1181 Citations
There is an urgent need to develop new methods for male contraception however a major barrier to drug discovery has been the lack of validated targets and the absence of an effective high-throughput phenotypic screening system To address this deficit we developed a fully-automated robotic screening platform that provided quantitative evaluation of compound activity against two key attributes of human sperm function motility and acrosome reaction In order to accelerate contraceptive development we screened the comprehensive collection of molecules that make up the ReFRAME repurposing library comprising nearly all the small molecules that have been approved or have undergone clinical ... More
There is an urgent need to develop new methods for male contraception, however a major barrier to drug discovery has been the lack of validated targets and the absence of an effective high-throughput phenotypic screening system. To address this deficit, we developed a fully-automated robotic screening platform that provided quantitative evaluation of compound activity against two key attributes of human sperm function: motility and acrosome reaction. In order to accelerate contraceptive development, we screened the comprehensive collection of 12,000 molecules that make up the ReFRAME repurposing library, comprising nearly all the small molecules that have been approved or have undergone clinical development, or have significant preclinical profiling. We identified several compounds that potently inhibit motility representing either novel drug candidates or routes to target identification. This platform will now allow for major drug discovery programmes that address the critical gap in the contraceptive portfolio as well as uncover novel human sperm biology. Less
Carbapenem-resistant Enterobacteriaceae CRE represent an urgent threat to human health Here we report the application of several complementary whole-genome sequencing WGS technologies to characterise a hospital outbreak of blaIMP- carbapenemase-producing E hormaechei Using Illumina sequencing we determined that all outbreak strains were sequence type ST and near-identical Comparison to publicly available data linked all outbreak isolates to a isolate from the same ward suggesting an environmental source in the hospital Using Pacific Biosciences sequencing we resolved the complete context of the blaIMP- gene on a large IncHI plasmid carried by all IMP- -producing strains across different hospitals Shotgun metagenomic sequencing ... More
Carbapenem-resistant Enterobacteriaceae (CRE) represent an urgent threat to human health. Here we report the application of several complementary whole-genome sequencing (WGS) technologies to characterise a hospital outbreak of blaIMP-4 carbapenemase-producing E. hormaechei. Using Illumina sequencing, we determined that all outbreak strains were sequence type 90 (ST90) and near-identical. Comparison to publicly available data linked all outbreak isolates to a 2013 isolate from the same ward, suggesting an environmental source in the hospital. Using Pacific Biosciences sequencing, we resolved the complete context of the blaIMP-4 gene on a large IncHI2 plasmid carried by all IMP-4-producing strains across different hospitals. Shotgun metagenomic sequencing of environmental samples also found evidence of ST90 E. hormaechei and the IncHI2 plasmid within the hospital plumbing. Finally, Oxford Nanopore sequencing rapidly resolved the true relationship of subsequent isolates to the initial outbreak. Overall, our strategic application of three WGS technologies provided an in-depth analysis of the outbreak. Less
Developing antibody agonists targeting the human apelin receptor APJ is a promising therapeutic approach for the treatment of chronic heart failure Here we report the structure-guided discovery of a single-domain antibody sdAb agonist JN - based on the cocrystal structure of APJ with an sdAb antagonist JN the first cocrystal structure of a class A G protein coupled receptor GPCR with a functional antibody As revealed by the structure JN binds to the extracellular side of APJ makes critical contacts with the second extracellular loop and inserts the CDR into the ligand-binding pocket We converted JN into a full agonist ... More
Developing antibody agonists targeting the human apelin receptor (APJ) is a promising therapeutic approach for the treatment of chronic heart failure. Here, we report the structure-guided discovery of a single-domain antibody (sdAb) agonist JN241-9, based on the cocrystal structure of APJ with an sdAb antagonist JN241, the first cocrystal structure of a class A G protein–coupled receptor (GPCR) with a functional antibody. As revealed by the structure, JN241 binds to the extracellular side of APJ, makes critical contacts with the second extracellular loop, and inserts the CDR3 into the ligand-binding pocket. We converted JN241 into a full agonist JN241-9 by inserting a tyrosine into the CDR3. Modeling and molecular dynamics simulation shed light on JN241-9–stimulated receptor activation, providing structural insights for finding agonistic antibodies against class A GPCRs. Less
Antimicrobial resistance is a major global threat that calls for new antibiotics Globomycin and myxovirescin are two natural antibiotics that target the lipoprotein-processing enzyme LspA thereby compromising the integrity of the bacterial cell envelope As part of a project aimed at understanding their mechanism of action and for drug development we provide high-resolution crystal structures of the enzyme from the human pathogen methicillin-resistant Staphylococcus aureus MRSA complexed with globomycin and with myxovirescin Our results reveal an instance of convergent evolution The two antibiotics possess different molecular structures Yet they appear to inhibit identically as non-cleavable tetrahedral intermediate analogs Remarkably the ... More
Antimicrobial resistance is a major global threat that calls for new antibiotics. Globomycin and myxovirescin are two natural antibiotics that target the lipoprotein-processing enzyme, LspA, thereby compromising the integrity of the bacterial cell envelope. As part of a project aimed at understanding their mechanism of action and for drug development, we provide high-resolution crystal structures of the enzyme from the human pathogen methicillin-resistant Staphylococcus aureus (MRSA) complexed with globomycin and with myxovirescin. Our results reveal an instance of convergent evolution. The two antibiotics possess different molecular structures. Yet, they appear to inhibit identically as non-cleavable tetrahedral intermediate analogs. Remarkably, the two antibiotics superpose along nineteen contiguous atoms that interact similarly with LspA. This 19-atom motif recapitulates a part of the substrate lipoprotein in its proposed binding mode. Incorporating this motif into a scaffold with suitable pharmacokinetic properties should enable the development of effective antibiotics with built-in resistance hardiness. Less
The most abundant member of the collagen protein family collagen I also known as type I collagen COL is composed of one unique chain B and two similar chain A polypeptides that self-assemble with one amino acid offset into a heterotrimeric triple helix Given the offset chain B can occupy either the leading BAA middle ABA or trailing AAB position of the triple helix yielding three isomeric biomacromolecules with different protein recognition properties Despite five decades of intensive research there is no consensus on the position of chain B in COL Here three triple-helical heterotrimers that each contain a putative ... More
The most abundant member of the collagen protein family, collagen I (also known as type I collagen; COL1), is composed of one unique (chain B) and two similar (chain A) polypeptides that self-assemble with one amino acid offset into a heterotrimeric triple helix. Given the offset, chain B can occupy either the leading (BAA), middle (ABA) or trailing (AAB) position of the triple helix, yielding three isomeric biomacromolecules with different protein recognition properties. Despite five decades of intensive research, there is no consensus on the position of chain B in COL1. Here, three triple-helical heterotrimers that each contain a putative von Willebrand factor (VWF) and discoidin domain receptor (DDR) recognition sequence from COL1 were designed with chain B permutated in all three positions. AAB demonstrated a strong preference for both VWF and DDR, and also induced higher levels of cellular DDR phosphorylation. Thus, we resolve this long-standing mystery and show that COL1 adopts an AAB register. Less
This chapter discusses that high-throughput HT macromolecular X-ray crystallography MX plays in the drug discovery process It focuses on the pivotal element in MX namely the diffraction experiment and describes the evolution of the main protagonists viz the crystal and the beam the means of producing each of them and of manipulating them so as to make them interact most efficiently and productively and the ways of deriving structure-based drug discovery-relevant structural information from observations of that interaction The chapter describes the all-human nine-step workflow that prevailed before systematic efforts were made to move from interactivity to automation serving as ... More
This chapter discusses that high-throughput (HT) macromolecular X-ray crystallography (MX) plays in the drug discovery process. It focuses on the pivotal element in MX, namely, the diffraction experiment, and describes the evolution of the main protagonists, viz. the crystal and the beam, the means of producing each of them and of manipulating them so as to make them interact most efficiently and productively, and the ways of deriving structure-based drug discovery-relevant structural information from observations of that interaction. The chapter describes the "all-human" nine-step workflow that prevailed before systematic efforts were made to move from interactivity to automation, serving as a "baseline" against which to motivate and describe those subsequent efforts. The ability of a crystalline specimen to yield Bragg reflections corresponding to atomic-scale detail is called its "diffraction quality" - higher quality meaning higher resolution limits. Less
Direct soaking of protein crystals with small-molecule fragments grouped into complementary clusters is a useful technique when assessing the potential of a new crystal system to support structure-guided drug discovery It provides a robustness check prior to any extensive crystal screening a double check for assay binding cutoffs and structural data for binding pockets that may or may not be picked out in assay measurements The structural output from this technique for three novel fragment molecules identified to bind to the antibacterial target Acinetobacter baumannii undecaprenyl pyrophosphate synthase are reported and the different physicochemical requirements of a successful antibiotic are ... More
Direct soaking of protein crystals with small-molecule fragments grouped into complementary clusters is a useful technique when assessing the potential of a new crystal system to support structure-guided drug discovery. It provides a robustness check prior to any extensive crystal screening, a double check for assay binding cutoffs and structural data for binding pockets that may or may not be picked out in assay measurements. The structural output from this technique for three novel fragment molecules identified to bind to the antibacterial target Acinetobacter baumannii undecaprenyl pyrophosphate synthase are reported, and the different physicochemical requirements of a successful antibiotic are compared with traditional medicines. Less
Serial crystallography at both synchrotron and X-ray free-electron laser light sources is becoming increasingly popular However the tools in the majority of crystallization laboratories are focused on producing large single crystals by vapour diffusion that fit the cryo-cooled paradigm of modern synchrotron crystallography This paper presents several case studies and some ideas and strategies on how to perform the conversion from a single crystal grown by vapour diffusion to the many thousands of micro-crystals required for modern serial crystallography grown by batch crystallization These case studies aim to show i how vapour diffusion conditions can be converted into batch by ... More
Serial crystallography, at both synchrotron and X-ray free-electron laser light sources, is becoming increasingly popular. However, the tools in the majority of crystallization laboratories are focused on producing large single crystals by vapour diffusion that fit the cryo-cooled paradigm of modern synchrotron crystallography. This paper presents several case studies and some ideas and strategies on how to perform the conversion from a single crystal grown by vapour diffusion to the many thousands of micro-crystals required for modern serial crystallography grown by batch crystallization. These case studies aim to show (i) how vapour diffusion conditions can be converted into batch by optimizing the length of time crystals take to appear; (ii) how an understanding of the crystallization phase diagram can act as a guide when designing batch crystallization protocols; and (iii) an accessible methodology when attempting to scale batch conditions to larger volumes. These methods are needed to minimize the sample preparation gap between standard rotation crystallography and dedicated serial laboratories, ultimately making serial crystallography more accessible to all crystallographers. Less
NEMO is a scaffolding protein which plays an essential role in the NF- B pathway by assembling the IKK-complex with the kinases IKK and IKK Upon activation the IKK complex phosphorylates the I B molecules leading to NF- B nuclear translocation and activation of target genes Inhibition of the NEMO IKK interaction is an attractive therapeutic paradigm for the modulation of NF- B pathway activity making NEMO a target for inhibitors design and discovery To facilitate the process of discovery and optimization of NEMO inhibitors we engineered an improved construct of the IKK-binding domain of NEMO that would allow for ... More
NEMO is a scaffolding protein which plays an essential role in the NF-κB pathway by assembling the IKK-complex with the kinases IKKα and IKKβ. Upon activation, the IKK complex phosphorylates the IκB molecules leading to NF-κB nuclear translocation and activation of target genes. Inhibition of the NEMO/IKK interaction is an attractive therapeutic paradigm for the modulation of NF-κB pathway activity, making NEMO a target for inhibitors design and discovery. To facilitate the process of discovery and optimization of NEMO inhibitors, we engineered an improved construct of the IKK-binding domain of NEMO that would allow for structure determination of the protein in the apo form and while bound to small molecular weight inhibitors. Here, we present the strategy utilized for the design, expression and structural characterization of the IKK-binding domain of NEMO. The protein is expressed in E. coli cells, solubilized under denaturing conditions and purified through three chromatographic steps. We discuss the protocols for obtaining crystals for structure determination and describe data acquisition and analysis strategies. The protocols will find wide applicability to the structure determination of complexes of NEMO and small molecule inhibitors. Less
The hormone melatonin secreted from the pineal gland mediates multiple physiological effects including modulation of Wnt -catenin signalling The Wnt palmitoleate lipid modification is essential for its signalling activity while the carboxylesterase Notum can remove the lipid from Wnt and inactivate it Notum enzyme inhibition can therefore upregulate Wnt signalling While searching for Notum inhibitors by crystallographic fragment screening a hit compound N- - -fluoro- H-indol- -yl ethyl acetamide that is structurally similar to melatonin came to our attention We then soaked melatonin and its precursor N-acetylserotonin into Notum crystals and obtained high-resolution structures of their complexes In each of ... More
The hormone melatonin, secreted from the pineal gland, mediates multiple physiological effects including modulation of Wnt/β-catenin signalling. The Wnt palmitoleate lipid modification is essential for its signalling activity, while the carboxylesterase Notum can remove the lipid from Wnt and inactivate it. Notum enzyme inhibition can therefore upregulate Wnt signalling. While searching for Notum inhibitors by crystallographic fragment screening, a hit compound N-[2-(5-fluoro-1H-indol-3-yl)ethyl]acetamide that is structurally similar to melatonin came to our attention. We then soaked melatonin and its precursor N-acetylserotonin into Notum crystals and obtained high-resolution structures (≤1.5 Å) of their complexes. In each of the structures, two compound molecules bind with Notum: one at the enzyme's catalytic pocket, overlapping the space occupied by the acyl tail of the Wnt palmitoleate lipid, and the other at the edge of the pocket opposite the substrate entrance. Although the inhibitory activity of melatonin shown by in vitro enzyme assays is low (IC50 75 µmol/L), the structural information reported here provides a basis for the design of potent and brain accessible drugs for neurodegenerative diseases such as Alzheimer's disease, in which upregulation of Wnt signalling may be beneficial. Less
Sex is a key modifier of neurological disease outcomes Microglia are implicated in neurological diseases and modulated by microRNAs but it is unknown whether microglial microRNAs have sex-specific influences on disease We show in mice that microglial microRNA expression differs in males and females and that loss of microRNAs leads to sex-specific changes in the microglial transcriptome and tau pathology These findings suggest that microglial microRNAs influence tau pathogenesis in a sex-specific manner
In tropical iron ore regions biologically mediated reduction of crystalline iron oxides drives ongoing iron cycling that contributes to the stability of surface duricrusts This represents a biotechnological opportunity with respect to post-mining rehabilitation attempts requiring re-formation of these duricrusts However cultivated dissimilatory iron reducing bacteria typically reduce crystalline iron oxides quite poorly A glucose-fermenting microbial consortium capable of reducing at least mmol L goethite was enriched from an iron duricrust region Metagenome analysis led to the recovery of a metagenome assembled genome MAG of an iron reducer belonging to the alphaproteobacterial genus Telmatospirillum This is the first report of ... More
In tropical iron ore regions, biologically mediated reduction of crystalline iron oxides drives ongoing iron cycling that contributes to the stability of surface duricrusts. This represents a biotechnological opportunity with respect to post-mining rehabilitation attempts, requiring re-formation of these duricrusts. However, cultivated dissimilatory iron reducing bacteria typically reduce crystalline iron oxides quite poorly. A glucose-fermenting microbial consortium capable of reducing at least 27 mmol/L goethite was enriched from an iron duricrust region. Metagenome analysis led to the recovery of a metagenome assembled genome (MAG) of an iron reducer belonging to the alphaproteobacterial genus Telmatospirillum. This is the first report of iron reduction within the Telmatospirillum and the first reported genome of an iron-reducing, neutrophilic member of the Alphaproteobacteria. The Telmatospirillum MAG encodes putative metal transfer reductases (MtrA, MtrB) and a novel, multi-heme outer membrane cytochrome for extracellular electron transfer. In the presence of goethite, short chain fatty acid production shifted significantly in favor of acetate rather than propionate, indicating goethite is a hydrogen sink in the culture. Therefore, the presence of fermentative bacteria likely promotes iron reduction via hydrogen production. Stimulating microbial fermentation has potential to drive reduction of crystalline iron oxides, the rate limiting step for iron duricrust re-formation. Less
Shiga toxin Stx is the major virulence factor of Shiga toxin-producing Escherichia coli STEC Stx evolves rapidly and as such new subtypes continue to emerge that challenge the efficacy of existing disease management and surveillance strategies A new subtype Stx k was recently identified in E coli isolated from a wide range of sources including diarrheal patients animals and raw meats and was poorly detected by existing immunoassays In this study the structure of Stx kE Q was determined at resolution and the conservation of structure with Stx a was revealed A novel polyclonal antibody capable of neutralizing Stx k ... More
Shiga toxin (Stx) is the major virulence factor of Shiga toxin-producing Escherichia coli (STEC). Stx evolves rapidly and, as such, new subtypes continue to emerge that challenge the efficacy of existing disease management and surveillance strategies. A new subtype, Stx2k, was recently identified in E. coli isolated from a wide range of sources including diarrheal patients, animals, and raw meats, and was poorly detected by existing immunoassays. In this study, the structure of Stx2kE167Q was determined at 2.29 Å resolution and the conservation of structure with Stx2a was revealed. A novel polyclonal antibody capable of neutralizing Stx2k and an immunoassay, with a 10-fold increase in sensitivity compared to assays using extant antibodies, were developed. Stx2k is less toxic than Stx2a in Vero cell assays but is similar to Stx2a in receptor-binding preference, thermostability, and acid tolerance. Although Stx2k does not appear to be as potent as Stx2a to Vero cells, the wide distribution and blended virulence profiles of the Stx2k-producing strains suggest that horizontal gene transfer through Stx2k-converting phages could result in the emergence of new and highly virulent pathogens. This study provides useful information and tools for early detection and control of Stx2k-producing E. coli, which could reduce public risk of infection by less-known STECs. Less
Membrane integral ATP synthases produce adenosine triphosphate the universal energy currency of most organisms However important details of proton driven energy conversion are still unknown We present the first high-resolution structure of the in meso crystallized c-ring of subunits from spinach chloroplasts The structure reveals molecular mechanisms of intersubunit contacts in the c -ring and it shows additional electron densities inside the c-ring which form circles parallel to the membrane plane Similar densities were found in all known high-resolution structures of c-rings of F FO ATP synthases from archaea and bacteria to eukaryotes The densities might originate from isoprenoid quinones ... More
Membrane integral ATP synthases produce adenosine triphosphate, the universal “energy currency” of most organisms. However, important details of proton driven energy conversion are still unknown. We present the first high-resolution structure (2.3 Å) of the in meso crystallized c-ring of 14 subunits from spinach chloroplasts. The structure reveals molecular mechanisms of intersubunit contacts in the c14-ring, and it shows additional electron densities inside the c-ring which form circles parallel to the membrane plane. Similar densities were found in all known high-resolution structures of c-rings of F1FO ATP synthases from archaea and bacteria to eukaryotes. The densities might originate from isoprenoid quinones (such as coenzyme Q in mitochondria and plastoquinone in chloroplasts) that is consistent with differential UV-Vis spectroscopy of the c-ring samples, unusually large distance between polar/apolar interfaces inside the c-ring and universality among different species. Although additional experiments are required to verify this hypothesis, coenzyme Q and its analogues known as electron carriers of bioenergetic chains may be universal cofactors of ATP synthases, stabilizing c-ring and prevent ion leakage through it. Less
Cysteinyl leukotriene G protein-coupled receptors CysLT and CysLT regulate pro-inflammatory responses associated with allergic disorders While selective inhibition of CysLT R has been used for treating asthma and associated diseases for over two decades CysLT R has recently started to emerge as a potential drug target against atopic asthma brain injury and central nervous system disorders as well as several types of cancer Here we describe four crystal structures of CysLT R in complex with three dual CysLT R CysLT R antagonists The reported structures together with the results of comprehensive mutagenesis and computer modeling studies shed light on molecular ... More
Cysteinyl leukotriene G protein-coupled receptors CysLT1 and CysLT2 regulate pro-inflammatory responses associated with allergic disorders. While selective inhibition of CysLT1R has been used for treating asthma and associated diseases for over two decades, CysLT2R has recently started to emerge as a potential drug target against atopic asthma, brain injury and central nervous system disorders, as well as several types of cancer. Here, we describe four crystal structures of CysLT2R in complex with three dual CysLT1R/CysLT2R antagonists. The reported structures together with the results of comprehensive mutagenesis and computer modeling studies shed light on molecular determinants of CysLTR ligand selectivity and specific effects of disease-related single nucleotide variants. Less
The availability of whole-genome sequence data made possible by significant advances in DNA sequencing technology led to the emergence of structural genomics projects in the late s These projects not only significantly increased the number of D structures deposited in the Protein Data Bank in the last two decades but also influenced present crystallographic strategies by introducing automation and high-throughput approaches in the structure-determination pipeline Today dedicated crystallization facilities many of which are open to the general user community routinely set up and track thousands of crystallization screening trials per day Here we review the current methods for high-throughput crystallization ... More
The availability of whole-genome sequence data, made possible by significant advances in DNA sequencing technology, led to the emergence of structural genomics projects in the late 1990s. These projects not only significantly increased the number of 3D structures deposited in the Protein Data Bank in the last two decades, but also influenced present crystallographic strategies by introducing automation and high-throughput approaches in the structure-determination pipeline. Today, dedicated crystallization facilities, many of which are open to the general user community, routinely set up and track thousands of crystallization screening trials per day. Here, we review the current methods for high-throughput crystallization and procedures to obtain crystals suitable for X-ray diffraction studies, and we describe the crystallization pipeline implemented in the medium-scale crystallography platform at the Institut Pasteur (Paris) as an example. Less
Adrenergic receptors are highly homologous while at the same time display a wide diversity of ligand and G-protein binding and understanding this diversity is key for designing selective or biased drugs for them Here we determine two crystal structures of the a A adrenergic receptor a AAR in complex with a partial agonist and an antagonist Key non-conserved residues from the ligand-binding pocket Phe and Tyr to G-protein coupling region Ile and Lys are discovered to play a key role in the interplay between partial agonism and biased signaling of a AAR which provides insights into the diversity of ligand ... More
Adrenergic receptors are highly homologous while at the same time display a wide diversity of ligand and G-protein binding, and understanding this diversity is key for designing selective or biased drugs for them. Here, we determine two crystal structures of the a2A adrenergic receptor (a2AAR) in complex with a partial agonist and an antagonist. Key non-conserved residues from the ligand-binding pocket (Phe7.39 and Tyr6.55) to G-protein coupling region (Ile34.51 and Lys34.56) are discovered to play a key role in the interplay between partial agonism and biased signaling of a2AAR, which provides insights into the diversity of ligand binding and G-protein coupling preference of adrenergic receptors and lays the foundation for the discovery of next-generation drugs targeting these receptors. Less
Broadly HIV- neutralizing VRC class antibodies target the CD -binding site of Env They are derived from VH - antibody heavy chains paired with rare light chains expressing -amino acid-long CDRL s They have been isolated from infected subjects but have not yet been elicited by immunization Env-derived immunogens capable of binding the germline forms of VRC B cell receptors on naive B cells have been designed and evaluated in knockin mice However the elicited antibodies cannot bypass glycans present on the conserved position N of Env which restricts access to the CD -binding site Efforts to guide the appropriate ... More
Broadly HIV-1 neutralizing VRC01 class antibodies target the CD4-binding site of Env. They are derived from VH1-2*02 antibody heavy chains paired with rare light chains expressing 5-amino acid-long CDRL3s. They have been isolated from infected subjects but have not yet been elicited by immunization. Env-derived immunogens capable of binding the germline forms of VRC01 B cell receptors on naive B cells have been designed and evaluated in knockin mice. However, the elicited antibodies cannot bypass glycans present on the conserved position N276 of Env, which restricts access to the CD4-binding site. Efforts to guide the appropriate maturation of these antibodies by sequential immunization have not yet been successful. Here, we report on a two-step immunization scheme that leads to the maturation of VRC01-like antibodies capable of accommodating the N276 glycan and displaying autologous tier 2 neutralizing activities. Our results are relevant to clinical trials aiming to elicit VRC01 antibodies. Less
Adrenergic G-protein-coupled receptors GPCRs mediate different cellular signaling pathways in the presence of endogenous catecholamines and play important roles in both physiological and pathological conditions Extensive studies have been carried out to investigate the structure and function of adrenergic receptors ARs However the structure of a adrenergic receptors aARs remains to be determined Here we report the structure of the human a C adrenergic receptor a CAR with the non-selective antagonist RS at Our structure mutations modeling and functional experiments indicate that a a CAR-specific D ECL -R ECL -Y network plays a role in determining a adrenergic subtype selectivity ... More
Adrenergic G-protein-coupled receptors (GPCRs) mediate different cellular signaling pathways in the presence of endogenous catecholamines and play important roles in both physiological and pathological conditions. Extensive studies have been carried out to investigate the structure and function of � adrenergic receptors (�ARs). However, the structure of a adrenergic receptors (aARs) remains to be determined. Here, we report the structure of the human a2C adrenergic receptor (a2CAR) with the non-selective antagonist, RS79948, at 2.8 �. Our structure, mutations, modeling, and functional experiments indicate that a a2CAR-specific D206ECL2-R409ECL3-Y4056.58 network plays a role in determining a2 adrenergic subtype selectivity. Furthermore, our results show that a specific loosened helix at the top of TM4 in a2CAR is involved in receptor activation. Together, our structure of human a2CAR-RS79948 provides key insight into the mechanism underlying the a2 adrenergic receptor activation and subtype selectivity. Less
Crystallization processes have been widely used in the pharmaceutical industry for the manufacture storage and delivery of small-molecule and small protein therapeutics However the identification of crystallization processes for biologics particularly monoclonal antibodies has been prohibitive due to the size and the flexibility of their overall structure There remains a challenge and an opportunity to utilize the benefits of crystallization of biologics The research laboratories of Merck Sharp Dome Corp MSD in collaboration with the International Space Station ISS National Laboratory performed crystallization experiments with pembrolizumab Keytruda on the SpaceX-Commercial Resupply Services- mission to the ISS By leveraging microgravity effects ... More
Crystallization processes have been widely used in the pharmaceutical industry for the manufacture, storage, and delivery of small-molecule and small protein therapeutics. However, the identification of crystallization processes for biologics, particularly monoclonal antibodies, has been prohibitive due to the size and the flexibility of their overall structure. There remains a challenge and an opportunity to utilize the benefits of crystallization of biologics. The research laboratories of Merck Sharp & Dome Corp. (MSD) in collaboration with the International Space Station (ISS) National Laboratory performed crystallization experiments with pembrolizumab (Keytruda�) on the SpaceX-Commercial Resupply Services-10 mission to the ISS. By leveraging microgravity effects such as reduced sedimentation and minimal convection currents, conditions producing crystalline suspensions of homogeneous monomodal particle size distribution (39 �m) in high yield were identified. In contrast, the control ground experiments produced crystalline suspensions with a heterogeneous bimodal distribution of 13 and 102 �m particles. In addition, the flight crystalline suspensions were less viscous and sedimented more uniformly than the comparable ground-based crystalline suspensions. These results have been applied to the production of crystalline suspensions on earth, using rotational mixers to reduce sedimentation and temperature gradients to induce and control crystallization. Using these techniques, we have been able to produce uniform crystalline suspensions (1�5 �m) with acceptable viscosity (<12 cP), rheological, and syringeability properties suitable for the preparation of an injectable formulation. The results of these studies may help widen the drug delivery options to improve the safety, adherence, and quality of life for patients and caregivers. Less
Reaching the full potential of optoelectronic materials is often hindered by the years of necessary trial-and-error Perovskites are an example of materials having exceptional optoelectronic properties but require improvement with respect to stability and toxicity as they approach commercialization Exploring new types of perovskites is key to achieving these goals In this thesis I develop an accelerated materials discovery pipeline aimed at discovering new perovskite materials This pipeline incorporates image recognition that detects crystals via convolutional neural networks with accuracy and uses parameter exploration to predict an optimal material with experimental data With this framework I discovered a new type ... More
Reaching the full potential of optoelectronic materials is often hindered by the years of necessary trial-and-error. Perovskites are an example of materials having exceptional optoelectronic properties, but require improvement with respect to stability and toxicity as they approach commercialization. Exploring new types of perovskites is key to achieving these goals. In this thesis I develop an accelerated materials discovery pipeline aimed at discovering new perovskite materials. This pipeline incorporates image recognition that detects crystals via convolutional neural networks with 95% accuracy and uses parameter exploration to predict an optimal material with experimental data. With this framework, I discovered a new type of perovskite single crystal, (3-PLA)2PbCl4, that employs a new ligand, 3-PLA, offering avenues to higher efficiency and more stable devices. This work develops a framework for discovering and optimizing materials in a wide chemical space and provides the groundwork for identifying new materials that lie beyond known chemical spaces. Less
Selective activation of the -opioid receptor DOP has great potential for the treatment of chronic pain benefitting from ancillary anxiolytic and antidepressant-like effects Moreover DOP agonists show reduced adverse effects as compared to -opioid receptor MOP agonists that are in the spotlight of the current opioid crisis Here we report the first crystal structures of the DOP in an activated state in complex with two relevant and structurally diverse agonists the potent opioid agonist peptide KGCHM and the small-molecule agonist DPI- at and resolution respectively Our study identifies key determinants for agonist recognition receptor activation and DOP selectivity revealing crucial ... More
Selective activation of the δ-opioid receptor (DOP) has great potential for the treatment of chronic pain, benefitting from ancillary anxiolytic and antidepressant-like effects. Moreover, DOP agonists show reduced adverse effects as compared to μ-opioid receptor (MOP) agonists that are in the spotlight of the current “opioid crisis.” Here, we report the first crystal structures of the DOP in an activated state, in complex with two relevant and structurally diverse agonists: the potent opioid agonist peptide KGCHM07 and the small-molecule agonist DPI-287 at 2.8 and 3.3 Å resolution, respectively. Our study identifies key determinants for agonist recognition, receptor activation, and DOP selectivity, revealing crucial differences between both agonist scaffolds. Our findings provide the first investigation into atomic-scale agonist binding at the DOP, supported by site-directed mutagenesis and pharmacological characterization. These structures will underpin the future structure-based development of DOP agonists for an improved pain treatment with fewer adverse effects. Less
The release of synthetic chemical pollutants in the environment is posing serious health risks Enzymes including oxygenases play a crucial role in xenobiotic degradation In the present study we employed a functional metagenomics approach to overcome the limitation of cultivability of microbes under standard laboratory conditions in order to isolate novel dioxygenases capable of degrading recalcitrant pollutants Fosmid clones possessing dioxygenase activity were further sequenced and their genes were identified using bioinformatics tools Two positive fosmid clones SD and RW suggested the presence of -dihydroxybiphenyl -dioxygenase BphC-SD and catechol -dioxygenase C O-RW respectively Recombinant versions of these enzymes were purified ... More
The release of synthetic chemical pollutants in the environment is posing serious health risks. Enzymes, including oxygenases, play a crucial role in xenobiotic degradation. In the present study, we employed a functional metagenomics approach to overcome the limitation of cultivability of microbes under standard laboratory conditions in order to isolate novel dioxygenases capable of degrading recalcitrant pollutants. Fosmid clones possessing dioxygenase activity were further sequenced, and their genes were identified using bioinformatics tools. Two positive fosmid clones, SD3 and RW1, suggested the presence of 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC-SD3) and catechol 2,3-dioxygenase (C23O-RW1), respectively. Recombinant versions of these enzymes were purified to examine their pollutant-degrading abilities. The crystal structure of BphC-SD3 was determined at 2.6-Å resolution, revealing a two-domain architecture, i.e., N-terminal and C-terminal domains, with the sequential arrangement of βαβββ in each domain, characteristic of Fe-dependent class II type I extradiol dioxygenases. The structure also reveals the presence of conserved amino acids lining the catalytic pocket and Fe3+ metal ion in the large funnel-shaped active site in the C-terminal domain. Further studies suggest that Fe3+ bound in the BphC-SD3 active site probably imparts aerobic stability. We further demonstrate the potential application of BphC-SD3 in biosensing of catecholic compounds. The halotolerant and oxygen-resistant properties of these enzymes reported in this study make them potential candidates for bioremediation and biosensing applications. Less
Staphylococcus aureus strains produce a unique family of immunostimulatory exotoxins termed as bacterial superantigens SAgs which cross-link major histocompatibility complex class II MHC II molecule and T-cell receptor TCR to stimulate large numbers of T cells at extremely low concentrations SAgs are associated with food poisoning and toxic shock syndrome To date genetically distinct staphylococcal SAgs have been reported This study reports the first X-ray structure of newly characterized staphylococcal enterotoxin N SEN SEN possesses the classical two domain architecture that includes an N-terminal oligonucleotide-binding fold and a C-terminal -grasp domain Amino acid and structure alignments revealed that several critical ... More
Staphylococcus aureus strains produce a unique family of immunostimulatory exotoxins termed as bacterial superantigens (SAgs), which cross-link major histocompatibility complex class II (MHC II) molecule and T-cell receptor (TCR) to stimulate large numbers of T cells at extremely low concentrations. SAgs are associated with food poisoning and toxic shock syndrome. To date, 26 genetically distinct staphylococcal SAgs have been reported. This study reports the first X-ray structure of newly characterized staphylococcal enterotoxin N (SEN). SEN possesses the classical two domain architecture that includes an N-terminal oligonucleotide-binding fold and a C-terminal β-grasp domain. Amino acid and structure alignments revealed that several critical amino acids that are proposed to be responsible for MHC II and TCR molecule engagements are variable in SEN, suggesting that SEN may adopt a different binding mode to its cellular receptors. This work helps better understand the mechanisms of action of SAgs. Less
The selective downregulation of activated intracellular proteins is a key challenge in cell biology RHO small GTPases switch between a guanosine diphosphate GDP -bound and a guanosine triphosphate GTP -bound state that drives downstream signaling At present no tool is available to study endogenous RHO-GTPinduced conformational changes in live cells Here we established a cell-based screen to selectively degrade RHOB-GTP using F-box-intracellular single-domain antibody fusion We identified one intracellular antibody intrabody that shows selective targeting of endogenous RHOB-GTP mediated by interactions between the CDR loop of the domain antibody and the GTP-binding pocket of RHOB Our results suggest that while ... More
The selective downregulation of activated intracellular proteins is a key challenge in cell biology. RHO small GTPases switch between a guanosine diphosphate (GDP)-bound and a guanosine triphosphate (GTP)-bound state that drives downstream signaling. At present, no tool is available to study endogenous RHO-GTPinduced conformational changes in live cells. Here, we established a cell-based screen to selectively degrade RHOB-GTP using F-box-intracellular single-domain antibody fusion. We identified one intracellular antibody (intrabody) that shows selective targeting of endogenous RHOB-GTP mediated by interactions between the CDR3 loop of the domain antibody and the GTP-binding pocket of RHOB. Our results suggest that, while RHOB is highly regulated at the expression level, only the GTP-bound pool, but not its global expression, mediates RHOB functions in genomic instability and in cell invasion. The F-box/intrabody-targeted protein degradation represents a unique approach to knock down the active form of small GTPases or other proteins with multiple cellular activities. Less
Short-term parameters correlating to long-term protein stability such as the protein cloud point temperature Tcloud are of interest to improve efficiency during protein product development Such efficiency is reached if short-term parameters are obtained in a low volume and high-throughput HT manner This study presents a low volume HT detection method for sub-zero Tcloud determination of lysozyme as such an experimental method is not available yet The setup consists of a cryogenic device with an automated imaging system Measurement reproducibility median absolute deviation of C and literature-based parameter validation Pearson correlation coefficient of were shown by a robustness and validation ... More
Short-term parameters correlating to long-term protein stability, such as the protein cloud point temperature (Tcloud), are of interest to improve efficiency during protein product development. Such efficiency is reached if short-term parameters are obtained in a low volume and high-throughput (HT) manner. This study presents a low volume HT detection method for (sub-zero) Tcloud determination of lysozyme, as such an experimental method is not available yet. The setup consists of a cryogenic device with an automated imaging system. Measurement reproducibility (median absolute deviation of 0.2 °C) and literature-based parameter validation (Pearson correlation coefficient of 0.996) were shown by a robustness and validation study. The subsequent case study demonstrated a partial correlation between the obtained apparent Tcloud parameter and long-term protein stability as a function of lysozyme concentration, ion type, ionic strength, and freeze/thaw stress. The presented experimental setup demonstrates its ability to advance short-term strategies for efficient protein formulation development. Less
The over-expression and aggregation of -synuclein Syn are linked to the onset and pathology of Parkinson s disease Native monomeric Syn exists in an intrinsically disordered ensemble of interconverting conformations which has made its therapeutic targeting by small molecules highly challenging Nonetheless here we successfully target the monomeric structural ensemble of Syn and thereby identify novel drug-like small molecules that impact multiple pathogenic processes Using a surface plasmon resonance high-throughput screen in which monomeric Syn is incubated with microchips arrayed with tethered compounds we identified novel Syn interacting drug-like compounds Because these small molecules could impact a variety of Syn ... More
The over-expression and aggregation of α-synuclein (αSyn) are linked to the onset and pathology of Parkinson’s disease. Native monomeric αSyn exists in an intrinsically disordered ensemble of interconverting conformations, which has made its therapeutic targeting by small molecules highly challenging. Nonetheless, here we successfully target the monomeric structural ensemble of αSyn and thereby identify novel drug-like small molecules that impact multiple pathogenic processes. Using a surface plasmon resonance high-throughput screen, in which monomeric αSyn is incubated with microchips arrayed with tethered compounds, we identified novel αSyn interacting drug-like compounds. Because these small molecules could impact a variety of αSyn forms present in the ensemble, we tested representative hits for impact on multiple αSyn malfunctions in vitro and in cells including aggregation and perturbation of vesicular dynamics. We thereby identified a compound that inhibits αSyn misfolding and is neuroprotective, multiple compounds that restore phagocytosis impaired by αSyn overexpression, and a compound blocking cellular transmission of αSyn. Our studies demonstrate that drug-like small molecules that interact with native αSyn can impact a variety of its pathological processes. Thus, targeting the intrinsically disordered ensemble of αSyn offers a unique approach to the development of small molecule research tools and therapeutics for Parkinson’s disease. Less
In vitro models cell culture models microtissue organ-on-a-chip microfabrication micropumps membrane manufacturing filtration vascularization perfusion microfluidics channels cleanroom silicon glass polydimethylsiloxane additive manufacturing bioprinting bioprinter three-dimensional printing bioreactor stimulation biointerfaces scaffold barrier junction sterilization biodegradable polymers functionalization hydrogels coatings etching photolithography microinjection fluid control automation disposable autoclavable media exchange sampling pipetting viscous drag pump peristaltic pump capillary pump valves bubble traps flow sensor pressure sensor noninvasive monitoring oxygen sensor pH sensor mechanotransduction shear stress compression tensile stress gauge sensor TEER electrochemical sensor biosensor calibration immunosensor ion-sensitive field-effect transistor electrical impedance spectroscopy label-free sensor lactate sensor glucose sensor temperature sensor
The elicitation of broadly neutralizing antibodies bNAbs against the HIV- envelope glycoprotein Env trimer remains a major vaccine challenge Most cross-conserved protein determinants are occluded by self-N-glycan shielding limiting B cell recognition of the underlying polypeptide surface The exceptions to the contiguous glycan shield include the conserved receptor CD binding site CD bs and glycoprotein gp elements proximal to the furin cleavage site Accordingly we performed heterologous trimer-liposome prime boosting in rabbits to drive B cells specific for cross-conserved sites To preferentially expose the CD bs to B cells we eliminated proximal N-glycans while maintaining the native-like state of the ... More
The elicitation of broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope glycoprotein (Env) trimer remains a major vaccine challenge. Most cross-conserved protein determinants are occluded by self-N-glycan shielding, limiting B cell recognition of the underlying polypeptide surface. The exceptions to the contiguous glycan shield include the conserved receptor CD4 binding site (CD4bs) and glycoprotein (gp)41 elements proximal to the furin cleavage site. Accordingly, we performed heterologous trimer-liposome prime:boosting in rabbits to drive B cells specific for cross-conserved sites. To preferentially expose the CD4bs to B cells, we eliminated proximal N-glycans while maintaining the native-like state of the cleavage-independent NFL trimers, followed by gradual N-glycan restoration coupled with heterologous boosting. This approach successfully elicited CD4bs-directed, cross-neutralizing Abs, including one targeting a unique glycan-protein epitope and a bNAb (87% breadth) directed to the gp120:gp41 interface, both resolved by high-resolution cryoelectron microscopy. This study provides proof-of-principle immunogenicity toward eliciting bNAbs by vaccination Less
Hit-to-lead optimization is a critical phase in drug discovery Herein we report on the fragment-based discovery and optimization of -aminopyridine derivatives as a novel lead-like structure for the treatment of the dangerous opportunistic pathogen Pseudomonas aeruginosa We pursue an innovative treatment strategy by interfering with the Pseudomonas quinolone signal PQS quorum sensing QS system leading to an abolishment of bacterial pathogenicity Our compounds act on the PQS receptor PqsR a key transcription factor controlling the expression of various pathogenicity determinants In this target-driven approach we made use of biophysical screening via surface plasmon resonance SPR followed by isothermal titration calorimetry ... More
Hit-to-lead optimization is a critical phase in drug discovery. Herein, we report on the fragment-based discovery and optimization of 2-aminopyridine derivatives as a novel lead-like structure for the treatment of the dangerous opportunistic pathogen Pseudomonas aeruginosa. We pursue an innovative treatment strategy by interfering with the Pseudomonas quinolone signal (PQS) quorum sensing (QS) system leading to an abolishment of bacterial pathogenicity. Our compounds act on the PQS receptor (PqsR), a key transcription factor controlling the expression of various pathogenicity determinants. In this target-driven approach, we made use of biophysical screening via surface plasmon resonance (SPR) followed by isothermal titration calorimetry (ITC)-enabled enthalpic efficiency (EE) evaluation. Hit optimization then involved growth vector identification and exploitation. Astonishingly, the latter was successfully achieved by introducing flexible linkers rather than rigid motifs leading to a boost in activity on the target receptor and anti-virulence potency. Less
Well-ordered HIV- envelope glycoprotein Env trimers are prioritized for clinical evaluation and there is a need for an improved understanding about how elicited B cell responses evolve following immunization To accomplish this we prime-boosted rhesus macaques with clade C NFL trimers and identified unique Ab lineages from single-sorted Env-specific memory B cells We traced all lineages in high-throughput heavy chain HC repertoire Rep-seq data generated from multiple immune compartments and time points and expressed several as monoclonal Abs mAbs Our results revealed broad dissemination and high levels of somatic hypermutation SHM of most lineages including tier virus neutralizing lineages following ... More
Well-ordered HIV-1 envelope glycoprotein (Env) trimers are prioritized for clinical evaluation, and there is a need for an improved understanding about how elicited B cell responses evolve following immunization. To accomplish this, we prime-boosted rhesus macaques with clade C NFL trimers and identified 180 unique Ab lineages from ∼1,000 single-sorted Env-specific memory B cells. We traced all lineages in high-throughput heavy chain (HC) repertoire (Rep-seq) data generated from multiple immune compartments and time points and expressed several as monoclonal Abs (mAbs). Our results revealed broad dissemination and high levels of somatic hypermutation (SHM) of most lineages, including tier 2 virus neutralizing lineages, following boosting. SHM was highest in the Ab complementarity determining regions (CDRs) but also surprisingly high in the framework regions (FRs), especially FR3. Our results demonstrate the capacity of the immune system to affinity-mature large numbers of Env-specific B cell lineages simultaneously, supporting the use of regimens consisting of repeated boosts to improve each Ab, even those belonging to less expanded lineages. Less
A multiprotein complex polarisome nucleates actin cables for polarized cell growth in budding yeast and filamentous fungi However the dynamic regulations of polarisome proteins in polymerizing actin under physiological and stress conditions remains unknown We identify a previously functionally unknown polarisome member actin-interacting-protein Aip which promotes actin assembly synergistically with formin Bni Aip -C terminus is responsible for its activities by interacting with G-actin and Bni Through N-terminal intrinsically disordered region Aip forms high-order oligomers and generate cytoplasmic condensates under the stresses conditions The molecular dynamics and reversibility of Aip condensates are regulated by scaffolding protein Spa via liquid-liquid phase ... More
A multiprotein complex polarisome nucleates actin cables for polarized cell growth in budding yeast and filamentous fungi. However, the dynamic regulations of polarisome proteins in polymerizing actin under physiological and stress conditions remains unknown. We identify a previously functionally unknown polarisome member, actin-interacting-protein 5 (Aip5), which promotes actin assembly synergistically with formin Bni1. Aip5-C terminus is responsible for its activities by interacting with G-actin and Bni1. Through N-terminal intrinsically disordered region, Aip5 forms high-order oligomers and generate cytoplasmic condensates under the stresses conditions. The molecular dynamics and reversibility of Aip5 condensates are regulated by scaffolding protein Spa2 via liquid-liquid phase separation both in vitro and in vivo. In the absence of Spa2, Aip5 condensates hamper cell growth and actin cable structures under stress treatment. The present study reveals the mechanisms of actin assembly for polarity establishment and the adaptation in stress conditions to protect actin assembly by protein phase separation. Less
The investigational drugs E indisulam and tasisulam aryl-sulfonamides promote the degradation of the splicing factor RBM in a proteasome-dependent mechanism While the activity critically depends on the Cullin RING ligase substrate receptor DCAF the molecular details remain elusive Here we present the cryo-EM structure of the DDB -DCAF -DDA core ligase complex bound to RBM and E at resolution together with crystal structures of engineered subcomplexes We show that DCAF adopts a novel fold stabilized by DDA and that extensive protein-protein contacts between the ligase and substrate mitigate low affinity interactions between aryl-sulfonamides and DCAF Our data demonstrates how aryl-sulfonamides ... More
The investigational drugs E7820, indisulam and tasisulam (aryl-sulfonamides) promote the degradation of the splicing factor RBM39 in a proteasome-dependent mechanism. While the activity critically depends on the Cullin RING ligase substrate receptor DCAF15, the molecular details remain elusive. Here we present the cryo-EM structure of the DDB1-DCAF15-DDA1 core ligase complex bound to RBM39 and E7820 at 4.4 Å resolution, together with crystal structures of engineered subcomplexes. We show that DCAF15 adopts a novel fold stabilized by DDA1, and that extensive protein-protein contacts between the ligase and substrate mitigate low affinity interactions between aryl-sulfonamides and DCAF15. Our data demonstrates how aryl-sulfonamides neo-functionalize a shallow, non-conserved pocket on DCAF15 to selectively bind and degrade RBM39 and the closely related splicing factor RBM23 without the requirement for a high affinity ligand, which has broad implications for the de novo discovery of molecular glue degraders. Less
Indanomycin is biosynthesized by a hybrid nonribosomal peptide synthase polyketide synthase NRPS PKS followed by a number of tailoring' steps to form the two ring systems that are present in the mature product It had previously been hypothesized that the indane ring of indanomycin was formed by the action of IdmH using a Diels Alder reaction Here the crystal structure of a selenomethionine-labelled truncated form of IdmH IdmH- was solved using single-wavelength anomalous dispersion SAD phasing This truncated variant allows consistent and easy crystallization but importantly the structure was used as a search model in molecular replacement allowing the full-length ... More
Indanomycin is biosynthesized by a hybrid nonribosomal peptide synthase/polyketide synthase (NRPS/PKS) followed by a number of `tailoring' steps to form the two ring systems that are present in the mature product. It had previously been hypothesized that the indane ring of indanomycin was formed by the action of IdmH using a Diels–Alder reaction. Here, the crystal structure of a selenomethionine-labelled truncated form of IdmH (IdmH-Δ99–107) was solved using single-wavelength anomalous dispersion (SAD) phasing. This truncated variant allows consistent and easy crystallization, but importantly the structure was used as a search model in molecular replacement, allowing the full-length IdmH structure to be determined to 2.7 Å resolution. IdmH is a homodimer, with the individual protomers consisting of an α+β barrel. Each protomer contains a deep hydrophobic pocket which is proposed to constitute the active site of the enzyme. To investigate the reaction catalysed by IdmH, 88% of the backbone NMR resonances were assigned, and using chemical shift perturbation of [15N]-labelled IdmH it was demonstrated that indanomycin binds in the active-site pocket. Finally, combined quantum mechanical/molecular mechanical (QM/MM) modelling of the IdmH reaction shows that the active site of the enzyme provides an appropriate environment to promote indane-ring formation, supporting the assignment of IdmH as the key Diels–Alderase catalysing the final step in the biosynthesis of indanomycin through a similar mechanism to other recently characterized Diels–Alderases involved in polyketide-tailoring reactions. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at https://proteopedia.org/w/Journal:IUCrJ:S2052252519012399. Less
The class B family of G-protein-coupled receptors GPCRs has long been a paradigm for peptide hormone recognition and signal transduction One class B GPCR the glucagon-like peptide- receptor GLP- R has been considered as an anti-diabetes drug target and there are several peptidic drugs available for the treatment of this overwhelming disease The previously determined structures of inactive GLP- R in complex with two negative allosteric modulators include ten thermal-stabilizing mutations that were selected from a total of designed mutations Here we systematically summarize all mutations we have tested and the results suggest that the mutagenesis strategy that strengthens inter-helical ... More
The class B family of G-protein-coupled receptors (GPCRs) has long been a paradigm for peptide hormone recognition and signal transduction. One class B GPCR, the glucagon-like peptide-1 receptor (GLP-1R), has been considered as an anti-diabetes drug target and there are several peptidic drugs available for the treatment of this overwhelming disease. The previously determined structures of inactive GLP-1R in complex with two negative allosteric modulators include ten thermal-stabilizing mutations that were selected from a total of 98 designed mutations. Here we systematically summarize all 98 mutations we have tested and the results suggest that the mutagenesis strategy that strengthens inter-helical hydrophobic interactions shows the highest success rate. We further investigate four back mutations by thermal-shift assay, crystallization and molecular dynamic simulations, and conclude that mutation I1962.66bF increases thermal stability intrinsically and that mutation S2714.47bA decreases crystal packing entropy extrinsically, while mutations S1932.63bC and M2333.36bC may be dispensable since these two cysteines are not disulfide-linked. Our results indicate intrinsic connections between different regions of GPCR transmembrane helices and the current data suggest a general mutagenesis principle for structural determination of GPCRs and other membrane proteins. Less
Redesigning existing food protein formulations is necessary in situations where food authorities propose dose adjustments or removal of currently employed additives Redesigning formulations involves evaluating substitute additives to obtain similar long-term physical stability as the original formulation Such formulation screening experiments benefit from comprehensive data visualization understanding the effects of substitute additives on long-term physical stability and identification of short-term optimization targets This work employs empirical phase diagrams to reach these benefits by combining multidimensional long-term protein physical stability data with short-term empirical protein properties A case study was performed where multidimensional protein phase diagrams formulations allowed for identification of ... More
Redesigning existing food protein formulations is necessary in situations where food authorities propose dose adjustments or removal of currently employed additives. Redesigning formulations involves evaluating substitute additives to obtain similar long-term physical stability as the original formulation. Such formulation screening experiments benefit from comprehensive data visualization, understanding the effects of substitute additives on long-term physical stability, and identification of short-term optimization targets. This work employs empirical phase diagrams to reach these benefits by combining multidimensional long-term protein physical stability data with short-term empirical protein properties. A case study was performed where multidimensional protein phase diagrams (1152 formulations) allowed for identification of stabilizing effects as a result of pH, methionine, sugars, salt, and minimized glycerol content. Corresponding empirical protein property diagrams (144 formulations) resulted in the identification of normalized surface tension as a short-term empirical protein property to reach long-term physical stability presumably similar to the original product, namely via preferential hydration. Additionally, changes in pH and salt were identified as environmental optimization targets to reach stability via repulsive electrostatic forces. This case study shows the applicability of the empirical phase diagram method to rationally perform formulation redesign screenings, while simultaneously expanding knowledge on protein long-term physical stability. Less
Recently two groups of rhodopsin genes were identified in large double-stranded DNA viruses The structure and function of viral rhodopsins are unknown We present functional characterization and high-resolution structure of an Organic Lake Phycodnavirus rhodopsin II OLPVRII of group It forms a pentamer with a symmetrical bottle-like central channel with the narrow vestibule in the cytoplasmic part covered by a ring of arginines whereas phenylalanines form a hydrophobic barrier in its exit The proton donor E is placed in the helix B The structure is unique among the known rhodopsins Structural and functional data and molecular dynamics suggest that OLPVRII ... More
Recently, two groups of rhodopsin genes were identified in large double-stranded DNA viruses. The structure and function of viral rhodopsins are unknown. We present functional characterization and high-resolution structure of an Organic Lake Phycodnavirus rhodopsin II (OLPVRII) of group 2. It forms a pentamer, with a symmetrical, bottle-like central channel with the narrow vestibule in the cytoplasmic part covered by a ring of 5 arginines, whereas 5 phenylalanines form a hydrophobic barrier in its exit. The proton donor E42 is placed in the helix B. The structure is unique among the known rhodopsins. Structural and functional data and molecular dynamics suggest that OLPVRII might be a light-gated pentameric ion channel analogous to pentameric ligand-gated ion channels, however, future patch clamp experiments should prove this directly. The data shed light on a fundamentally distinct branch of rhodopsins and may contribute to the understanding of virus-host interactions in ecologically important marine protists. Less
Purpose The overall goal of this study was to investigate the dissolution performance and crystallization kinetics of amorphous solid dispersions ASDs of a weakly basic compound posaconazole dispersed in a pH-sensitive polymeric matrix consisting of hydroxypropyl methylcellulose acetate succinate HPMC-AS using fasted-state simulated media Methods ASDs with three different drug loadings and wt and the commercially available tablets were exposed to acidic media pH followed by transfer to and dissolution in intestinal media pH Parallel single stage dissolution experiments in only simulated intestinal media were also performed to better understand the impact of the gastric stage Different analytical methods including ... More
Purpose
The overall goal of this study was to investigate the dissolution performance and crystallization kinetics of amorphous solid dispersions (ASDs) of a weakly basic compound, posaconazole, dispersed in a pH-sensitive polymeric matrix consisting of hydroxypropyl methylcellulose acetate succinate (HPMC-AS), using fasted-state simulated media.
Methods
ASDs with three different drug loadings, 10, 25 and 50 wt.%, and the commercially available tablets were exposed to acidic media (pH 1.6), followed by transfer to, and dissolution in, intestinal media (pH 6.5). Parallel single stage dissolution experiments in only simulated intestinal media were also performed to better understand the impact of the gastric stage. Different analytical methods, including nanoparticle tracking analysis, powder x-ray diffraction, second harmonic generation and two-photon excitation ultraviolet fluorescence microscopy, were used to characterize the phase behavior of these systems at different stages of dissolution.
Results
Results revealed that all ASDs exhibited some degree of drug release upon suspension in acidic media, and were also vulnerable to matrix crystallization. Upon transfer to intestinal media conditions, supersaturation was observed. This was short-lived for some dispersions due to the release of the crystals formed in the acid immersion stage which acted as seeds for crystal growth. Lower drug loading ASDs also exhibited transient formation of amorphous nanodroplets prior to crystallization.
Conclusions
This work emphasizes the significance of assessing the impact of pH change on dissolution and provides a fundamental basis of understanding the phase behavior kinetics of ASDs of weakly basic drugs when formulated with pH sensitive polymers. Less
The overall goal of this study was to investigate the dissolution performance and crystallization kinetics of amorphous solid dispersions (ASDs) of a weakly basic compound, posaconazole, dispersed in a pH-sensitive polymeric matrix consisting of hydroxypropyl methylcellulose acetate succinate (HPMC-AS), using fasted-state simulated media.
Methods
ASDs with three different drug loadings, 10, 25 and 50 wt.%, and the commercially available tablets were exposed to acidic media (pH 1.6), followed by transfer to, and dissolution in, intestinal media (pH 6.5). Parallel single stage dissolution experiments in only simulated intestinal media were also performed to better understand the impact of the gastric stage. Different analytical methods, including nanoparticle tracking analysis, powder x-ray diffraction, second harmonic generation and two-photon excitation ultraviolet fluorescence microscopy, were used to characterize the phase behavior of these systems at different stages of dissolution.
Results
Results revealed that all ASDs exhibited some degree of drug release upon suspension in acidic media, and were also vulnerable to matrix crystallization. Upon transfer to intestinal media conditions, supersaturation was observed. This was short-lived for some dispersions due to the release of the crystals formed in the acid immersion stage which acted as seeds for crystal growth. Lower drug loading ASDs also exhibited transient formation of amorphous nanodroplets prior to crystallization.
Conclusions
This work emphasizes the significance of assessing the impact of pH change on dissolution and provides a fundamental basis of understanding the phase behavior kinetics of ASDs of weakly basic drugs when formulated with pH sensitive polymers. Less
Macrophage migration inhibitory factor MIF is a pleiotropic cytokine that increasingly is being studied in cancers and inflammatory diseases Though murine models have been instrumental in understanding the functional role of MIF in different pathological conditions the information obtained from these models is biased towards a specific species In experimental science results obtained from multiple clinically relevant animal models always provide convincing data that might recapitulate in humans Syrian golden hamster Mesocricetus auratus is a clinically relevant animal model for multiple human diseases Hence the major objectives of this study were to characterize the structure and function of Mesocricetus auratus ... More
Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that increasingly is being studied in cancers and inflammatory diseases. Though murine models have been instrumental in understanding the functional role of MIF in different pathological conditions, the information obtained from these models is biased towards a specific species. In experimental science, results obtained from multiple clinically relevant animal models always provide convincing data that might recapitulate in humans. Syrian golden hamster (Mesocricetus auratus), is a clinically relevant animal model for multiple human diseases. Hence, the major objectives of this study were to characterize the structure and function of Mesocricetus auratus MIF (MaMIF) and finally evaluate its effect on pancreatic tumor growth in vivo. Initially, the recombinant MaMIF was cloned, expressed and purified in a bacterial expression system. The MaMIF primary sequence, biochemical properties, and crystal structure analysis showed greater similarity with human MIF. The crystal structure of MaMIF illustrates that it forms a homotrimer as known in human and mouse. However, MaMIF exhibits some minor structural variations when compared to human and mouse MIF. The in vitro functional studies show that MaMIF has tautomerase activity and enhances activation and migration of hamster peripheral blood mononuclear cells (PBMCs). Interestingly, injection of MaMIF into HapT1 pancreatic tumor-bearing hamsters significantly enhanced the tumor growth and tumor-associated angiogenesis. Together, the current study shows a structural and functional similarity between the hamster and human MIF. Moreover, it has demonstrated that a high level of circulating MIF originating from non-tumor cells might also promote pancreatic tumor growth in vivo. Less
Macrophage migration inhibitory factor MIF is a pleiotropic cytokine that increasingly is being studied in cancers and inflammatory diseases Though murine models have been instrumental in understanding the functional role of MIF in different pathological conditions the information obtained from these models is biased towards a specific species In experimental science results obtained from multiple clinically relevant animal models always provide convincing data that might recapitulate in humans Syrian golden hamster Mesocricetus auratus is a clinically relevant animal model for multiple human diseases Hence the major objectives of this study were to characterize structure and function of hamster MIF and ... More
Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that increasingly is being studied in cancers and inflammatory diseases. Though murine models have been instrumental in understanding the functional role of MIF in different pathological conditions, the information obtained from these models is biased towards a specific species. In experimental science, results obtained from multiple clinically relevant animal models always provide convincing data that might recapitulate in humans. Syrian golden hamster (Mesocricetus auratus), is a clinically relevant animal model for multiple human diseases. Hence, the major objectives of this study were to characterize structure and function of hamster MIF, and finally evaluate its effect on pancreatic tumor growth in vivo. Initially, the recombinant hamster MIF (rha-MIF) was cloned, expressed and purified in bacterial expression system. The rha-MIF primary sequence, biochemical properties and crystal structure analysis showed a greater similarity with human MIF. The crystal structure of hamster MIF illustrates that it forms a homotrimer as known in human and mouse. However, hamster MIF exhibits some minor structural variations when compared to human and mouse MIF. The in vitro functional studies show that rha-MIF has tautomerase activity and enhances activation and migration of hamster peripheral blood mononuclear cells (PBMCs). Interestingly, injection of rha-MIF into HapT1 pancreatic tumor bearing hamsters significantly enhanced the tumor growth and tumor associated angiogenesis. Together, the current study shows a structural and functional similarity between hamster and human MIF. Moreover, it has demonstrated that a high-level of circulating MIF originating from non-tumor cells might also promote pancreatic tumor growth in vivo. Less
Rational structure-based drug design SBDD relies on the availability of a large number of co-crystal structures to map the ligand-binding pocket of the target protein and use this information for lead-compound optimization via an iterative process While SBDD has proven successful for many drug-discovery projects its application to G protein-coupled receptors GPCRs has been limited owing to extreme difficulties with their crystallization Here a method is presented for the rapid determination of multiple co-crystal structures for a target GPCR in complex with various ligands taking advantage of the serial femtosecond crystallography approach which obviates the need for large crystals and ... More
Rational structure-based drug design (SBDD) relies on the availability of a large number of co-crystal structures to map the ligand-binding pocket of the target protein and use this information for lead-compound optimization via an iterative process. While SBDD has proven successful for many drug-discovery projects, its application to G protein-coupled receptors (GPCRs) has been limited owing to extreme difficulties with their crystallization. Here, a method is presented for the rapid determination of multiple co-crystal structures for a target GPCR in complex with various ligands, taking advantage of the serial femtosecond crystallography approach, which obviates the need for large crystals and requires only submilligram quantities of purified protein. The method was applied to the human β2-adrenergic receptor, resulting in eight room-temperature co-crystal structures with six different ligands, including previously unreported structures with carvedilol and propranolol. The generality of the proposed method was tested with three other receptors. This approach has the potential to enable SBDD for GPCRs and other difficult-to-crystallize membrane proteins. Less
Co-inhibitory immune receptors can contribute to T cell dysfunction in patients with cancer Blocking antibodies against cytotoxic T-lymphocyte-associated protein CTLA- and programmed cell death PD- partially reverse this effect and are becoming standard of care in an increasing number of malignancies However many of the other axes by which tumours become inhospitable to T cells are not fully understood Here we report that V-domain immunoglobulin suppressor of T cell activation VISTA engages and suppresses T cells selectively at acidic pH such as that found in tumour microenvironments Multiple histidine residues along the rim of the VISTA extracellular domain mediate binding ... More
Co-inhibitory immune receptors can contribute to T cell dysfunction in patients with cancer1,2. Blocking antibodies against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) partially reverse this effect and are becoming standard of care in an increasing number of malignancies3. However, many of the other axes by which tumours become inhospitable to T cells are not fully understood. Here we report that V-domain immunoglobulin suppressor of T cell activation (VISTA) engages and suppresses T cells selectively at acidic pH such as that found in tumour microenvironments. Multiple histidine residues along the rim of the VISTA extracellular domain mediate binding to the adhesion and co-inhibitory receptor P-selectin glycoprotein ligand-1 (PSGL-1). Antibodies engineered to selectively bind and block this interaction in acidic environments were sufficient to reverse VISTA-mediated immune suppression in vivo. These findings identify a mechanism by which VISTA may engender resistance to anti-tumour immune responses, as well as an unexpectedly determinative role for pH in immune co-receptor engagement. Less
The tricarboxylic acid cycle intermediate succinate is involved in metabolic processes and plays a crucial role in the homeostasis of mitochondrial reactive oxygen species The receptor responsible for succinate signalling SUCNR also known as GPR is a member of the G-protein-coupled-receptor family and links succinate signalling to renin-induced hypertension retinal angiogenesis and inflammation Because SUCNR senses succinate as an immunological danger signal which has relevance for diseases including ulcerative colitis liver fibrosis diabetes and rheumatoid arthritis it is of interest as a therapeutic target Here we report the high-resolution crystal structure of rat SUCNR in complex with an intracellular binding ... More
The tricarboxylic acid cycle intermediate succinate is involved in metabolic processes and plays a crucial role in the homeostasis of mitochondrial reactive oxygen species1. The receptor responsible for succinate signalling, SUCNR1 (also known as GPR91), is a member of the G-protein-coupled-receptor family2 and links succinate signalling to renin-induced hypertension, retinal angiogenesis and inflammation3,4,5. Because SUCNR1 senses succinate as an immunological danger signal6—which has relevance for diseases including ulcerative colitis, liver fibrosis7, diabetes and rheumatoid arthritis3,8—it is of interest as a therapeutic target. Here we report the high-resolution crystal structure of rat SUCNR1 in complex with an intracellular binding nanobody in the inactive conformation. Structure-based mutagenesis and radioligand-binding studies, in conjunction with molecular modelling, identified key residues for species-selective antagonist binding and enabled the determination of the high-resolution crystal structure of a humanized rat SUCNR1 in complex with a high-affinity, human-selective antagonist denoted NF-56-EJ40. We anticipate that these structural insights into the architecture of the succinate receptor and its antagonist selectivity will enable structure-based drug discovery and will further help to elucidate the function of SUCNR1 in vitro and in vivo. Less
Influenza A virus IAV nonstructural protein NS a potent antagonist of the host immune response is capable of interacting with RNA and a wide range of cellular proteins NS consists of an RNA-binding domain RBD and an effector domain ED separated by a flexible linker region LR H N -NS has a characteristic -residue deletion in the LR with either G minor group or E major group at the st position and non-H N -NS contains E with an intact linker Based on the orientation of the ED with respect to the RBD previous crystallographic studies have shown that minor ... More
Influenza A virus (IAV) nonstructural protein 1 (NS1), a potent antagonist of the host immune response, is capable of interacting with RNA and a wide range of cellular proteins. NS1 consists of an RNA-binding domain (RBD) and an effector domain (ED) separated by a flexible linker region (LR). H5N1-NS1 has a characteristic 5-residue deletion in the LR, with either G (minor group) or E (major group) at the 71st position, and non-H5N1-NS1 contains E71 with an intact linker. Based on the orientation of the ED with respect to the RBD, previous crystallographic studies have shown that minor group H5N1-NS1(G71), a non-H5N1-NS1 [H6N6-NS1(E71)], and the LR deletion mutant H6N6-NS1(Δ80-84/E71) mimicking the major group H5N1-NS1 exhibit “open,” “semiopen,” and “closed” conformations, respectively, suggesting that NS1 exhibits a strain-dependent conformational preference. Here we report the first crystal structure of a naturally occurring H5N1-NS1(E71) and show that it adopts an open conformation similar to that of the minor group of H5N1-NS1 [H5N1-NS1(G71)]. We also show that H6N6-NS1(Δ80-84/E71) under a different crystallization condition and H6N6-NS1(Δ80-84/G71) also exhibit open conformations, suggesting that NS1 can adopt an open conformation irrespective of E or G at the 71st position. Our single-molecule fluorescence resonance energy transfer (FRET) analysis to investigate the conformational preference of NS1 in solution showed that all NS1 constructs predominantly exist in an open conformation. Further, our coimmunoprecipitation and binding studies showed that they all bind to cellular factors with similar affinities. Taken together, our studies suggest that NS1 exhibits strain-independent structural plasticity that allows it to interact with a wide variety of cellular ligands during viral infection. Less
Musashi- MSI belongs to Musashi family of RNA binding proteins RBP Like Musashi- MSI it is overexpressed in a variety of cancers and is a promising therapeutic target Both MSI proteins contain two N-terminal RNA recognition motifs and play roles in posttranscriptional regulation of target mRNAs Previously we have identified several inhibitors of MSI all of which bind to MSI as well In order to design MSI -specific inhibitors and compare the differences of binding mode of the inhibitors we set out to solve the structure of MSI -RRM the key motif that is responsible for the binding Here we ... More
Musashi-2 (MSI2) belongs to Musashi family of RNA binding proteins (RBP). Like Musashi-1 (MSI1), it is overexpressed in a variety of cancers and is a promising therapeutic target. Both MSI proteins contain two N-terminal RNA recognition motifs and play roles in posttranscriptional regulation of target mRNAs. Previously, we have identified several inhibitors of MSI1, all of which bind to MSI2 as well. In order to design MSI2-specific inhibitors and compare the differences of binding mode of the inhibitors, we set out to solve the structure of MSI2-RRM1, the key motif that is responsible for the binding. Here, we report the crystal structure and the first NMR solution structure of MSI2-RRM1, and compare these to the structures of MSI1-RBD1 and other RBPs. A high degree of structural similarity was observed between the crystal and solution NMR structures. MSI2-RRM1 shows a highly similar overall folding topology to MSI1-RBD1 and other RBPs. The structural information of MSI2-RRM1 will be helpful for understanding MSI2-RNA interaction and for guiding rational drug design of MSI2-specific inhibitors. Less
The G protein coupled cysteinyl leukotriene receptor CysLT R mediates inflammatory processes and plays a major role in numerous disorders including asthma allergic rhinitis cardiovascular disease and cancer Selective CysLT R antagonists are widely prescribed as antiasthmatic drugs however these drugs demonstrate low effectiveness in some patients and exhibit a variety of side effects To gain deeper understanding into the functional mechanisms of CysLTRs we determined the crystal structures of CysLT R bound to two chemically distinct antagonists zafirlukast and pranlukast The structures reveal unique ligand-binding modes and signaling mechanisms including lateral ligand access to the orthosteric pocket between transmembrane ... More
The G protein–coupled cysteinyl leukotriene receptor CysLT1R mediates inflammatory processes and plays a major role in numerous disorders, including asthma, allergic rhinitis, cardiovascular disease, and cancer. Selective CysLT1R antagonists are widely prescribed as antiasthmatic drugs; however, these drugs demonstrate low effectiveness in some patients and exhibit a variety of side effects. To gain deeper understanding into the functional mechanisms of CysLTRs, we determined the crystal structures of CysLT1R bound to two chemically distinct antagonists, zafirlukast and pranlukast. The structures reveal unique ligand-binding modes and signaling mechanisms, including lateral ligand access to the orthosteric pocket between transmembrane helices TM4 and TM5, an atypical pattern of microswitches, and a distinct four-residue–coordinated sodium site. These results provide important insights and structural templates for rational discovery of safer and more effective drugs. Less
Protein trafficking requires coat complexes that couple recognition of sorting motifs in transmembrane cargoes with biogenesis of transport carriers The mechanisms of cargo transport through the endosomal network are poorly understood Here we identify a sorting motif for endosomal recycling of cargoes including the cation-independent mannose- -phosphate receptor and semaphorin C by the membrane tubulating BAR domain-containing sorting nexins SNX and SNX Crystal structures establish that this motif folds into a -hairpin which binds a site in the SNX SNX phox homology domains Over sixty cargoes share this motif and require SNX SNX for their recycling These include cargoes involved ... More
Protein trafficking requires coat complexes that couple recognition of sorting motifs in transmembrane cargoes with biogenesis of transport carriers. The mechanisms of cargo transport through the endosomal network are poorly understood. Here, we identify a sorting motif for endosomal recycling of cargoes, including the cation-independent mannose-6-phosphate receptor and semaphorin 4C, by the membrane tubulating BAR domain-containing sorting nexins SNX5 and SNX6. Crystal structures establish that this motif folds into a β-hairpin, which binds a site in the SNX5/SNX6 phox homology domains. Over sixty cargoes share this motif and require SNX5/SNX6 for their recycling. These include cargoes involved in neuronal migration and a Drosophila snx6 mutant displays defects in axonal guidance. These studies identify a sorting motif and provide molecular insight into an evolutionary conserved coat complex, the ‘Endosomal SNX–BAR sorting complex for promoting exit 1’ (ESCPE-1), which couples sorting motif recognition to the BAR-domain-mediated biogenesis of cargo-enriched tubulo-vesicular transport carriers. Less
Protein-crystallization imaging and classification is a labor-intensive process typically performed either by humans or by instruments that currently cost well over This cost puts the use of crystallization-trial imaging outside the reach of most academic laboratories and also start-up biotechnology firms where resources are scarce An imaging system has been designed and prototyped which automatically captures images from multi-well protein-crystallization experiments using both standard and fluorescent imaging techniques at a cost times lower than current market rates The machine uses a Panowin F D printer as a base and controls it using G-code commands sent from a Python script running ... More
Protein-crystallization imaging and classification is a labor-intensive process typically performed either by humans or by instruments that currently cost well over $100 000. This cost puts the use of crystallization-trial imaging outside the reach of most academic laboratories, and also start-up biotechnology firms, where resources are scarce. An imaging system has been designed and prototyped which automatically captures images from multi-well protein-crystallization experiments using both standard and fluorescent imaging techniques at a cost 28 times lower than current market rates. The machine uses a Panowin F1 3D printer as a base and controls it using G-code commands sent from a Python script running on a desktop computer. A graphical user interface (GUI) was developed to enable users to control the machine and facilitate image capture, classification and editing. A 488 nm laser diode and a 525 nm filter were incorporated to allow in situ fluorescent imaging of proteins trace-labeled with a fluorophore, Alexa Fluor 488. The instrument was primarily designed using a 3D printer and augmented using commercially available parts, and this publication aims to serve as a guide for comparable in-laboratory robotics projects. Less
The influence of process parameters during freeze thaw FT operations is essential for the preservation of the protein stability activity during production and storage processes in the biopharmaceutical industry Process parameters such as FT ramps the final storage time and temperature affect the occurring FT stress onto the target protein in different ways FT stress includes cold denaturation freeze concentration and ice crystal formation which can result in protein aggregation To visualize the impact of variations in FT ramps descriptors such as solubility phase behavior and crystal morphology were evaluated The phase diagram-based toolbox in combination with an HTS-compatible cryo-device ... More
The influence of process parameters during freeze/thaw (FT) operations is essential for the preservation of the protein stability/activity during production and storage processes in the biopharmaceutical industry. Process parameters, such as FT ramps, the final storage time and temperature, affect the occurring FT stress onto the target protein in different ways. FT stress includes cold denaturation, freeze concentration, and ice crystal formation which can result in protein aggregation. To visualize the impact of variations in FT ramps, descriptors such as solubility, phase behavior and crystal morphology were evaluated. The phase diagram-based toolbox in combination with an HTS-compatible cryo-device allowed the identification of suitable ramping schemes during FT operations. It could be clearly shown that rapid operations are needed above the glass transition temperature of the target protein to circumvent precipitation during FT cycles. Finally, a stability index is introduced which allows ranking of the systems investigated. Less
Microfluidic devices have been increasingly used for low-volume liquid handling operations However laboratory automation of such delicate devices has lagged behind due to the lack of world-to-chip macro-to-micro interfaces In this paper we have presented the first pipette-free robotic microfluidic interface using a microfluidic-embedded container cap referred to as a microfluidic cap-to-dispense CD to achieve a seamless integration of liquid handling and robotic automation without any traditional pipetting steps The CD liquid handling platform offers a generic and modular way to connect the robotic device to standard liquid containers It utilizes the high accuracy and high flexibility of the robotic ... More
Microfluidic devices have been increasingly used for low-volume liquid handling operations. However, laboratory automation of such delicate devices has lagged behind due to the lack of world-to-chip (macro-to-micro) interfaces. In this paper, we have presented the first pipette-free robotic–microfluidic interface using a microfluidic-embedded container cap, referred to as a microfluidic cap-to-dispense (μCD), to achieve a seamless integration of liquid handling and robotic automation without any traditional pipetting steps. The μCD liquid handling platform offers a generic and modular way to connect the robotic device to standard liquid containers. It utilizes the high accuracy and high flexibility of the robotic system to recognize, capture and position; and then using microfluidic adaptive printing it can achieve high-precision on-demand volume distribution. With its modular connectivity, nanoliter processability, high adaptability, and multitask capacity, μCD shows great potential as a generic robotic–microfluidic interface for complete pipette-free liquid handling automation. Less