meso in situ serial X-ray crystallography IMISX method uses thin plastic windowed plates that minimize LCP crystal manipulation The method which is compatible with high-throughput in situ measurements allows systematic diffraction screening and rapid data collection from hundreds of microcrystals in in meso crystallization wells without direct crystal harvesting In this chapter we describe an ... More
The lipid cubic phases (LCP) have enabled the determination of many important high-resolution structures of membrane proteins such as G-protein-coupled receptors, photosensitive proteins, enzymes, channels, and transporters. However, harvesting the crystals from the glass or plastic plates in which crystals grow is challenging. The in meso in situ serial X-ray crystallography (IMISX) method uses thin plastic windowed plates that minimize LCP crystal manipulation. The method, which is compatible with high-throughput in situ measurements, allows systematic diffraction screening and rapid data collection from hundreds of microcrystals in in meso crystallization wells without direct crystal harvesting. In this chapter, we describe an IMISX protocol for in situ serial X-ray data collection of LCP-grown crystals at both cryogenic and room temperatures which includes the crystallization setup, sample delivery, automated serial diffraction data collection, and experimental phasing. We also detail how the IMISX method was applied successfully for the structure determination of two novel targets—the undecaprenyl-pyrophosphate phosphatase BacA and the chemokine G-protein-coupled receptor CCR2A. Less
Two-component signaling systems TCSs are a large and important class of sensory systems in bacteria archaea and some eukaryotes yet their mechanism of action is still not fully understood from the structural point of view Many TCS receptors are elongated flexible proteins with transmembrane TM regions and are difficult to work with Consequently truncated fragments of the receptors are often used in structural studies However it is not fully clear whether the structures of the fragments correspond well to their native structures in the context of full-length proteins Recently we crystallized a fragment of Escherichia coli nitrate nitrite sensor histidine ... More
Two-component signaling systems (TCSs) are a large and important class of sensory systems in bacteria, archaea, and some eukaryotes, yet their mechanism of action is still not fully understood from the structural point of view. Many TCS receptors are elongated flexible proteins with transmembrane (TM) regions, and are difficult to work with. Consequently, truncated fragments of the receptors are often used in structural studies. However, it is not fully clear whether the structures of the fragments correspond well to their native structures in the context of full-length proteins. Recently, we crystallized a fragment of Escherichia coli nitrate/nitrite sensor histidine kinase, NarQ, encompassing the sensor, TM, and HAMP domains. Here we report that a smaller proteolytic fragment consisting of the sensor and TM domains can also be crystallized using the in meso approach. The structure of the fragment is similar to the previously determined one, with minor differences in the vicinity of the truncation site. The results show that the crystallization of such sensor–TM fragments can be accomplished and can provide information on the packing of transmembrane helices, albeit limited, and that the proteolysis may or may not be a problem during crystallization. Less
Rhodopsins are the most abundant light-harvesting proteins A new family of rhodopsins heliorhodopsins HeRs has recently been discovered Unlike in the known rhodopsins in HeRs the N termini face the cytoplasm The function of HeRs remains unknown We present the structures of the bacterial HeR- C in two states at the resolution of which highlight its remarkable difference from all known rhodopsins The interior of HeR s extracellular part is completely hydrophobic while the cytoplasmic part comprises a cavity Schiff base cavity SBC surrounded by charged amino acids and containing a cluster of water molecules presumably being a primary proton ... More
Rhodopsins are the most abundant light-harvesting proteins. A new family of rhodopsins, heliorhodopsins (HeRs), has recently been discovered. Unlike in the known rhodopsins, in HeRs the N termini face the cytoplasm. The function of HeRs remains unknown. We present the structures of the bacterial HeR-48C12 in two states at the resolution of 1.5 Å, which highlight its remarkable difference from all known rhodopsins. The interior of HeR’s extracellular part is completely hydrophobic, while the cytoplasmic part comprises a cavity (Schiff base cavity [SBC]) surrounded by charged amino acids and containing a cluster of water molecules, presumably being a primary proton acceptor from the Schiff base. At acidic pH, a planar triangular molecule (acetate) is present in the SBC. Structure-based bioinformatic analysis identified 10 subfamilies of HeRs, suggesting their diverse biological functions. The structures and available data suggest an enzymatic activity of HeR-48C12 subfamily and their possible involvement in fundamental redox biological processes. Less
Histone lysine specific demethylase LSD has become a potential therapeutic target for the treatment of cancer Discovery and develop novel and potent LSD inhibitors is a challenge although several of them have already entered into clinical trials Herein for the first time we reported the discovery of a series of -cyano- -phenylpyrimidine derivatives as LSD inhibitors using flavin adenine dinucleotide FAD similarity-based designing strategy of which compound q was finally identified to repress LSD with IC nmol L Docking analysis suggested that compound q fitted well into the FAD-binding pocket Further mechanism studies showed that compound q may inhibit LSD ... More
Histone lysine specific demethylase 1 (LSD1) has become a potential therapeutic target for the treatment of cancer. Discovery and develop novel and potent LSD1 inhibitors is a challenge, although several of them have already entered into clinical trials. Herein, for the first time, we reported the discovery of a series of 5-cyano-6-phenylpyrimidine derivatives as LSD1 inhibitors using flavin adenine dinucleotide (FAD) similarity-based designing strategy, of which compound 14q was finally identified to repress LSD1 with IC50 = 183 nmol/L. Docking analysis suggested that compound 14q fitted well into the FAD-binding pocket. Further mechanism studies showed that compound 14q may inhibit LSD1 activity competitively by occupying the FAD binding sites of LSD1 and inhibit cell migration and invasion by reversing epithelial to mesenchymal transition (EMT). Overall, these findings showed that compound 14q is a suitable candidate for further development of novel FAD similarity-based LSD1 inhibitors. Less
Human muscarinic receptor M belongs to the class A subfamily of the G-protein-coupled receptors GPCRs M has emerged as an attractive drug target for the treatment of Alzheimer s disease and schizophrenia Recent results showed that M -mediated cholinergic transmission is related to motor symptoms in Parkinson s disease Selective ligand design for the five muscarinic acetylcholine receptor mAchR subtypes currently remains challenging owing to the high sequence and structural similarity of their orthosteric binding pockets In order to obtain M -selective antagonists a new approach was tried to lock M into an inactive form by rationally designing an N ... More
Human muscarinic receptor M4 belongs to the class A subfamily of the G-protein-coupled receptors (GPCRs). M4 has emerged as an attractive drug target for the treatment of Alzheimer’s disease and schizophrenia. Recent results showed that M4-mediated cholinergic transmission is related to motor symptoms in Parkinson’s disease. Selective ligand design for the five muscarinic acetylcholine receptor (mAchR) subtypes currently remains challenging owing to the high sequence and structural similarity of their orthosteric binding pockets. In order to obtain M4-selective antagonists, a new approach was tried to lock M4 into an inactive form by rationally designing an N4497.49R mutation, which mimics the allosteric sodium binding in the conserved sodium site usually found in class A GPCRs. In addition, the crystal structure of the mutation-induced inactive M4 was determined. By comparative analysis with other mAchR structures, followed by functional assays, the N4497.49R mutation was shown to stabilize M4 into an inactive state. Virtual screening of a focused ligand library using the crystal structure showed that the inactive M4 prefers antagonists much more than agonists. This study provides a powerful mutation strategy to stabilize GPCRs in inactive states and facilitate their structure determination. Less
GPR is a class-A orphan G-protein-coupled receptor that is highly expressed in the brain and represents a promising therapeutic target for the treatment of Huntington s disease and several psychiatric disorders Pathological malfunction of GPR signalling occurs primarily through the heterotrimeric Gs protein but it is unclear how GPR and Gs couple for signal transduction and whether a native ligand or other activating input is required Here we present the high-resolution structures of human GPR in three states a ligand-free state a Gs-coupled self-activation state and a potential allosteric ligand-bound state Together our structures reveal that extracellular loop occupies the ... More
GPR52 is a class-A orphan G-protein-coupled receptor that is highly expressed in the brain and represents a promising therapeutic target for the treatment of Huntington’s disease and several psychiatric disorders1,2. Pathological malfunction of GPR52 signalling occurs primarily through the heterotrimeric Gs protein2, but it is unclear how GPR52 and Gs couple for signal transduction and whether a native ligand or other activating input is required. Here we present the high-resolution structures of human GPR52 in three states: a ligand-free state, a Gs-coupled self-activation state and a potential allosteric ligand-bound state. Together, our structures reveal that extracellular loop 2 occupies the orthosteric binding pocket and operates as a built-in agonist, conferring an intrinsically high level of basal activity to GPR523. A fully active state is achieved when Gs is coupled to GPR52 in the absence of an external agonist. The receptor also features a side pocket for ligand binding. These insights into the structure and function of GPR52 could improve our understanding of other self-activated GPCRs, enable the identification of endogenous and tool ligands, and guide drug discovery efforts that target GPR52. Less
Toxoplasma gondii a protozoan parasite undergoes a complex and poorly understood developmental process that is critical for establishing a chronic infection in its intermediate hosts Here we applied single-cell RNA-sequencing scRNA-seq on Toxoplasma in both tachyzoite and bradyzoite stages using three widely studied strains to construct a comprehensive atlas of cell-cycle and asexual development revealing hidden states and transcriptional factors associated with each developmental stage Analysis of SAG -related sequence SRS antigenic repertoire reveals a highly heterogeneous sporadic expression pattern unexplained by measurement noise cell cycle or asexual development Furthermore we identified AP IX- as a transcription factor that controls ... More
Toxoplasma gondii, a protozoan parasite, undergoes a complex and poorly understood developmental process that is critical for establishing a chronic infection in its intermediate hosts. Here, we applied single-cell RNA-sequencing (scRNA-seq) on >5,400 Toxoplasma in both tachyzoite and bradyzoite stages using three widely studied strains to construct a comprehensive atlas of cell-cycle and asexual development, revealing hidden states and transcriptional factors associated with each developmental stage. Analysis of SAG1-related sequence (SRS) antigenic repertoire reveals a highly heterogeneous, sporadic expression pattern unexplained by measurement noise, cell cycle, or asexual development. Furthermore, we identified AP2IX-1 as a transcription factor that controls the switching from the ubiquitous SAG1 to rare surface antigens not previously observed in tachyzoites. In addition, comparative analysis between Toxoplasma and Plasmodium scRNA-seq results reveals concerted expression of gene sets, despite fundamental differences in cell division. Lastly, we built an interactive data-browser for visualization of our atlas resource. Less
The determination of distances between specific points in nucleic acids is essential to understanding their behaviour at the molecular level The ability to measure distances of nm is particularly important deformations arising from protein binding commonly fall within this range but the reliable measurement of such distances for a conformational ensemble remains a significant challenge Using several techniques we show that electron paramagnetic resonance EPR spectroscopy of oligonucleotides spin-labelled with triazole-appended nitroxides at the position offers a robust and minimally perturbing tool for obtaining such measurements For two nitroxides we present results from EPR spectroscopy X-ray crystal structures of B-form ... More
The determination of distances between specific points in nucleic acids is essential to understanding their behaviour at the molecular level. The ability to measure distances of 2–10 nm is particularly important: deformations arising from protein binding commonly fall within this range, but the reliable measurement of such distances for a conformational ensemble remains a significant challenge. Using several techniques, we show that electron paramagnetic resonance (EPR) spectroscopy of oligonucleotides spin-labelled with triazole-appended nitroxides at the 2′ position offers a robust and minimally perturbing tool for obtaining such measurements. For two nitroxides, we present results from EPR spectroscopy, X-ray crystal structures of B-form spin-labelled DNA duplexes, molecular dynamics simulations and nuclear magnetic resonance spectroscopy. These four methods are mutually supportive, and pinpoint the locations of the spin labels on the duplexes. In doing so, this work establishes 2′-alkynyl nitroxide spin-labelling as a minimally perturbing method for probing DNA conformation. Less
Trematode infections such as schistosomiasis and fascioliasis cause significant morbidity in an estimated million people worldwide and the associated agricultural losses are estimated at more than US billion per year Current chemotherapy is limited Triosephosphate isomerase TIM an enzyme of the glycolytic pathway has emerged as a useful drug target in many parasites including Fasciola hepatica TIM FhTIM We identified novel compounds that selectively inhibit this enzyme Using microscale thermophoresis we explored the interaction between target and compounds and identified a potent interaction between the sulfonyl- -thiadiazole compound and FhTIM which showed an IC of M and a Kd of ... More
Trematode infections such as schistosomiasis and fascioliasis cause significant morbidity in an estimated 250 million people worldwide and the associated agricultural losses are estimated at more than US$ 6 billion per year. Current chemotherapy is limited. Triosephosphate isomerase (TIM), an enzyme of the glycolytic pathway, has emerged as a useful drug target in many parasites, including Fasciola hepatica TIM (FhTIM). We identified 21 novel compounds that selectively inhibit this enzyme. Using microscale thermophoresis we explored the interaction between target and compounds and identified a potent interaction between the sulfonyl-1,2,4-thiadiazole (compound 187) and FhTIM, which showed an IC50 of 5 µM and a Kd of 66 nM. In only 4 hours, this compound killed the juvenile form of F. hepatica with an IC50 of 3 µM, better than the reference drug triclabendazole (TCZ). Interestingly, we discovered in vitro inhibition of FhTIM by TCZ, with an IC50 of 7 µM suggesting a previously uncharacterized role of FhTIM in the mechanism of action of this drug. Compound 187 was also active against various developmental stages of Schistosoma mansoni. The low toxicity in vitro in different cell types and lack of acute toxicity in mice was demonstrated for this compound, as was demonstrated the efficacy of 187 in vivo in F. hepatica infected mice. Finally, we obtained the first crystal structure of FhTIM at 1.9 Å resolution which allows us using docking to suggest a mechanism of interaction between compound 187 and TIM. In conclusion, we describe a promising drug candidate to control neglected trematode infections in human and animal health. Less
Phospholipase D enzymes PLDs are ubiquitous phosphodiesterases that produce phosphatidic acid PA a key second messenger and biosynthetic building block Although an orthologous bacterial Streptomyces sp strain PMF PLD structure was solved two decades ago the molecular basis underlying the functions of the human PLD enzymes hPLD remained unclear based on this structure due to the low homology between these sequences Here we describe the first crystal structures of hPLD and hPLD catalytic domains and identify novel structural elements and functional differences between the prokaryotic and eukaryotic enzymes Furthermore structure-based mutation studies and structures of inhibitor hPLD complexes allowed us ... More
Phospholipase D enzymes (PLDs) are ubiquitous phosphodiesterases that produce phosphatidic acid (PA), a key second messenger and biosynthetic building block. Although an orthologous bacterial Streptomyces sp. strain PMF PLD structure was solved two decades ago, the molecular basis underlying the functions of the human PLD enzymes (hPLD) remained unclear based on this structure due to the low homology between these sequences. Here, we describe the first crystal structures of hPLD1 and hPLD2 catalytic domains and identify novel structural elements and functional differences between the prokaryotic and eukaryotic enzymes. Furthermore, structure-based mutation studies and structures of inhibitor–hPLD complexes allowed us to elucidate the binding modes of dual and isoform-selective inhibitors, highlight key determinants of isoenzyme selectivity and provide a basis for further structure-based drug discovery and functional characterization of this therapeutically important superfamily of enzymes. Less
Rhodopsins are the most abundant light-harvesting proteins A new family of rhodopsins heliorhodopsins HeRs was recently discovered In opposite to the known rhodopsins their N-termini face the cytoplasm HeRs structure and function remain unknown We present structures of two HeR- C states at showing its remarkable difference from all known rhodopsins Its internal extracellular part is completely hydrophobic while the cytoplasmic part comprises a cavity active site surrounded by charged amino acids and containing a cluster of water molecules presumably being a primary proton acceptor from the Schiff base At acidic pH a planar triangle molecule acetate is present in ... More
Rhodopsins are the most abundant light-harvesting proteins. A new family of rhodopsins, heliorhodopsins (HeRs), was recently discovered. In opposite to the known rhodopsins their N-termini face the cytoplasm. HeRs structure and function remain unknown. We present structures of two HeR-48C12 states at 1.5 Å showing its remarkable difference from all known rhodopsins. Its internal extracellular part is completely hydrophobic, while the cytoplasmic part comprises a cavity (’active site’), surrounded by charged amino acids and containing a cluster of water molecules, presumably being a primary proton acceptor from the Schiff base. At acidic pH a planar triangle molecule (acetate) is present in the ‘active site’ which demonstrated its ability to maintain such anions as carbonate or nitrate. Structure-based bioinformatic analysis identified 10 subfamilies of HeRs suggesting their diverse biological functions. The structures and available data suggest an enzymatic activity of HeR-48C12 subfamily and their possible involvement into fundamental redox biological processes. Less
The process of macromolecular crystallisation almost always begins by setting up crystallisation trials using commercial or other premade screens followed by cycles of optimisation where the crystallisation cocktails are focused towards a particular small region of chemical space The screening process is relatively straightforward but still requires an understanding of the plethora of commercially available screens Optimisation is complicated by requiring both the design and preparation of the appropriate secondary screens Software has been developed in the C lab to aid the process of choosing initial screens to analyse the results of the initial trials and to design and describe ... More
The process of macromolecular crystallisation almost always begins by setting up crystallisation trials using commercial or other premade screens, followed by cycles of optimisation where the crystallisation cocktails are focused towards a particular small region of chemical space. The screening process is relatively straightforward, but still requires an understanding of the plethora of commercially available screens. Optimisation is complicated by requiring both the design and preparation of the appropriate secondary screens. Software has been developed in the C3 lab to aid the process of choosing initial screens, to analyse the results of the initial trials, and to design and describe how to prepare optimisation screens. Less
In high-throughput systems the crystallization experiments require the inspection and analysis of a large number of trial images The visualization and analysis tools are needed to view and analyze the experimental results and recommend novel crystalline conditions by analyzing prior results It is essential to integrate all these components into a single system Therefore we developed Visual-X an interactive visualization software developed to aid the user for quick and efficient visualization and analysis of the results of the experiments Visual-X has a number of useful features for visualization and analysis dual plate view thumbnail and symbolic detailed well view with ... More
In high-throughput systems, the crystallization experiments require the inspection and analysis of a large number of trial images. The visualization and analysis tools are needed to view and analyze the experimental results, and recommend novel crystalline conditions by analyzing prior results. It is essential to integrate all these components into a single system. Therefore, we developed Visual-X2, an interactive visualization software developed to aid the user for quick and efficient visualization and analysis of the results of the experiments. Visual-X2 has a number of useful features for visualization and analysis: dual plate view (thumbnail and symbolic), detailed well view with scoring option, multiple-scan and time-course views, support for screening analysis based on multiple screens, three novel screen analysis methods (associative experimental design, GenScreen, and novelty methods), and generating pipetting file with a family of conditions varying concentrations based on stock concentration. Less
Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB and CB Their high sequence similarity low agonist selectivity and lack of activation and G protein-coupling knowledge have hindered the development of therapeutic applications Importantly missing structural information has significantly held back the development of promising CB -selective agonist drugs for treating inflammatory and neuropathic pain without the psychoactivity of CB Here we report the cryoelectron microscopy structures of synthetic cannabinoid-bound CB and CB in complex with Gi as well as agonist-bound CB crystal structure Of important scientific and therapeutic benefit our results reveal a ... More
Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB1 and CB2. Their high sequence similarity, low agonist selectivity, and lack of activation and G protein-coupling knowledge have hindered the development of therapeutic applications. Importantly, missing structural information has significantly held back the development of promising CB2-selective agonist drugs for treating inflammatory and neuropathic pain without the psychoactivity of CB1. Here, we report the cryoelectron microscopy structures of synthetic cannabinoid-bound CB2 and CB1 in complex with Gi, as well as agonist-bound CB2 crystal structure. Of important scientific and therapeutic benefit, our results reveal a diverse activation and signaling mechanism, the structural basis of CB2-selective agonists design, and the unexpected interaction of cholesterol with CB1, suggestive of its endogenous allosteric modulating role. Less
Amorphous solid dispersions ASDs are single-phase amorphous systems where drug molecules are molecularly dispersed dissolved in a polymer matrix The molecular dispersion of the drug molecules is responsible for their improved dissolution properties Unambiguously establishing the phase behavior of the ASDs is of utmost importance In this paper we focused on the complementary nature of modulated differential scanning calorimetry m DSC and X-ray powder diffraction XRPD to elucidate the phase behavior of ASDs as demonstrated by a critical discussion of practical real-life examples observed in our research group The ASDs were manufactured by either applying a solvent-based technique spray drying ... More
Amorphous solid dispersions (ASDs) are single-phase amorphous systems, where drug molecules are molecularly dispersed (dissolved) in a polymer matrix. The molecular dispersion of the drug molecules is responsible for their improved dissolution properties. Unambiguously establishing the phase behavior of the ASDs is of utmost importance. In this paper, we focused on the complementary nature of (modulated) differential scanning calorimetry ((m)DSC) and X-ray powder diffraction (XRPD) to elucidate the phase behavior of ASDs as demonstrated by a critical discussion of practical real-life examples observed in our research group. The ASDs were manufactured by either applying a solvent-based technique (spray drying), a heat-based technique (hot melt extrusion) or mechanochemical activation (cryo-milling). The encountered limiting factors of XRPD were the lack of sensitivity for small traces of crystallinity, the impossibility to differentiate between distinct amorphous phases and its impossibility to detect nanocrystals in a polymer matrix. In addition, the limiting factors of (m)DSC were defined as the well-described heat-induced sample alteration upon heating, the interfering of residual solvent evaporation with other thermal events and the coinciding of enthalpy recovery with melting events. In all of these cases, the application of a single analytical technique would have led to erroneous conclusions, whilst the combination of (m)DSC and XRPD elucidated the true phases of the ASD. Less