901 Citations
Subcutaneous SC administration is a desired route for monoclonal antibodies mAbs However formulating mAbs for small injection volumes at high concentrations with suitable stability and injectability is a significant challenge Here this work presents a platform technology that combines the stability of crystalline antibodies with injectability and tunability of soft hydrogel particles Composite alginate hydrogel particles are generated via a gentle centrifugal encapsulation process which avoids use of chemical reactions or an external organic phase Crystalline suspension of anti-programmed cell death protein PD- antibody pembrolizumab is utilized as a model therapeutic antibody Crystalline forms of the mAb encapsuled in the ... More
Subcutaneous (SC) administration is a desired route for monoclonal antibodies (mAbs). However, formulating mAbs for small injection volumes at high concentrations with suitable stability and injectability is a significant challenge. Here, this work presents a platform technology that combines the stability of crystalline antibodies with injectability and tunability of soft hydrogel particles. Composite alginate hydrogel particles are generated via a gentle centrifugal encapsulation process which avoids use of chemical reactions or an external organic phase. Crystalline suspension of anti-programmed cell death protein 1 (PD-1) antibody (pembrolizumab) is utilized as a model therapeutic antibody. Crystalline forms of the mAb encapsuled in the hydrogel particles lead to stable, high concentration, and injectable formulations. Formulation concentrations as high as 315 mg mL−1 antibody are achieved with encapsulation efficiencies in the range of 89–97%, with no perceivable increase in the number of antibody aggregates. Bioanalytical studies confirm superior maintained quality of the antibody in comparison with formulation approaches involving organic phases and chemical reactions. This work illustrates tuning the alginate particles’ disintegration by using partially oxide alginates. Crystalline mAb-laden particles are evaluated for their biocompatibility using cell-based in vitro assays. Furthermore, the pharmacokinetics (PK) of the subcutaneously delivered human anti-PD-1 mAb in crystalline antibody-laden alginate hydrogel particles in Wistar rats is evaluated. Less
PT Formulatrix is one of the industries that produce automation tools or robots therefore companies are required to produce automation products that meet the needs of the world market PT Formulatrix has production divisions LH Liquid Handling and RI Rock Imager Examples of the production of automation equipment include NT Formulator for liquid handling and RI RI RI for rock imagers The difference lies in the shape and function of each product The problem focuses on the rock imager production process where production is made with a make to order system and the assembly process is parallel making the assembly ... More
PT. Formulatrix is one of the industries that produce automation tools or robots, therefore companies are required to produce automation products that meet the needs of the world market. PT. Formulatrix has 2 production divisions LH (Liquid Handling) and RI (Rock Imager). Examples of the production of automation equipment include, NT8, Formulator for liquid handling and RI 1000, RI 182, RI 54 for rock imagers. The difference lies in the shape and function of each product. The problem focuses on the rock imager production process, where production is made with a make to order system and the assembly process is parallel, making the assembly not smooth because you have to choose components, waiting for replacement components because they are damaged or missing to assemble. To improve the effective and efficient production process, replace the better production process. While production in liquid handling is said to be smooth because the production is small and the product is small compared to the rock imager. Assembling in parallel makes assemblers a hassle when assembling robots, because they have to sort out robot components in one container. Making assembly inefficient and ineffective. After collecting 5S data to support the kitting process in the rock imager assembling division, the process of assembling a one-table robot with components. As for the application of the 5S method applied at PT. Formulatrix Indonesia. The seiri method is applied to every division without exception, because in each division many mechanical equipment is found scattered on tables or on the floor. Seiton is a continuation of seiri, where the sorting results that have been carried out will be followed by the process of arranging the sorted equipment. Seiso at this stage, what is being done is the cleaning process. The cleaning that is done is cleaning the work area, such as the floor of the equipment used for the production process. At this stage it is more directed at the process of monitoring the 5S method that has been implemented. This stage is the last part of the 5S method. This section focuses more on how to get used to the application of this method. With the kitting process and the 5S method, it is hoped that assembling robots will be more efficient and effective, because waiting, sorting out parts, and looking for tools to assemble will be more optimal than the old process. With the kitting process, the efficiency value is better than the old process, from the previous 83% to 92%, which the researchers got from a trial at PT. Formulatrix Indonesia. Keywords: Rock Imager production process, 5S, Kitting. Less
The use of periodically structured illumination coupled with spatial Fourier-transform fluorescence recovery after photobleaching FT-FRAP was shown to support diffusivity mapping within segmented domains of arbitrary shape Periodic comb-bleach patterning of the excitation beam during photobleaching encoded spatial maps of diffusion onto harmonic peaks in the spatial Fourier transform Diffusion manifests as a simple exponential decay of a given harmonic improving the signal to noise ratio and simplifying mathematical analysis Image segmentation prior to Fourier transformation was shown to support pooling for signal to noise enhancement for regions of arbitrary shape expected to exhibit similar diffusivity within a domain Following ... More
The use of periodically structured illumination coupled with spatial Fourier-transform fluorescence recovery after photobleaching (FT-FRAP) was shown to support diffusivity mapping within segmented domains of arbitrary shape. Periodic “comb-bleach” patterning of the excitation beam during photobleaching encoded spatial maps of diffusion onto harmonic peaks in the spatial Fourier transform. Diffusion manifests as a simple exponential decay of a given harmonic, improving the signal to noise ratio and simplifying mathematical analysis. Image segmentation prior to Fourier transformation was shown to support pooling for signal to noise enhancement for regions of arbitrary shape expected to exhibit similar diffusivity within a domain. Following proof-of-concept analyses based on simulations with known ground-truth maps, diffusion imaging by FT-FRAP was used to map spatially-resolved diffusion differences within phase-separated domains of model amorphous solid dispersion spin-cast thin films. Notably, multi-harmonic analysis by FT-FRAP was able to definitively discriminate and quantify the roles of internal diffusion and exchange to higher mobility interfacial layers in modeling the recovery kinetics within thin amorphous/amorphous phase-separated domains, with interfacial diffusion playing a critical role in recovery. These results have direct implications for the design of amorphous systems for stable storage and efficacious delivery of therapeutic molecules. Less
Enzymatic pockets such as those of histone deacetylases HDACs are among the most favored targets for drug development However enzymatic inhibitors often exhibit low selectivity and high toxicity due to targeting multiple enzyme paralogs which are often involved in distinct multisubunit complexes Here we report the discovery and characterization of a non-enzymatic small molecule inhibitor of HDAC transcriptional repression functions with comparable anti-tumor activity to the enzymatic HDAC inhibitor Vorinostat and anti-psychedelic activity of an HDAC knockout in vivo We highlight that these phenotypes are achieved while modulating the expression of - and -fold fewer genes than enzymatic and genetic ... More
Enzymatic pockets such as those of histone deacetylases (HDACs) are among the most favored targets for drug development. However, enzymatic inhibitors often exhibit low selectivity and high toxicity due to targeting multiple enzyme paralogs, which are often involved in distinct multisubunit complexes. Here, we report the discovery and characterization of a non-enzymatic small molecule inhibitor of HDAC transcriptional repression functions with comparable anti-tumor activity to the enzymatic HDAC inhibitor Vorinostat, and anti-psychedelic activity of an HDAC2 knockout in vivo. We highlight that these phenotypes are achieved while modulating the expression of 20- and 80-fold fewer genes than enzymatic and genetic inhibition in the respective models. Thus, by achieving the same biological outcomes as established therapeutics while impacting a dramatically smaller number of genes, inhibitors of protein-protein interactions can offer important advantages in improving the selectivity of epigenetic modulators. Less
Opioid-related fatal overdoses have reached epidemic proportions Because existing treatments for opioid use disorders offer limited long-term protection accelerating the development of newer approaches is critical Monoclonal antibodies mAbs are an emerging treatment strategy that targets and sequesters selected opioids in the bloodstream reducing drug distribution across the blood-brain barrier thus preventing or reversing opioid toxicity We previously identified a series of murine mAbs with high affinity and selectivity for oxycodone morphine fentanyl and nicotine To determine their binding mechanism we used X-ray crystallography to solve the structures of mAbs bound to their respective targets to resolution or higher Structural ... More
Opioid-related fatal overdoses have reached epidemic proportions. Because existing treatments for opioid use disorders offer limited long-term protection, accelerating the development of newer approaches is critical. Monoclonal antibodies (mAbs) are an emerging treatment strategy that targets and sequesters selected opioids in the bloodstream, reducing drug distribution across the blood-brain barrier, thus preventing or reversing opioid toxicity. We previously identified a series of murine mAbs with high affinity and selectivity for oxycodone, morphine, fentanyl, and nicotine. To determine their binding mechanism, we used X-ray crystallography to solve the structures of mAbs bound to their respective targets, to 2.2 Å resolution or higher. Structural analysis showed a critical convergent hydrogen bonding mode that is dependent on a glutamic acid residue in the mAbs’ heavy chain and a tertiary amine of the ligand. Characterizing drug-mAb complexes represents a significant step toward rational antibody engineering and future manufacturing activities to support clinical evaluation. Less
Chemical modifications of RNA have key roles in many biological processes N -methylguanosine m G is required for integrity and stability of a large subset of tRNAs The methyltransferase WD repeat-containing protein METTL WDR complex is the methyltransferase that modifies G in the variable loop of certain tRNAs and its dysregulation drives tumorigenesis in numerous cancer types Mutations in WDR cause human developmental phenotypes including microcephaly How METTL WDR modifies tRNA substrates and is regulated remains elusive Here we show through structural biochemical and cellular studies of human METTL WDR that WDR serves as a scaffold for METTL and the ... More
Chemical modifications of RNA have key roles in many biological processes1,2,3. N7-methylguanosine (m7G) is required for integrity and stability of a large subset of tRNAs4,5,6,7. The methyltransferase 1–WD repeat-containing protein 4 (METTL1–WDR4) complex is the methyltransferase that modifies G46 in the variable loop of certain tRNAs, and its dysregulation drives tumorigenesis in numerous cancer types8,9,10,11,12,13,14. Mutations in WDR4 cause human developmental phenotypes including microcephaly15,16,17. How METTL1–WDR4 modifies tRNA substrates and is regulated remains elusive18. Here we show, through structural, biochemical and cellular studies of human METTL1–WDR4, that WDR4 serves as a scaffold for METTL1 and the tRNA T-arm. Upon tRNA binding, the αC region of METTL1 transforms into a helix, which together with the α6 helix secures both ends of the tRNA variable loop. Unexpectedly, we find that the predicted disordered N-terminal region of METTL1 is part of the catalytic pocket and essential for methyltransferase activity. Furthermore, we reveal that S27 phosphorylation in the METTL1 N-terminal region inhibits methyltransferase activity by locally disrupting the catalytic centre. Our results provide a molecular understanding of tRNA substrate recognition and phosphorylation-mediated regulation of METTL1–WDR4, and reveal the presumed disordered N-terminal region of METTL1 as a nexus of methyltransferase activity. Less
Linear IgE epitopes play essential roles in persistent allergies including peanut and tree nut allergies Using chemically synthesized peptides attached to membranes and microarray experiments is one approach for determining predominant epitopes that has seen success However the overall expense of this approach and the inherent challenges in scaling up the production and purification of synthetic peptides precludes the general application of this approach To overcome this problem we have constructed a plasmid vector for expressing peptides sandwiched between an N-terminal His-tag and a trimeric protein The vector was used to make overlapping peptides derived from peanut allergens Ara h ... More
Linear IgE epitopes play essential roles in persistent allergies, including peanut and tree nut allergies. Using chemically synthesized peptides attached to membranes and microarray experiments is one approach for determining predominant epitopes that has seen success. However, the overall expense of this approach and the inherent challenges in scaling up the production and purification of synthetic peptides precludes the general application of this approach. To overcome this problem, we have constructed a plasmid vector for expressing peptides sandwiched between an N-terminal His-tag and a trimeric protein. The vector was used to make overlapping peptides derived from peanut allergens Ara h 2. All the peptides were successfully expressed and purified. The resulting peptides were applied to identify IgE binding epitopes of Ara h 2 using four sera samples from individuals with known peanut allergies. New and previously defined dominant IgE binding epitopes of Ara h 2 were identified. This system may be readily applied to produce agents for component- and epitope-resolved food allergy diagnosis. Less
X-ray crystallography has long been a key method in solving the three-dimensional structure of proteins Structural information is essential for unraveling the molecular function of proteins and structure-based drug design However there are several obstacles associated with the structural determination of proteins using X-ray crystallography such as the generation of a large amount of protein samples instability of purified proteins and difficulty in obtaining large and well-diffracting crystals all of which can prolong the process of determining the crystal structure from months to years Over the past decade new techniques and strategies have been developed to assist X-ray crystallographers in ... More
X-ray crystallography has long been a key method in solving the three-dimensional structure of proteins. Structural information is essential for unraveling the molecular function of proteins and structure-based drug design. However, there are several obstacles associated with the structural determination of proteins using X-ray crystallography, such as the generation of a large amount of protein samples, instability of purified proteins, and difficulty in obtaining large and well-diffracting crystals, all of which can prolong the process of determining the crystal structure, from months to years. Over the past decade, new techniques and strategies have been developed to assist X-ray crystallographers in overcoming some of these obstacles. In this chapter, we discuss some of these technological advances. Familiarity with these new developments would benefit researchers in both academic and industrial environments who study macromolecular structural dynamics using X-ray crystallography. Less
The advent of SARS-CoV- the causative agent of COVID- and its worldwide impact on global health have provided the impetus for the development of effective countermeasures that can be deployed against the virus including vaccines monoclonal antibodies and direct-acting antivirals DAAs Despite these efforts the current paucity of DAAs has created an urgent need for the creation of an enhanced and diversified portfolio of broadly acting agents with different mechanisms of action that can effectively abrogate viral infection SARS-CoV- C-like protease CLpro an enzyme essential for viral replication is a validated target for the discovery of SARS-CoV- therapeutics In this ... More
The advent of SARS-CoV-2, the causative agent of COVID-19, and its worldwide impact on global health, have provided the impetus for the development of effective countermeasures that can be deployed against the virus, including vaccines, monoclonal antibodies, and direct-acting antivirals (DAAs). Despite these efforts, the current paucity of DAAs has created an urgent need for the creation of an enhanced and diversified portfolio of broadly acting agents with different mechanisms of action that can effectively abrogate viral infection. SARS-CoV-2 3C-like protease (3CLpro), an enzyme essential for viral replication, is a validated target for the discovery of SARS-CoV-2 therapeutics. In this report, we describe the structure-guided utilization of the cyclopropane moiety in the design of highly potent inhibitors of SARS-CoV-2 3CLpro, SARS-CoV-1 3CLpro, and MERS-CoV 3CLpro. High-resolution cocrystal structures were used to identify the structural determinants associated with the binding of the inhibitors to the active site of the enzyme and unravel the mechanism of action. Aldehydes 5c and 11c inhibited SARS-CoV-2 replication with EC50 values of 12 and 11 nM, respectively. Furthermore, the corresponding aldehyde bisulfite adducts 5d and 11d were equipotent with EC50 values of 13 and 12 nM, respectively. The safety index (SI) values for compounds 5c/11c and 5d/11d ranged between 7692 and 9090. Importantly, aldehydes 5c/11c and bisulfite adducts 5d/11d potently inhibited MERS-CoV 3CLpro with IC50 values of 80 and 120 nM, and 70 and 70 nM, respectively. Likewise, compounds 5c/11c and 5d/11d inhibited SARS-CoV-1 with IC50 values of 960 and 350 nM and 790 and 240 nM, respectively. Taken together, these studies suggest that the inhibitors described herein have low cytotoxicity and high potency and are promising candidates for further development as broad-spectrum direct-acting antivirals against highly pathogenic coronaviruses. Less
Enzymatic late-stage diversification of small molecules has the potential to rapidly generate diversity in compound libraries dedicated to drug discovery In this context freestanding Fe II -ketoglutarate-dependent halogenases have raised particular interest as this enzyme family allows the otherwise difficult regio- and stereoselective halogenation of unactivated C sp H bonds Here we report the development of two engineered variants of the halogenase WelO for the racemic resolution of a mixture of stereoisomers generated in the synthesis of a bioactive martinelline-derived fragment By screening a -site combinatorial variant library we could identify two variants exhibiting exquisite substrate selectivity towards the desired ... More
Enzymatic late-stage diversification of small molecules has the potential to rapidly generate diversity in compound libraries dedicated to drug discovery. In this context, freestanding Fe(II)/α-ketoglutarate-dependent halogenases have raised particular interest as this enzyme family allows the otherwise difficult regio- and stereoselective halogenation of unactivated C(sp3)−H bonds. Here, we report the development of two engineered variants of the halogenase WelO5* for the racemic resolution of a mixture of stereoisomers generated in the synthesis of a bioactive martinelline-derived fragment. By screening a 3-site combinatorial variant library, we could identify two variants exhibiting exquisite substrate selectivity towards the desired enantiomers. Strikingly, the inversion of substrate stereopreference between the halogenase variants was achieved by varying only three residues in the active site. Protein crystallization and subsequent structure elucidation of the wildtype enzyme and a WelO5* variant shed light on the factors governing substrate acceptance and selectivity. Less
Crystallization of proteins is a critical step in structural biology biopharmaceutical industry and materials science Microfluidic technology has emerged as a promising tool for screening the crystallization conditions with advantages of increased throughput reduced consumption of reagents and lower cost However current mirofluidic approches generally lack the module for high-speed data analysis ignore the time-resolved changes of protein crystalline states or morphologies in the crystallization process and suffer from inconsistency after scaling up due to the subnano- nano-liter-scaled volume To address the issues we propose a deep learning-aided programmable microliter-droplet system which allows the high-throughput screening of time-resolved protein crystallization ... More
Crystallization of proteins is a critical step in structural biology, biopharmaceutical industry and materials science. Microfluidic technology has emerged as a promising tool for screening the crystallization conditions with advantages of increased throughput, reduced consumption of reagents and lower cost. However, current mirofluidic approches generally lack the module for high-speed data analysis, ignore the time-resolved changes of protein crystalline states or morphologies in the crystallization process and suffer from inconsistency after scaling up due to the subnano-/nano-liter-scaled volume. To address the issues, we propose a deep learning-aided programmable microliter-droplet system, which allows the high-throughput screening of time-resolved protein crystallization in microliter scale. Based on the system, a series of temporal phase diagrams are acquired, which reveal the time-resolved crystallization of target proteins under different crystallization conditions. They provide precise guidance on the scale-up experiment (∼93 % in consistency), and help gain insight into the kinetic characteristics of protein crystallization. Less
Coproporphyrin ferrochelatases CpfCs are enzymes catalyzing the penultimate step in the coproporphyrin-dependent CPD heme biosynthesis pathway which is mainly utilized by monoderm bacteria Ferrochelatases insert ferrous iron into a porphyrin macrocycle and have been studied for many decades nevertheless many mechanistic questions remain unanswered to date Especially CpfCs which are found in the CPD pathway are currently in the spotlight of research This pathway was identified in and revealed that the correct substrate for these ferrochelatases is coproporphyrin III cpIII instead of protoporphyrin IX as believed prior the discovery of the CPD pathway The chemistry of cpIII which has four ... More
Coproporphyrin ferrochelatases (CpfCs) are enzymes catalyzing the penultimate step in the coproporphyrin-dependent (CPD) heme biosynthesis pathway, which is mainly utilized by monoderm bacteria. Ferrochelatases insert ferrous iron into a porphyrin macrocycle and have been studied for many decades, nevertheless many mechanistic questions remain unanswered to date. Especially CpfCs, which are found in the CPD pathway, are currently in the spotlight of research. This pathway was identified in 2015 and revealed that the correct substrate for these ferrochelatases is coproporphyrin III (cpIII) instead of protoporphyrin IX, as believed prior the discovery of the CPD pathway. The chemistry of cpIII, which has four propionates, differs significantly from protoporphyrin IX, which features two propionate and two vinyl groups. These findings let us to thoroughly describe the physiological cpIII-ferrochelatase complex in solution and in the crystal phase. Here, we present the first crystallographic structure of the CpfC from the representative monoderm pathogen Listeria monocytogenes bound to its physiological substrate, cpIII, together with the in-solution data obtained by resonance Raman and UV–vis spectroscopy, for wild-type ferrochelatase and variants, analyzing propionate interactions. The results allow us to evaluate the porphyrin distortion and provide an in-depth characterization of the catalytically-relevant binding mode of cpIII prior to iron insertion. Our findings are discussed in the light of the observed structural restraints and necessities for this porphyrin-enzyme complex to catalyze the iron insertion process. Knowledge about this initial situation is essential for understanding the preconditions for iron insertion in CpfCs and builds the basis for future studies. Less
Mitogen-activated protein kinase MAPK phosphatase MKP is responsible for regulating the activity of the stress-responsive MAPKs and has been put forth as a potential therapeutic target for a number of diseases including dystrophic muscle disease a fatal rare disease which has neither a treatment nor cure In previous work we identified Compound -dimethyl- - - methylthio - -dihydrothieno -h quinazolin- -yl thio butan- -one as the lead compound of a novel class of MKP inhibitors In this work we explore the structure-activity relationship for inhibition of MKP through modifications to the scaffold and functional groups present in A series of ... More
Mitogen-activated protein kinase (MAPK) phosphatase 5 (MKP5) is responsible for regulating the activity of the stress-responsive MAPKs and has been put forth as a potential therapeutic target for a number of diseases, including dystrophic muscle disease a fatal rare disease which has neither a treatment nor cure. In previous work, we identified Compound 1 (3,3-dimethyl-1-((9-(methylthio)-5,6-dihydrothieno[3,4-h]quinazolin-2-yl)thio)butan-2-one) as the lead compound of a novel class of MKP5 inhibitors. In this work, we explore the structure-activity relationship for inhibition of MKP5 through modifications to the scaffold and functional groups present in 1. A series of derivative compounds was designed, synthesized, and evaluated for inhibition of MKP5. In addition, the X-ray crystal structures of six enzyme-inhibitor complexes were solved, further elucidating the necessary requirements for MKP5 inhibition. We found that the parallel-displaced π-π interaction between the inhibitor three-ring core and Tyr435 is critical for modulating potency, and that modifications to the core and functionalization at the C-9 position are essential for ensuring proper positioning of the core for this interaction. These results lay the foundation from which more potent MKP5 allosteric inhibitors can be developed for potential therapeutics towards the treatment of dystrophic muscle disease. Less
Applying rational design we developed kDa cyanobacteriochrome-based near-infrared NIR-I fluorescent protein miRFP nano miRFP nano efficiently binds endogenous biliverdin chromophore and brightly fluoresces in mammalian cells and tissues miRFP nano has maximal emission at nm and an emission tail in the short-wave infrared SWIR region allowing deep-penetrating off-peak fluorescence imaging in vivo The miRFP nano structure reveals the molecular basis of its red shift We demonstrate superiority of miRFP nano-enabled SWIR imaging over NIR-I imaging of microbes in the mouse digestive tract mammalian cells injected into the mouse mammary gland and NF-kB activity in a mouse model of liver inflammation
Coelenterazine-v CTZ-v a synthetic vinylene-bridged -extended derivative is able to significantly alter bioluminescence spectra of different CTZ-dependent luciferases and photoproteins by shifting them towards longer wavelengths However Ca -regulated photoproteins activated with CTZ-v display very low bioluminescence activities that hampers its usage as a substrate of photoprotein bioluminescence Here we report the crystal structure of semi-synthetic Ca -discharged obelin-v bound with the reaction product determined at resolution Comparison of the crystal structure of Ca -discharged obelin-v with those of other obelins before and after bioluminescence reaction reveals no considerable changes in the overall structure However the drastic changes in CTZ-binding ... More
Coelenterazine-v (CTZ-v), a synthetic vinylene-bridged π-extended derivative, is able to significantly alter bioluminescence spectra of different CTZ-dependent luciferases and photoproteins by shifting them towards longer wavelengths. However, Ca2+-regulated photoproteins activated with CTZ-v display very low bioluminescence activities that hampers its usage as a substrate of photoprotein bioluminescence. Here, we report the crystal structure of semi-synthetic Ca2+-discharged obelin-v bound with the reaction product determined at 2.1 Å resolution. Comparison of the crystal structure of Ca2+-discharged obelin-v with those of other obelins before and after bioluminescence reaction reveals no considerable changes in the overall structure. However, the drastic changes in CTZ-binding cavity are observed owing to the completely different reaction product, coelenteramine-v (CTM-v). Since CTM-v is certainly the main product of obelin-v bioluminescence and is considered to be a product of the “dark” pathway of dioxetanone intermediate decomposition, it explains the low bioluminescence activity of obelin and apparently of other photoproteins with CTZ-v. Less
The past fifty years have been marked by the surge of neurodegenerative diseases Unfortunately current treatments are only symptomatic Hence the search for new and innovative therapeutic targets for curative treatments becomes a major challenge Among these targets the adenosine A A receptor A AAR has been the subject of much research in recent years In this paper we report the design synthesis and pharmacological analysis of quinazoline derivatives as A AAR antagonists with high ligand efficiency This class of molecules has been discovered by a virtual screening and bears no structural semblance with reference antagonist ZM- More precisely we ... More
The past fifty years have been marked by the surge of neurodegenerative diseases. Unfortunately, current treatments are only symptomatic. Hence, the search for new and innovative therapeutic targets for curative treatments becomes a major challenge. Among these targets, the adenosine A2A receptor (A2AAR) has been the subject of much research in recent years. In this paper, we report the design, synthesis and pharmacological analysis of quinazoline derivatives as A2AAR antagonists with high ligand efficiency. This class of molecules has been discovered by a virtual screening and bears no structural semblance with reference antagonist ZM-241385. More precisely, we identified a series of 2-aminoquinazoline as promising A2AAR antagonists. Among them, one compound showed a high affinity towards A2AAR (21a, Ki = 20 nM). We crystallized this ligand in complex with A2AAR, confirming one of our predicted docking poses and opening up possibilities for further optimization to derive selective ligands for specific adenosine receptor subtypes. Less
Transmembrane ion transport is a key process in living cells Active transport of ions is carried out by various ion transporters including microbial rhodopsins MRs MRs perform diverse functions such as active and passive ion transport photo-sensing and others In particular MRs can pump various monovalent ions like Na K Cl I NO The only characterized MR proposed to pump sulfate in addition to halides belongs to the cyanobacterium Synechocystis sp PCC and is named Synechocystis halorhodopsin SyHR The structural study of SyHR may help to understand what makes an MR pump divalent ions Here we present the crystal structure ... More
Transmembrane ion transport is a key process in living cells. Active transport of ions is carried out by various ion transporters including microbial rhodopsins (MRs). MRs perform diverse functions such as active and passive ion transport, photo-sensing, and others. In particular, MRs can pump various monovalent ions like Na+, K+, Cl−, I−, NO3−. The only characterized MR proposed to pump sulfate in addition to halides belongs to the cyanobacterium Synechocystis sp. PCC 7509 and is named Synechocystis halorhodopsin (SyHR). The structural study of SyHR may help to understand what makes an MR pump divalent ions. Here we present the crystal structure of SyHR in the ground state, the structure of its sulfate-bound form as well as two photoreaction intermediates, the K and O states. These data reveal the molecular origin of the unique properties of the protein (exceptionally strong chloride binding and proposed pumping of divalent anions) and sheds light on the mechanism of anion release and uptake in cyanobacterial halorhodopsins. The unique properties of SyHR highlight its potential as an optogenetics tool and may help engineer different types of anion pumps with applications in optogenetics. Less
The transcription factor TEAD together with its coactivator YAP TAZ is a key transcriptional modulator of the Hippo pathway Activation of TEAD transcription by YAP has been implicated in a number of malignancies and this complex represents a promising target for drug discovery However both YAP and its extensive binding interfaces to TEAD have been difficult to address using small molecules mainly due to a lack of druggable pockets TEAD is post-translationally modified by palmitoylation that targets a conserved cysteine at a central pocket which provides an opportunity to develop cysteine-directed covalent small molecules for TEAD inhibition Here we employed ... More
The transcription factor TEAD, together with its coactivator YAP/TAZ, is a key transcriptional modulator of the Hippo pathway. Activation of TEAD transcription by YAP has been implicated in a number of malignancies, and this complex represents a promising target for drug discovery. However, both YAP and its extensive binding interfaces to TEAD have been difficult to address using small molecules, mainly due to a lack of druggable pockets. TEAD is post-translationally modified by palmitoylation that targets a conserved cysteine at a central pocket, which provides an opportunity to develop cysteine-directed covalent small molecules for TEAD inhibition. Here, we employed covalent fragment screening approach followed by structure-based design to develop an irreversible TEAD inhibitor MYF-03–69. Using a range of in vitro and cell-based assays we demonstrated that through a covalent binding with TEAD palmitate pocket, MYF-03–69 disrupts YAP-TEAD association, suppresses TEAD transcriptional activity and inhibits cell growth of Hippo signaling defective malignant pleural mesothelioma (MPM). Further, a cell viability screening with a panel of 903 cancer cell lines indicated a high correlation between TEAD-YAP dependency and the sensitivity to MYF-03–69. Transcription profiling identified the upregulation of proapoptotic BMF gene in cancer cells that are sensitive to TEAD inhibition. Further optimization of MYF-03–69 led to an in vivo compatible compound MYF-03–176, which shows strong antitumor efficacy in MPM mouse xenograft model via oral administration. Taken together, we disclosed a story of the development of covalent TEAD inhibitors and its high therapeutic potential for clinic treatment for the cancers that are driven by TEAD-YAP alteration. Less
Histone deacetylase HDAC is an atypical lysine deacetylase with tandem catalytic domains and an ubiquitin-binding zinc finger domain HDAC is involved in various biological processes such as cell motility or stress responses and has been implicated in pathologies ranging from cancer to neurodegeneration Due to this broad range of functions there has been considerable interest in developing HDAC -specific small molecule inhibitors several of which are already available The crystal structure of the tandem catalytic domains of zebrafish HDAC has revealed an arrangement with twofold symmetry and extensive surface interaction between the catalytic domains Further dissection of the biochemical properties ... More
Histone deacetylase 6 (HDAC6) is an atypical lysine deacetylase with tandem catalytic domains and an ubiquitin-binding zinc finger domain. HDAC6 is involved in various biological processes, such as cell motility or stress responses, and has been implicated in pathologies ranging from cancer to neurodegeneration. Due to this broad range of functions, there has been considerable interest in developing HDAC6-specific small molecule inhibitors, several of which are already available. The crystal structure of the tandem catalytic domains of zebrafish HDAC6 has revealed an arrangement with twofold symmetry and extensive surface interaction between the catalytic domains. Further dissection of the biochemical properties of HDAC6 and the development of novel inhibitors will benefit from being able to routinely express high-quality protein. We present here our optimized protocol for expression and crystallization of the zebrafish tandem catalytic domains. Less
Colibactin a DNA cross-linking agent produced by gut bacteria is implicated in colorectal cancer Its biosynthesis uses a prodrug resistance mechanism a non-toxic precursor assembled in the cytoplasm is activated after export to the periplasm This activation is mediated by ClbP an inner-membrane peptidase with an N-terminal periplasmic catalytic domain and a C-terminal three-helix transmembrane domain Although the transmembrane domain is required for colibactin activation its role in catalysis is unclear Our structure of full-length ClbP bound to a product analog reveals an interdomain interface important for substrate binding and enzyme stability and interactions that explain the selectivity of ClbP ... More
Colibactin, a DNA cross-linking agent produced by gut bacteria, is implicated in colorectal cancer. Its biosynthesis uses a prodrug resistance mechanism: a non-toxic precursor assembled in the cytoplasm is activated after export to the periplasm. This activation is mediated by ClbP, an inner-membrane peptidase with an N-terminal periplasmic catalytic domain and a C-terminal three-helix transmembrane domain. Although the transmembrane domain is required for colibactin activation, its role in catalysis is unclear. Our structure of full-length ClbP bound to a product analog reveals an interdomain interface important for substrate binding and enzyme stability and interactions that explain the selectivity of ClbP for the N-acyl-d-asparagine prodrug motif. Based on structural and biochemical evidence, we propose that ClbP dimerizes to form an extended substrate-binding site that can accommodate a pseudodimeric precolibactin with its two terminal prodrug motifs in the two ClbP active sites, thus enabling the coordinated activation of both electrophilic warheads. Less
The human gut bacterial genotoxin colibactin is a possible key driver of colorectal cancer CRC development Understanding colibactin s biological effects remains difficult owing to the instability of the proposed active species and the complexity of the gut microbiota Here we report small molecule boronic acid inhibitors of colibactin biosynthesis Designed to mimic the biosynthetic precursor precolibactin these compounds potently inhibit the colibactin-activating peptidase ClbP Using biochemical assays and crystallography we show that they engage the ClbP binding pocket forming a covalent bond with the catalytic serine These inhibitors reproduce the phenotypes observed in a clbP deletion mutant and block ... More
The human gut bacterial genotoxin colibactin is a possible key driver of colorectal cancer (CRC) development. Understanding colibactin’s biological effects remains difficult owing to the instability of the proposed active species and the complexity of the gut microbiota. Here, we report small molecule boronic acid inhibitors of colibactin biosynthesis. Designed to mimic the biosynthetic precursor precolibactin, these compounds potently inhibit the colibactin-activating peptidase ClbP. Using biochemical assays and crystallography, we show that they engage the ClbP binding pocket, forming a covalent bond with the catalytic serine. These inhibitors reproduce the phenotypes observed in a clbP deletion mutant and block the genotoxic effects of colibactin on eukaryotic cells. The availability of ClbP inhibitors will allow precise, temporal control over colibactin production, enabling further study of its contributions to CRC. Finally, application of our inhibitors to related peptidase-encoding pathways highlights the power of chemical tools to probe natural product biosynthesis. Less
Voltage-gated sodium ion channels Nav are central to action potential initiation through regulating the entry of sodium ions Na into excitable cells including cardiomyocytes and neurones The -subunit of Nav consists of four homologous domains DI-DIV each consisting of six transmembrane helices S -S Helices S -S of each domain forms the lining of the central pore through which sodium ions Na enters the cell upon channel activation Helices S -S of each domain form the voltage sensor which becomes displaced in response to changes in intracellular potential Additionally Nav channels include an extracellular turret region whose role in channel ... More
Voltage-gated sodium ion channels (Nav) are central to action potential initiation through regulating the entry of sodium ions (Na+) into excitable cells including cardiomyocytes and neurones. The α-subunit of Nav consists of four homologous domains (DI-DIV), each consisting of six transmembrane helices (S1-S6). Helices S5-S6 of each domain forms the lining of the central pore through which sodium ions (Na+) enters the cell upon channel activation. Helices S1-S4 of each domain form the voltage sensor which becomes displaced in response to changes in intracellular potential. Additionally, Nav channels include an extracellular turret region, whose role in channel function is poorly understood. The C-terminal domain (CTD) of Nav, connected to the DIV-S6, interacts with various other proteins including calmodulin (CaM) and fibroblast growth factor (FGF13) and mediates various regulatory roles. The subtype Nav1.5 is primarily expressed in the heart where it initiates the cardiac action potential whereas Nav1.7 is found in the peripheral nervous system where it is associated with nociception.
Various Nav associated pathologies have been associated with mutations in the extracellular turret region; however, their molecular mechanism is not well understood. In the Nav1.5 structure determined by cryogenic electron microscopy (cryoEM), the wild-type residues that correspond to some of these mutants form a complex salt bridge at the interface between the DII and DIII turret loops. Furthermore, adjacent aromatic residues could potentially form cation-π interactions with the complex salt bridge. This region was examined using site-directed mutagenesis, electrophysiology and in silico modelling, confirming functional roles for the inter-domain salt-bridges and the aromatic residues. Evidence that disruption of these contacts perturbs the geometry of the DEKA selectivity ring and both the outer and inner pore vestibules that are crucial for sodium ion permeability were provided. These findings provide insights into a class of pathological mutations occurring not only in Nav1.5 but also in other sodium channel isoforms.
Further experiments performed preliminary studies that focussed on the CTD of Nav1.5 and Nav1.7, seeking to better understand the role of its regulation by Ca2+ and CaM, using various techniques such as ELISA, isothermal titration calorimetry (ITC) and Bio-Layer interferometry (BLI). The CTDs and CaM recombinant proteins were cloned using the Gateway cloning method, expressed in BL21 (DE3) cells using auto-induction, and purified via affinity chromatography and size exclusion chromatography. Also, attempts were made to determine the yet unresolved structure of Nav1.7 using x-ray crystallography. Finally, using an in-house phage display library of single chain fragment variable (scFv) antibodies, specific binders to the CTD of Nav1.5 and Nav1.7, were found and purified. These scFvs could have gating effects on their Nav channel targets, which might prove therapeutically applicable. Less
Various Nav associated pathologies have been associated with mutations in the extracellular turret region; however, their molecular mechanism is not well understood. In the Nav1.5 structure determined by cryogenic electron microscopy (cryoEM), the wild-type residues that correspond to some of these mutants form a complex salt bridge at the interface between the DII and DIII turret loops. Furthermore, adjacent aromatic residues could potentially form cation-π interactions with the complex salt bridge. This region was examined using site-directed mutagenesis, electrophysiology and in silico modelling, confirming functional roles for the inter-domain salt-bridges and the aromatic residues. Evidence that disruption of these contacts perturbs the geometry of the DEKA selectivity ring and both the outer and inner pore vestibules that are crucial for sodium ion permeability were provided. These findings provide insights into a class of pathological mutations occurring not only in Nav1.5 but also in other sodium channel isoforms.
Further experiments performed preliminary studies that focussed on the CTD of Nav1.5 and Nav1.7, seeking to better understand the role of its regulation by Ca2+ and CaM, using various techniques such as ELISA, isothermal titration calorimetry (ITC) and Bio-Layer interferometry (BLI). The CTDs and CaM recombinant proteins were cloned using the Gateway cloning method, expressed in BL21 (DE3) cells using auto-induction, and purified via affinity chromatography and size exclusion chromatography. Also, attempts were made to determine the yet unresolved structure of Nav1.7 using x-ray crystallography. Finally, using an in-house phage display library of single chain fragment variable (scFv) antibodies, specific binders to the CTD of Nav1.5 and Nav1.7, were found and purified. These scFvs could have gating effects on their Nav channel targets, which might prove therapeutically applicable. Less
Pathogenic bacteria utilize specialized macromolecular secretion systems to transport virulence factors across membrane s and manipulate their infected host To date secretion systems have been identified including the type IX secretion system T SS associated with human avian and farmed-fish diseases As a bacterial secretion system the T SS also facilitates gliding motility and the degradation of different macromolecules by the secretion of metabolic enzymes in nonpathogenic bacteria PorX is a highly conserved protein that regulates the transcription of essential T SS components and additionally mediates the function of T SS via direct interaction with PorL the rotary motor protein ... More
Pathogenic bacteria utilize specialized macromolecular secretion systems to transport virulence factors across membrane(s) and manipulate their infected host. To date, 11 secretion systems have been identified, including the type IX secretion system (T9SS) associated with human, avian and farmed-fish diseases. As a bacterial secretion system, the T9SS also facilitates gliding motility and the degradation of different macromolecules by the secretion of metabolic enzymes in nonpathogenic bacteria. PorX is a highly conserved protein that regulates the transcription of essential T9SS components and additionally mediates the function of T9SS via direct interaction with PorL, the rotary motor protein of the T9SS. PorX is also a member of a two-component system regulatory cascade, where it serves as the response regulator that relays a signal transduced from a conserved sensor histidine kinase, PorY, to a designated sigma factor. Here, the recombinant expression and purification of PorX homologous proteins from the pathogenic bacterium Porphyromonas gingivalis and the nonpathogenic bacterium Flavobacterium johnsoniae are reported. A bioinformatical characterization of the different domains comprising the PorX protein is also provided, and the crystallization and X-ray analysis of PorX from F. johnsoniae are reported. Less
Intrinsically disordered proteins IDPs often coordinate transient interactions with multiple proteins to mediate complex signals within large protein networks Among these the IDP hub protein G BP can form complexes with cytoplasmic phosphoprotein Caprin and ubiquitin peptidase USP the resulting control of USP activity contributes to a pathogenic virulence system that targets endocytic recycling of the ion channel CFTR However while the identities of protein interactors are known for many IDP hub proteins the relationship between pairwise affinities and the extent of protein recruitment and activity is not well understood Here we describe in vitro analysis of these G BP ... More
Intrinsically disordered proteins (IDPs) often coordinate transient interactions with multiple proteins to mediate complex signals within large protein networks. Among these, the IDP hub protein G3BP1 can form complexes with cytoplasmic phosphoprotein Caprin1 and ubiquitin peptidase USP10; the resulting control of USP10 activity contributes to a pathogenic virulence system that targets endocytic recycling of the ion channel CFTR. However, while the identities of protein interactors are known for many IDP hub proteins, the relationship between pairwise affinities and the extent of protein recruitment and activity is not well understood. Here we describe in vitro analysis of these G3BP1 affinities, and show tryptophan substitutions of specific G3BP1 residues reduce its affinity for both USP10 and Caprin1. We show that these same mutations reduce the stability of complexes between the full-length proteins, suggesting that co-purification can serve as a surrogate measure of interaction strength. The crystal structure of G3BP1 TripleW (F15W/F33W/F124W) mutant reveals a clear reorientation of the side chain of W33, creating a steric clash with USP10 and Caprin1. Furthermore, an amino-acid scan of USP10 and Caprin1 peptides reveals similarities and differences in the ability to substitute residues in the core motifs as well as specific substitutions with the potential to create higher affinity peptides. Taken together, these data show that small changes in component binding affinities can have significant effects on the composition of cellular interaction hubs. These specific protein mutations can be harnessed to manipulate complex protein networks, informing future investigations into roles of these networks in cellular processes. Less
The multifunctional human Parkinson s disease protein PARK DJ is an attractive therapeutic target due to its link with early-onset Parkinson s disease upregulation in various cancers and contribution to chemoresistance However only a few compounds have been identified to bind PARK due to the lack of a dedicated chemical toolbox We report the creation of such a toolbox and showcase the application of each of its components The selective PARK submicromolar inhibitor with a cyanimide reactive group covalently modifies the active site Cys Installment of different dyes onto the inhibitor delivered two PARK probes The Rhodamine probe provides a ... More
The multifunctional human Parkinson’s disease protein 7 (PARK7/DJ1) is an attractive therapeutic target due to its link with early-onset Parkinson’s disease, upregulation in various cancers, and contribution to chemoresistance. However, only a few compounds have been identified to bind PARK7 due to the lack of a dedicated chemical toolbox. We report the creation of such a toolbox and showcase the application of each of its components. The selective PARK7 submicromolar inhibitor with a cyanimide reactive group covalently modifies the active site Cys106. Installment of different dyes onto the inhibitor delivered two PARK7 probes. The Rhodamine110 probe provides a high-throughput screening compatible FP assay, showcased by screening a compound library (8000 molecules). The SulfoCy5-equipped probe is a valuable tool to assess the effect of PARK7 inhibitors in a cell lysate. Our work creates new possibilities to explore PARK7 function in a physiologically relevant setting and develop new and improved PARK7 inhibitors. Less
The main focus of this project was chicken protein MICAL which is involved in the semaphorin-plexin signalling pathway and has a significant effect on the rearrangement of the cytoskeleton The prominent role of the MICAL protein is primarily associated with axon guidance as it destabilizes actin filaments through its oxidative activity We focused on elucidating the molecular mechanisms of chicken MICAL autoinhibition using molecular and structural biology methods together with new protein structure prediction methods Chicken MICAL was produced in Sf insect cells using a baculovirus expression system and we produced both full-length and truncated versions of chicken MICAL protein ... More
The main focus of this project was chicken protein MICAL1, which is involved in the semaphorin-plexin signalling pathway and has a significant effect on the rearrangement of the cytoskeleton. The prominent role of the MICAL1 protein is primarily associated with axon guidance, as it destabilizes actin filaments through its oxidative activity. We focused on elucidating the molecular mechanisms of chicken MICAL1 autoinhibition using molecular and structural biology methods together with new protein structure prediction methods. Chicken MICAL1 was produced in Sf9 insect cells using a baculovirus expression system and we produced both full-length and truncated versions of chicken MICAL1 protein. We kinetically characterized the protein and determined its oligomeric state in solution. We made great efforts to solve the protein structure using crystallography, electron microscopy and protein structure prediction in Alphafold 2. Based on the results of these experiments and assays, we conclude that MICAL1 proteins are regulated through their C terminal domain, which interacts with the monooxygenase domain. The part of this interaction is the autoinhibition of chicken MICAL1. We excluded the possibility that chicken MICAL1 is regulated by changing its oligomeric state. The results of this master's thesis. Less
Autofluorescence-detected photothermal mid-infrared AF-PTIR microscopy was shown to enable parts-per-million detection of -indomethacin impurity in -indomethacin samples Subtle differences in the photothermal response of the UV-autofluorescence of two indomethacin crystal polymorphs were used for sub-micron chemical discrimination based on fingerprint region mid-IR spectroscopy The AF-PTIR assignment was independently confirmed by second harmonic generation SHG microscopy which was shown to reduce the total analysis time by rapidly identifying the suitable fields of view AF-PTIR microscopy has the potential to assist in the early identification of crystal form impurities in the solid dosage forms development pipeline
Neoantigens derived from somatic mutations are specific to cancer cells and are ideal targets for cancer immunotherapy KRAS is the most frequently mutated oncogene and drives the pathogenesis of several cancers Here we show the identification and development of an affinity-enhanced T cell receptor TCR that recognizes a peptide derived from the most common KRAS mutant KRASG D presented in the context of HLA-A The affinity of the engineered TCR is increased by over one million-fold yet fully able to distinguish KRASG D over KRASWT While crystal structures reveal few discernible differences in TCR interactions with KRASWT versus KRASG D ... More
Neoantigens derived from somatic mutations are specific to cancer cells and are ideal targets for cancer immunotherapy. KRAS is the most frequently mutated oncogene and drives the pathogenesis of several cancers. Here we show the identification and development of an affinity-enhanced T cell receptor (TCR) that recognizes a peptide derived from the most common KRAS mutant, KRASG12D, presented in the context of HLA-A*11:01. The affinity of the engineered TCR is increased by over one million-fold yet fully able to distinguish KRASG12D over KRASWT. While crystal structures reveal few discernible differences in TCR interactions with KRASWT versus KRASG12D, thermodynamic analysis and molecular dynamics simulations reveal that TCR specificity is driven by differences in indirect electrostatic interactions. The affinity enhanced TCR, fused to a humanized anti-CD3 scFv, enables selective killing of cancer cells expressing KRASG12D. Our work thus reveals a molecular mechanism that drives TCR selectivity and describes a soluble bispecific molecule with therapeutic potential against cancers harboring a common shared neoantigen. Less
Modulators of the G protein-coupled A A adenosine receptor A AAR have been considered promising agents to treat Parkinson s disease inflammation cancer and central nervous system disorders Herein we demonstrate that a thiophene modification at the C position in the common adenine scaffold converted an A AAR agonist into an antagonist We synthesized and characterized a novel A AAR antagonist LJ- with Ki nM X-ray crystallographic structures of in complex with two thermostabilized A AAR constructs were solved at and resolutions In contrast to A AAR agonists which simultaneously interact with both Ser and His only transiently contacts His ... More
Modulators of the G protein-coupled A2A adenosine receptor (A2AAR) have been considered promising agents to treat Parkinson’s disease, inflammation, cancer, and central nervous system disorders. Herein, we demonstrate that a thiophene modification at the C8 position in the common adenine scaffold converted an A2AAR agonist into an antagonist. We synthesized and characterized a novel A2AAR antagonist, 2 (LJ-4517), with Ki = 18.3 nM. X-ray crystallographic structures of 2 in complex with two thermostabilized A2AAR constructs were solved at 2.05 and 2.80 Å resolutions. In contrast to A2AAR agonists, which simultaneously interact with both Ser2777.42 and His2787.43, 2 only transiently contacts His2787.43, which can be direct or water-mediated. The n-hexynyl group of 2 extends into an A2AAR exosite. Structural analysis revealed that the introduced thiophene modification restricted receptor conformational rearrangements required for subsequent activation. This approach can expand the repertoire of adenosine receptor antagonists that can be designed based on available agonist scaffolds. Less
Nonribosomal peptide synthetase heterocyclization Cy domains generate biologically important oxazoline thiazoline groups found in natural products including pharmaceuticals and virulence factors such as some siderophores Cy domains catalyze consecutive condensation and cyclodehydration reactions although the mechanism is unknown To better understand Cy domain catalysis here we report the crystal structure of the second Cy domain Cy of yersiniabactin synthetase from the causative agent of the plague Yersinia pestis Our high-resolution structure of Cy adopts a conformation that enables exploration of interactions with the extended thiazoline-containing cyclodehydration intermediate and the acceptor carrier protein CP to which it is tethered We also ... More
Nonribosomal peptide synthetase heterocyclization (Cy) domains generate biologically important oxazoline/thiazoline groups found in natural products, including pharmaceuticals and virulence factors such as some siderophores. Cy domains catalyze consecutive condensation and cyclodehydration reactions, although the mechanism is unknown. To better understand Cy domain catalysis, here we report the crystal structure of the second Cy domain (Cy2) of yersiniabactin synthetase from the causative agent of the plague, Yersinia pestis. Our high-resolution structure of Cy2 adopts a conformation that enables exploration of interactions with the extended thiazoline-containing cyclodehydration intermediate and the acceptor carrier protein (CP) to which it is tethered. We also report complementary electrostatic interfaces between Cy2 and its donor CP that mediate donor binding. Finally, we explored domain flexibility through normal mode analysis and identified small-molecule fragment-binding sites that may inform future antibiotic design targeting Cy function. Our results suggest how CP binding may influence global Cy conformations, with consequences for active-site remodeling to facilitate the separate condensation and cyclodehydration steps as well as potential inhibitor development. Less
Chickpea is a crop that is known as a source of high-quality proteins CL-AI which belongs to the S globulin and cupin superfamily was initially identified in chickpea seeds CL-AI has recently been shown to inhibit various types of -amylases To determine its molecular mechanism the crystal structure of CL-AI was solved at a final resolution of Structural analysis indicated that each asymmetric unit contains three molecules with threefold symmetry and a head-to-tail association and each molecule is divided into an -chain and a -chain CL-AI has high structural similarity to other S globulins and canonical metal-dependent enzyme-related cupin proteins ... More
Chickpea is a crop that is known as a source of high-quality proteins. CL-AI, which belongs to the 11S globulin and cupin superfamily, was initially identified in chickpea seeds. CL-AI has recently been shown to inhibit various types of α-amylases. To determine its molecular mechanism, the crystal structure of CL-AI was solved at a final resolution of 2.2 Å. Structural analysis indicated that each asymmetric unit contains three molecules with threefold symmetry and a head-to-tail association, and each molecule is divided into an α-chain and a β-chain. CL-AI has high structural similarity to other 11S globulins and canonical metal-dependent enzyme-related cupin proteins, whereas its stimilarity to α-amylase inhibitor from Phaseolus vulgaris is quite low. The structure presented here will provide insight into the function of CL-AI. Less
Akt is a Ser Thr protein kinase that plays a central role in metabolism and cancer Regulation of Akt s activity involves an autoinhibitory intramolecular interaction between its pleckstrin homology PH domain and its kinase domain that can be relieved by C-tail phosphorylation PH domain mutant E K Akt is a well-established oncogene Previously we reported that the conformation of autoinhibited Akt may be shifted by small molecule allosteric inhibitors limiting the mechanistic insights from existing X-ray structures that have relied on such compounds Chu et al Here we discover unexpectedly that a single mutation R A Akt exhibits intensified ... More
Akt is a Ser/Thr protein kinase that plays a central role in metabolism and cancer. Regulation of Akt’s activity involves an autoinhibitory intramolecular interaction between its pleckstrin homology (PH) domain and its kinase domain that can be relieved by C-tail phosphorylation. PH domain mutant E17K Akt is a well-established oncogene. Previously, we reported that the conformation of autoinhibited Akt may be shifted by small molecule allosteric inhibitors limiting the mechanistic insights from existing X-ray structures that have relied on such compounds (Chu et al., 2020). Here, we discover unexpectedly that a single mutation R86A Akt exhibits intensified autoinhibitory features with enhanced PH domain-kinase domain affinity. Structural and biochemical analysis uncovers the importance of a key interaction network involving Arg86, Glu17, and Tyr18 that controls Akt conformation and activity. Our studies also shed light on the molecular basis for E17K Akt activation as an oncogenic driver. Less
The bioactive lysophospholipid sphingosine- -phosphate S P acts via five different subtypes of S P receptors S PRs - S P - S P is predominantly expressed in nervous and immune systems regulating the egress of natural killer cells from lymph nodes and playing a role in immune and neurodegenerative disorders as well as carcinogenesis Several S PR therapeutic drugs have been developed to treat these diseases however they lack receptor subtype selectivity which leads to side effects In this article we describe a resolution room temperature crystal structure of the human S P receptor in complex with a selective ... More
The bioactive lysophospholipid sphingosine-1-phosphate (S1P) acts via five different subtypes of S1P receptors (S1PRs) - S1P1-5. S1P5 is predominantly expressed in nervous and immune systems, regulating the egress of natural killer cells from lymph nodes and playing a role in immune and neurodegenerative disorders, as well as carcinogenesis. Several S1PR therapeutic drugs have been developed to treat these diseases; however, they lack receptor subtype selectivity, which leads to side effects. In this article, we describe a 2.2 Å resolution room temperature crystal structure of the human S1P5 receptor in complex with a selective inverse agonist determined by serial femtosecond crystallography (SFX) at the Pohang Accelerator Laboratory X-Ray Free Electron Laser (PAL-XFEL) and analyze its structure-activity relationship data. The structure demonstrates a unique ligand-binding mode, involving an allosteric sub-pocket, which clarifies the receptor subtype selectivity and provides a template for structure-based drug design. Together with previously published S1PR structures in complex with antagonists and agonists, our structure with S1P5-inverse agonist sheds light on the activation mechanism and reveals structural determinants of the inverse agonism in the S1PR family. Less
We studied the prevalent cytotoxic CD T cell response mounted against severe acute respiratory syndrome coronavirus SARS-CoV- Spike glycoprotein - epitope sequence YLQPRTFLL via the most frequent human leukocyte antigen HLA class I worldwide HLA A The Spike P L mutation that has arisen in at least different SARS-CoV- lineages to date including in lineages classified as variants of concern was not recognized by the large CD T cell response seen across cohorts of HLA A convalescent patients and individuals vaccinated against SARS-CoV- despite these responses comprising of over different individual T cell receptors Viral escape at prevalent T cell ... More
We studied the prevalent cytotoxic CD8 T cell response mounted against severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2) Spike glycoprotein269-277 epitope (sequence YLQPRTFLL) via the most frequent
human leukocyte antigen (HLA) class I worldwide, HLA A*02. The Spike P272L mutation that has arisen in at
least 112 different SARS-CoV-2 lineages to date, including in lineages classified as ‘‘variants of concern,’’
was not recognized by the large CD8 T cell response seen across cohorts of HLA A*02+ convalescent patients
and individuals vaccinated against SARS-CoV-2, despite these responses comprising of over 175 different
individual T cell receptors. Viral escape at prevalent T cell epitopes restricted by high frequency HLAs may
be particularly problematic when vaccine immunity is focused on a single protein such as SARS-CoV-2 Spike,
providing a strong argument for inclusion of multiple viral proteins in next generation vaccines and highlighting
the need for monitoring T cell escape in new SARS-CoV-2 variants. Less
coronavirus 2 (SARS-CoV-2) Spike glycoprotein269-277 epitope (sequence YLQPRTFLL) via the most frequent
human leukocyte antigen (HLA) class I worldwide, HLA A*02. The Spike P272L mutation that has arisen in at
least 112 different SARS-CoV-2 lineages to date, including in lineages classified as ‘‘variants of concern,’’
was not recognized by the large CD8 T cell response seen across cohorts of HLA A*02+ convalescent patients
and individuals vaccinated against SARS-CoV-2, despite these responses comprising of over 175 different
individual T cell receptors. Viral escape at prevalent T cell epitopes restricted by high frequency HLAs may
be particularly problematic when vaccine immunity is focused on a single protein such as SARS-CoV-2 Spike,
providing a strong argument for inclusion of multiple viral proteins in next generation vaccines and highlighting
the need for monitoring T cell escape in new SARS-CoV-2 variants. Less
We studied the prevalent cytotoxic CD T cell response mounted against severe acute respiratory syndrome coronavirus SARS-CoV- Spike glycoprotein - epitope sequence YLQPRTFLL via the most frequent human leukocyte antigen HLA class I worldwide HLA A The Spike P L mutation that has arisen in at least different SARS-CoV- lineages to date including in lineages classified as variants of concern was not recognized by the large CD T cell response seen across cohorts of HLA A convalescent patients and individuals vaccinated against SARS-CoV- despite these responses comprising of over different individual T cell receptors Viral escape at prevalent T cell ... More
We studied the prevalent cytotoxic CD8 T cell response mounted against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike glycoprotein269-277 epitope (sequence YLQPRTFLL) via the most frequent human leukocyte antigen (HLA) class I worldwide, HLA A∗02. The Spike P272L mutation that has arisen in at least 112 different SARS-CoV-2 lineages to date, including in lineages classified as “variants of concern,” was not recognized by the large CD8 T cell response seen across cohorts of HLA A∗02+ convalescent patients and individuals vaccinated against SARS-CoV-2, despite these responses comprising of over 175 different individual T cell receptors. Viral escape at prevalent T cell epitopes restricted by high frequency HLAs may be particularly problematic when vaccine immunity is focused on a single protein such as SARS-CoV-2 Spike, providing a strong argument for inclusion of multiple viral proteins in next generation vaccines and highlighting the need for monitoring T cell escape in new SARS-CoV-2 variants. Less
Continuous developments in cryogenic X-ray crystallography have provided most of our knowledge of D protein structures which has recently been further augmented by revolutionary advances in cryoEM However a single structural conformation identified at cryogenic temperatures may introduce a fictitious structure as a result of cryogenic cooling artefacts limiting the overview of inherent protein physiological dynamics which play a critical role in the biological functions of proteins Here a room-temperature X-ray crystallo graphic method using temperature as a trigger to record movie-like structural snapshots has been developed The method has been used to show how TL a Da fragment undergoes ... More
Continuous developments in cryogenic X-ray crystallography have provided most of our knowledge of 3D protein structures, which has recently been further augmented by revolutionary advances in cryoEM. However, a single structural conformation identified at cryogenic temperatures may introduce a fictitious structure as a result of cryogenic cooling artefacts, limiting the overview of inherent protein physiological dynamics, which play a critical role in the biological functions of proteins. Here, a room-temperature X-ray crystallographic method using temperature as a trigger to record movie-like structural snapshots has been developed. The method has been used to show how TL00150, a 175.15 Da fragment, undergoes binding-mode changes in endothiapepsin. A surprising fragment-binding discrepancy was observed between the cryo-cooled and physiological temperature structures, and multiple binding poses and their interplay with DMSO were captured. The observations here open up new promising prospects for structure determination and interpretation at physiological temperatures with implications for structure-based drug discovery. Less
N-Methylated amino acids N-MeAAs are privileged residues of naturally occurring peptides critical to bioactivity However de novo discovery from ribosome display is limited by poor incorporation of N-methylated amino acids into the nascent peptide chain attributed to a poor EF-Tu affinity for the N-methyl-aminoacyl-tRNA By reconfiguring the tRNA s T-stem region to compensate and tune the EF-Tu affinity we conducted Random nonstandard Peptides Integrated Discovery RaPID display of a macrocyclic peptide MCP library containing six different N-MeAAs We have here devised a pool-and-split enrichment strategy using the RaPID display and identified N-methylated MCPs against three species of prokaryotic metal-ion-dependent phosphoglycerate ... More
N-Methylated amino acids (N-MeAAs) are privileged residues of naturally occurring peptides critical to bioactivity. However, de novo discovery from ribosome display is limited by poor incorporation of N-methylated amino acids into the nascent peptide chain attributed to a poor EF-Tu affinity for the N-methyl-aminoacyl-tRNA. By reconfiguring the tRNA’s T-stem region to compensate and tune the EF-Tu affinity, we conducted Random nonstandard Peptides Integrated Discovery (RaPID) display of a macrocyclic peptide (MCP) library containing six different N-MeAAs. We have here devised a “pool-and-split” enrichment strategy using the RaPID display and identified N-methylated MCPs against three species of prokaryotic metal-ion-dependent phosphoglycerate mutases. The enriched MCPs reached 57% N-methylation with up to three consecutively incorporated N-MeAAs, rivaling natural products. Potent nanomolar inhibitors ranging in ortholog selectivity, strongly mediated by N-methylation, were identified. Co-crystal structures reveal an architecturally related Ce-2 Ipglycermide active-site metal-ion-coordinating Cys lariat MCP, functionally dependent on two cisN-MeAAs with broadened iPGM species selectivity over the original nematode-selective MCPs. Furthermore, the isolation of a novel metal-ion-independent Staphylococcus aureus iPGM inhibitor utilizing a phosphoglycerate mimetic mechanism illustrates the diversity of possible chemotypes encoded by the N-MeAA MCP library. Less
Microbial rhodopsins are light-sensitive transmembrane proteins evolutionary adapted by various organisms like archaea bacteria simple eukaryote and viruses to utilize solar energy for their survival A complete understanding of functional mechanisms of these proteins is not possible without the knowledge of their high-resolution structures which can be primarily obtained by X-ray crystallography This technique however requires high-quality crystals growing of which is a great challenge especially in case of membrane proteins In this chapter we summarize methods applied for crystallization of microbial rhodopsins with the emphasis on crystallization in lipidic mesophases also known as in meso approach In particular we ... More
Microbial rhodopsins are light-sensitive transmembrane proteins, evolutionary adapted by various organisms like archaea, bacteria, simple eukaryote, and viruses to utilize solar energy for their survival. A complete understanding of functional mechanisms of these proteins is not possible without the knowledge of their high-resolution structures, which can be primarily obtained by X-ray crystallography. This technique, however, requires high-quality crystals, growing of which is a great challenge especially in case of membrane proteins. In this chapter, we summarize methods applied for crystallization of microbial rhodopsins with the emphasis on crystallization in lipidic mesophases, also known as in meso approach. In particular, we describe in detail the methods of crystallization using lipidic cubic phase to grow both large crystals optimized for traditional crystallographic data collection and microcrystals for serial crystallography. Less
T-cell receptor TCR immunotherapy is becoming a viable modality in cancer treatment with efficacy in clinical trials The safety of patients is paramount so innovative cell engineering methods are being employed to exploit adaptive immunity while controlling the factors governing antigen receptor ie TCR specificity and cross-reactivity We recently reported a TCR engineering campaign and selectivity profiling assay X-scan targeting a melanoma antigen gene MAGE -A peptide This helped to distinguish between two well-performing TCRs based on cross-reactivity potential during preclinical drug evaluation allowing one to be advanced to T-cell immunotherapeutic clinical trials Here we present three-dimensional structural information on ... More
T-cell receptor (TCR) immunotherapy is becoming a viable modality in cancer treatment with efficacy in clinical trials. The safety of patients is paramount, so innovative cell engineering methods are being employed to exploit adaptive immunity while controlling the factors governing antigen receptor (ie, TCR) specificity and cross-reactivity. We recently reported a TCR engineering campaign and selectivity profiling assay (X-scan) targeting a melanoma antigen gene (MAGE)-A10 peptide. This helped to distinguish between two well-performing TCRs based on cross-reactivity potential during preclinical drug evaluation, allowing one to be advanced to T-cell immunotherapeutic clinical trials. Here, we present three-dimensional structural information on those TCRs, highlighting engineering improvements and molecular mechanisms likely underpinning differential selectivity. Less
Heavy-atom soaking has been a major method for experimental phasing but it has been difficult for membrane proteins partly owing to the lack of available sites in the scarce soluble domain for non-invasive heavy-metal binding The lipid cubic phase LCP has proven to be a successful method for membrane protein crystallization but experimental phasing with LCP-grown crystals remains difficult and so far only such structures were phased experimentally Here the selenourea was tested as a soaking reagent for the single-wavelength anomalous dispersion SAD phasing of crystals grown in LCP Using a single crystal the structure of the glycerol -phosphate acyltransferase ... More
Heavy-atom soaking has been a major method for experimental phasing, but it has been difficult for membrane proteins, partly owing to the lack of available sites in the scarce soluble domain for non-invasive heavy-metal binding. The lipid cubic phase (LCP) has proven to be a successful method for membrane protein crystallization, but experimental phasing with LCP-grown crystals remains difficult, and so far, only 68 such structures were phased experimentally. Here, the selenourea was tested as a soaking reagent for the single-wavelength anomalous dispersion (SAD) phasing of crystals grown in LCP. Using a single crystal, the structure of the glycerol 3-phosphate acyltransferase (PlsY, ~21 kDa), a very hydrophobic enzyme with 80% membrane-embedded residues, was solved. Remarkably, a total of 15 Se sites were found in the two monomers of PlsY, translating to one selenourea-binding site per every six residues in the accessible extramembrane protein. Structure analysis reveals that surface-exposed selenourea sites are mostly contributed by mainchain amides and carbonyls. This low-specificity binding pattern may explain its high loading ratio. Importantly, both the crystal diffraction quality and the LCP integrity were unaffected by selenourea soaking. Taken together, selenourea presents a promising and generally useful reagent for heavy-atom soaking of membrane protein crystals grown in LCP. Less
The technique of cryogenic-electron microscopy cryo-EM has revolutionized the field of membrane protein structure and function with a focus on the dominantly observed molecular species This report describes the structural characterization of a fully active human apelin receptor APJR complexed with heterotrimeric G protein observed in both and stoichiometric ratios We use cryo-EM single-particle analysis to determine the structural details of both species from the same sample preparation Protein preparations in the presence of the endogenous peptide ligand ELA or a synthetic small molecule both demonstrate these mixed stoichiometric states Structural differences in G protein engagement between dimeric and monomeric ... More
The technique of cryogenic-electron microscopy (cryo-EM) has revolutionized the field of membrane protein structure and function with a focus on the dominantly observed molecular species. This report describes the structural characterization of a fully active human apelin receptor (APJR) complexed with heterotrimeric G protein observed in both 2:1 and 1:1 stoichiometric ratios. We use cryo-EM single-particle analysis to determine the structural details of both species from the same sample preparation. Protein preparations, in the presence of the endogenous peptide ligand ELA or a synthetic small molecule, both demonstrate these mixed stoichiometric states. Structural differences in G protein engagement between dimeric and monomeric APJR suggest a role for the stoichiometry of G protein-coupled receptor- (GPCR-)G protein coupling on downstream signaling and receptor pharmacology. Furthermore, a small, hydrophobic dimer interface provides a starting framework for additional class A GPCR dimerization studies. Together, these findings uncover a mechanism of versatile regulation through oligomerization by which GPCRs can modulate their signaling. Less
Candida Als family adhesins mediate adhesion to biological and abiotic substrates as well as fungal cell aggregation fungal-bacterial co-aggregation and biofilm formation The activity of at least two family members Als and Als is dependent on amyloid-like protein aggregation that is initiated by shear force Each Als adhesin has a -residue N-terminal Ig-like invasin region The following -residue low complexity threonine-rich T domain unfolds under shear force to expose a critical amyloid-forming segment SNGIVIVATTRTV at the interface between the Ig-like invasin domain and the T domain of Candida albicans Als Amyloid prediction programs identified six potential amyloidogenic sequences in the ... More
Candida Als family adhesins mediate adhesion to biological and abiotic substrates, as well as fungal cell aggregation, fungal-bacterial co-aggregation and biofilm formation. The activity of at least two family members, Als5 and Als1, is dependent on amyloid-like protein aggregation that is initiated by shear force. Each Als adhesin has a ∼300-residue N-terminal Ig-like/invasin region. The following 108-residue, low complexity, threonine-rich (T) domain unfolds under shear force to expose a critical amyloid-forming segment 322SNGIVIVATTRTV334 at the interface between the Ig-like/invasin domain 2 and the T domain of Candida albicans Als5. Amyloid prediction programs identified six potential amyloidogenic sequences in the Ig-like/invasin region and three others in the T domain of C. albicans Als5. Peptides derived from four of these sequences formed fibrils that bound thioflavin T, the amyloid indicator dye, and three of these revealed atomic-resolution structures of cross-β spines. These are the first atomic-level structures for fungal adhesins. One of these segments, from the T domain, revealed kinked β-sheets, similarly to LARKS (Low-complexity, Amyloid-like, Reversible, Kinked segments) found in human functional amyloids. Based on the cross-β structures in Als proteins, we use evolutionary arguments to identify functional amyloidogenic sequences in other fungal adhesins, including adhesins from Candida auris. Thus, cross-β structures are often involved in fungal pathogenesis and potentially in antifungal therapy. Less
G protein-coupled receptors GPCRs play vital roles in human physiology and pathophysiology This makes the elucidation of the high-resolution blueprints of these high value membrane proteins of crucial importance for the structure-based design of novel therapeutics However the production and crystallization of GPCRs for structure determination comes with many challenges In this chapter we provide a comprehensive protocol for expressing and purifying the thromboxane A receptor TPR an attractive therapeutic target for use in structure studies Guidelines for crystallizing the TPR are also included Together these procedures provide a template for generating crystal structures of the TPR and indeed other ... More
G protein-coupled receptors (GPCRs) play vital roles in human physiology and pathophysiology. This makes the elucidation of the high-resolution blueprints of these high value membrane proteins of crucial importance for the structure-based design of novel therapeutics. However, the production and crystallization of GPCRs for structure determination comes with many challenges.
In this chapter, we provide a comprehensive protocol for expressing and purifying the thromboxane A2 receptor (TPR), an attractive therapeutic target, for use in structure studies. Guidelines for crystallizing the TPR are also included. Together, these procedures provide a template for generating crystal structures of the TPR and indeed other GPCRs in complex with pharmacologically interesting ligands. Less
In this chapter, we provide a comprehensive protocol for expressing and purifying the thromboxane A2 receptor (TPR), an attractive therapeutic target, for use in structure studies. Guidelines for crystallizing the TPR are also included. Together, these procedures provide a template for generating crystal structures of the TPR and indeed other GPCRs in complex with pharmacologically interesting ligands. Less
As discussed in previous chapters the methylation of specific arginine and lysine side chains is carried out by two families of histone methyltransferases the Protein Arginine Methyltransferase PRMT family for arginine and the SET domain family for lysine The methylation of H K by Dot is a notable outlier In all cases X-ray crystallography has been a powerful technique that has provided the framework for understanding the enzyme mechanism kinetics regulation and specificity of these enzymes and is now a platform for the design of compounds aimed to inhibit their activity either to further understand their function or in a ... More
As discussed in previous chapters, the methylation of specific arginine and lysine side chains is carried out by two families of histone methyltransferases, the Protein Arginine Methyltransferase (PRMT) family for arginine, and the SET domain family for lysine. The methylation of H3K79 by Dot1 is a notable outlier. In all cases, X-ray crystallography has been a powerful technique that has provided the framework for understanding the enzyme mechanism, kinetics, regulation and specificity of these enzymes and is now a platform for the design of compounds aimed to inhibit their activity either to further understand their function or in a therapeutic setting. Notably, in combination with the structures of the complementary recognition domains that recognize their products, these structures have provided an important insight into how integral the number of methyl groups added to the acceptor amine is to making histone methylation a key process in epigenetic regulation of gene transcription. Here the concepts applied to determine their structure by X-ray crystallography are outlined, with particular emphasis on lysine methylation by the SET domain. Less
The cysteine of HCD C in DYRK A is involved in disulfide bridge formation with a cysteine C in the DFGSSC sequence The purpose of this project was to investigate how the state of the disulfide bridge would affect enzyme catalytic and ligand binding properties of the protein kinase A mutant DYRK A C A was thus designed to eliminate the disulfide bridge The mutant was expressed and purified following the same protocol as for DYRK A wt including HisTrap purification TEV cleavage and size exclusion chromatography Crystallization trials were performed for both the wt and the mutant with the ... More
The cysteine of HCD (C286) in DYRK1A is involved in disulfide bridge formation with a cysteine (C312) in the DFGSSC sequence. The purpose of this project was to investigate how the state of the disulfide bridge would affect enzyme catalytic and ligand binding properties of the protein kinase. A mutant, DYRK1A C312A, was thus designed to eliminate the disulfide bridge. The mutant was expressed and purified following the same protocol as for DYRK1A wt, including HisTrap purification, TEV cleavage and size exclusion chromatography. Crystallization trials were performed for both the wt and the mutant with the kinase inhibitor Staurosporine. DYRK1A wt with STU crystallized and diffracted with at a resolution of 2.33 Å. The DYRK1A C312A mutant with STU crystallized and diffracted with a resolution of 2.59 Å. The structure was solved by molecular replacement in Molrep (CCP4) and refined by Refmac5 and Phenix. Molecular dynamics (MD) simulations (SCHRODINGER) were performed with the intent to compare diverse disulfide bridge states. Ligand binding and enzyme catalytic properties were analyzed using a combination of techniques, including activity assays, microscale thermophoresis, and isothermal calorimetry. The Thermofluor assay confirmed that both the wt and the mutant bind tightly to STU and AZ-191. It also showed that the mutant consistently has a slightly lower melting temperature than the wt, which would indicate that it is less stable. Solvent accessible surface area (SASA) analysis support the theory of accessibility to conserved cysteine residues. Less
The novel coronavirus pandemic whose first outbreak was reported in December in Wuhan China COVID- is caused by the severe acute respiratory syndrome coronavirus SARS-CoV- Tissue damage caused by the virus leads to a strong immune response and activation of antigen-presenting cells which can elicit acute respiratory distress syndrome ARDS characterized by the rapid onset of widespread inflammation the so-called cytokine storm In many viral infections the recruitment of monocytes into the lung and their differentiation to dendritic cells DCs are seen as a response to the viral infection DCs are critical players in the development of the acute lung ... More
The novel coronavirus pandemic, whose first outbreak was reported in December 2019 in Wuhan, China (COVID-19), is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Tissue damage caused by the virus leads to a strong immune response and activation of antigen-presenting cells, which can elicit acute respiratory distress syndrome (ARDS) characterized by the rapid onset of widespread inflammation, the so-called cytokine storm. In many viral infections the recruitment of monocytes into the lung and their differentiation to dendritic cells (DCs) are seen as a response to the viral infection. DCs are critical players in the development of the acute lung inflammation that causes ARDS. Here we focus on the interaction of the ORF8 protein, a specific SARS-CoV-2 open reading frame protein, with dendritic cells (DCs). We show that ORF8 binds to dendritic cells, causes a pre-maturation of differentiating DCs, and induces the secretion of multiple pro-inflammatory cytokines by these cells. In addition, we identified dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) as a possible interaction partner of ORF8 on dendritic cells. Blockade of ORF8 signaling leads to reduced production of IL-1β, IL-6, IL-12p70, TNF-α, MCP-1 (CCL2), and IL-10 by dendritic cells. Analysis of patient sera with high anti-ORF8 antibody titers showed that there was nearly no neutralization of the ORF8 protein and its function. Therefore, a neutralizing antibody that has the capacity of blocking the cytokine and chemokine response mediated by ORF8 protein might be an essential and novel additional step in the therapy of severe SARS-CoV-2 cases. Less
Antimicrobial resistance primarily caused by the overuse of antimicrobials such as antibiotics is becoming an increasing concern to public health To that end the global spread of the -lactamase OXA- is worrisome as it readily catalyzes the hydrolysis of -lactam drugs such as penicillins as well as our last resort carbapenems On the contrary OXA- exhibits only limited catalytic activity against rd generations cephalosporins like ceftazidime However naturally evolving variants and results from laboratory studies have shown that OXA- can expand its substrate profile conferring increased ceftazidime resistance Expansion of the substrate profile towards ceftazidime is seen to be accompanied ... More
Antimicrobial resistance, primarily caused by the overuse of antimicrobials such as antibiotics, is becoming an increasing concern to public health. To that end, the global spread of the -lactamase OXA-48 is worrisome, as it readily catalyzes the hydrolysis of -lactam drugs, such as penicillins as well as our “last resort” carbapenems. On the contrary, OXA-48 exhibits only limited catalytic activity against 3rd generations cephalosporins like ceftazidime. However, naturally evolving variants and results from laboratory studies have shown that OXA-48 can expand its substrate profile, conferring increased ceftazidime resistance. Expansion of the substrate profile towards ceftazidime is seen to be accompanied by a trade-off towards carbapenems and penicillins, greatly reducing OXA-48 ability to catalyze the hydrolysis of penicillins and carbapenems. Here, X-ray crystallography, steady-state enzyme kinetics and differential scanning fluorimetry were used to characterize and analyze wild type (wt) OXA-48:wt and two variants, OXA-48:F72L and OXA-48:A33V/K51E/F72L/S212A/T213A (OXA-48:Q5), where the latter two were evolved towards increased ceftazidime resistance. Steady-state enzyme kinetics revealed that the two mutants had increased catalytic ability to hydrolyze ceftazidime. Such increases in kcat/Km hypothesized to arise from increased flexibility of the -loop, which was observed in the OXA-48:Q5 X-ray crystal structure in complex with piperacillin, is in line with previous studies. Further supporting the hypothesis, urea dependent kinetics and thermostability measurements show that these mutants likely exhibit increased dynamical behavior that would aid ceftazidime binding. OXA-48:F72L showed a bigger urea dependence on the enzyme activity with no activity at 4 M urea, whereas OXA-48:wt and OXA-48:Q5 needed 6 M urea to become inactive. This suggested that OXA-48:F72L is more flexible, and that OXA-48:Q5 regains some resistance to chemical denaturing by urea. The pH dependency showed higher piperacillin activity at pH 7.2 compared to 5.2 and 9.2 for all three variants. The increase in ceftazidime activity came along with a functional trade-off against the penicillin piperacillin as well as reduced thermostability of (OXA-48:F72L: -6.5C/OXA-48:Q5: -6.4C) compared to wt OXA-48, which may be caused by sub-optimal substrate positioning within the active site of OXA-48:Q5. This work provides experimental evidence, that during evolution of OXA-48 towards increased ceftazidime activity, structural changes can arise, likely affecting the chemical environment within the active site, causing increased enzyme flexibility, and ultimately shaping functional trade-offs. Less
Chitin is a biopolymer of N-acetyl-d-glucosamine with - -bond and is the main component of arthropod exoskeletons and the cell walls of many fungi Chitinase EC is an enzyme that hydrolyzes the - -bond in chitin and degrades chitin into oligomers It has been found in a wide range of organisms Chitinase from Gazyumaru Ficus microcarpa latex exhibits antifungal activity by degrading chitin in the cell wall of fungi and is expected to be used in medical and agricultural fields However the enzyme s thermostability is an important factor chitinase is not thermostable enough to maintain its activity under the ... More
Chitin is a biopolymer of N-acetyl-d-glucosamine with β-1,4-bond and is the main component of arthropod exoskeletons and the cell walls of many fungi. Chitinase (EC 3.2.1.14) is an enzyme that hydrolyzes the β-1,4-bond in chitin and degrades chitin into oligomers. It has been found in a wide range of organisms. Chitinase from Gazyumaru (Ficus microcarpa) latex exhibits antifungal activity by degrading chitin in the cell wall of fungi and is expected to be used in medical and agricultural fields. However, the enzyme’s thermostability is an important factor; chitinase is not thermostable enough to maintain its activity under the actual application conditions. In addition to the fact that thermostable chitinases exhibiting antifungal activity can be used under various conditions, they have some advantages for the production process and long-term preservation, which are highly demanded in industrial use. We solved the crystal structure of chitinase to explore the target sites to improve its thermostability. We rationally introduced proline residues, a disulfide bond, and salt bridges in the chitinase using protein-engineering methods based on the crystal structure and sequence alignment among other chitinases. As a result, we successfully constructed the thermostable mutant chitinases rationally with high antifungal and specific activities. The results provide a useful strategy to enhance the thermostability of this enzyme family. Less
This work aims to determine a model of the autoinhibition mechanism of MICAL proteins using biochemical biophysical and bioinformatical approaches MICAL proteins are a group of flavin monooxygenases that play a key role in various cellular processes as they facilitate the reorganization of the actin cytoskeleton MICAL- has long been known for its vital role in axon guidance as an effector of repulsive signaling through oxidative destabilization of actin filaments However recent findings indicate that MICAL- can also serve as a signaling molecule using localized hydrogen peroxide production to regulate other downstream effectors Despite the consensus that MICAL- activity must ... More
This work aims to determine a model of the autoinhibition mechanism of MICAL proteins using biochemical, biophysical, and bioinformatical approaches. MICAL proteins are a group of flavin monooxygenases that play a key role in various cellular processes, as they facilitate the reorganization of the actin cytoskeleton. MICAL-1 has long been known for its vital role in axon guidance as an effector of repulsive signaling through oxidative destabilization of actin filaments. However, recent findings indicate that MICAL-1 can also serve as a signaling molecule, using localized hydrogen peroxide production to regulate other downstream effectors. Despite the consensus that MICAL-1 activity must be strictly regulated, the exact molecular mechanism of this regulation has not yet been described. In this work, we provide a novel model of MICAL-1 autoinibiton mechanism based on a comparison of steady-state kinetic experiments and molecular dynamics simulations between full-length MICAL-1 from Coturnix japonica and its truncated form lacking the C-terminal domain. In our model, we conclude that changes in MICAL-1 activity are the result of intramolecular protein interactions between the C-terminal and the monooxygenase domain. Furthermore, we rule out the role of MICAL-1 oligomerization in its activity regulation. Our work provides the basis for further research that will need to focus on a more detailed investigation of intramolecular interactions between the MICAL-1 domains. Less
The worldwide impact of the ongoing COVID- pandemic on public health has made imperative the discovery and development of direct-acting antivirals aimed at targeting viral and or host targets SARS-CoV- C-like protease CLpro has emerged as a validated target for the discovery of SARS-CoV- therapeutics because of the pivotal role it plays in viral replication We describe herein the structure-guided design of highly potent inhibitors of SARS-CoV- CLpro that incorporate in their structure novel spirocyclic design elements aimed at optimizing potency by accessing new chemical space Inhibitors of both SARS-CoV- CLpro and MERS-CoV CLpro that exhibit nM potency and high ... More
The worldwide impact of the ongoing COVID-19 pandemic on public health has made imperative the discovery and development of direct-acting antivirals aimed at targeting viral and/or host targets. SARS-CoV-2 3C-like protease (3CLpro) has emerged as a validated target for the discovery of SARS-CoV-2 therapeutics because of the pivotal role it plays in viral replication. We describe herein the structure-guided design of highly potent inhibitors of SARS-CoV-2 3CLpro that incorporate in their structure novel spirocyclic design elements aimed at optimizing potency by accessing new chemical space. Inhibitors of both SARS-CoV-2 3CLpro and MERS-CoV 3CLpro that exhibit nM potency and high safety indices have been identified. The mechanism of action of the inhibitors and the structural determinants associated with binding were established using high-resolution cocrystal structures. Less