High-throughput crystallographic fragment screening of Zika virus NS3 Helicase.

The Zika virus (ZIKV), discovered in Africa in 1947, swiftly spread across continents, causing significant concern due to its recent association with microcephaly in newborns and Guillain-Barré syndrome in adults. Despite a decrease in prevalence, the potential for a resurgence remains, necessitating urgent therapeutic interventions. Like other flaviviruses, ZIKV presents promising drug targets within its replication machinery, notably the NS3 helicase (NS3Hel) protein, which plays critical roles in viral replication. However, a lack of structural information impedes the development of specific inhibitors targeting NS3Hel. Here we applied high-throughput crystallographic fragment screening on ZIKV NS3Hel, which yielded structures that reveal 3D binding poses of 46 fragments at multiple sites of the protein, including 11 unique fragments in the RNA-cleft site. These fragment structures provide templates for direct design of hit compounds and should thus assist the development of novel direct-acting antivirals against ZIKV and related flaviviruses, thus opening a promising avenue for combating future outbreaks.

Discovery of PLpro and Mpro Inhibitors for SARS-CoV-2.

There are very few small-molecule antivirals for SARS-CoV-2 that are either currently approved (or emergency authorized) in the US or globally, including remdesivir, molnupiravir, and paxlovid. The increasing number of SARS-CoV-2 variants that have appeared since the outbreak began over three years ago raises the need for continual development of updated vaccines and orally available antivirals in order to fully protect or treat the population. The viral main protease (Mpro) and the papain-like protease (PLpro) are key for viral replication; therefore, they represent valuable targets for antiviral therapy. We herein describe an in vitro screen performed using the 2560 compounds from the Microsource Spectrum library against Mpro and PLpro in an attempt to identify additional small-molecule hits that could be repurposed for SARS-CoV-2. We subsequently identified 2 hits for Mpro and 8 hits for PLpro. One of these hits was the quaternary ammonium compound cetylpyridinium chloride with dual activity (IC50 = 2.72 ± 0.09 µM for PLpro and IC50 = 7.25 ± 0.15 µM for Mpro). A second inhibitor of PLpro was the selective estrogen receptor modulator raloxifene (IC50 = 3.28 ± 0.29 µM for PLpro and IC50 = 42.8 ± 6.7 µM for Mpro). We additionally tested several kinase inhibitors and identified olmutinib (IC50 = 0.54 ± 0.04 µM), bosutinib (IC50 = 4.23 ± 0.28 µM), crizotinib (IC50 = 3.81 ± 0.04 µM), and dacominitinib (IC50 = IC50 3.33 ± 0.06 µM) as PLpro inhibitors for the first time. In some cases, these molecules have also been tested by others for antiviral activity for this virus, or we have used Calu-3 cells infected with SARS-CoV-2. The results suggest that approved drugs can be identified with promising activity against these proteases, and in several cases we or others have validated their antiviral activity. The additional identification of known kinase inhibitors as molecules targeting PLpro may provide new repurposing opportunities or starting points for chemical optimization.

The protein disulfide isomerase inhibitor 3-methyltoxoflavin inhibits Chikungunya virus.

Chikungunya virus (CHIKV) is the etiological agent of chikungunya fever, a (re)emerging arbovirus infection, that causes severe and often persistent arthritis, as well as representing a serious health concern worldwide for which no antivirals are currently available. Despite efforts over the last decade to identify and optimize new inhibitors or to reposition existing drugs, no compound has progressed to clinical trials for CHIKV and current prophylaxis is based on vector control, which has shown limited success in containing the virus. Our efforts to rectify this situation were initiated by screening 36 compounds using a replicon system and ultimately identified the natural product derivative 3-methyltoxoflavin with activity against CHIKV using a cell-based assay (EC50 200 nM, SI = 17 in Huh-7 cells). We have additionally screened 3-methyltoxoflavin against a panel of 17 viruses and showed that it only additionally demonstrated inhibition of the yellow fever virus (EC50 370 nM, SI = 3.2 in Huh-7 cells). We have also showed that 3-methyltoxoflavin has excellent in vitro human and mouse microsomal metabolic stability, good solubility and high Caco-2 permeability and it is not likely to be a P-glycoprotein substrate. In summary, we demonstrate that 3-methyltoxoflavin has activity against CHIKV, good in vitro absorption, distribution, metabolism and excretion (ADME) properties as well as good calculated physicochemical properties and may represent a valuable starting point for future optimization to develop inhibitors for this and other related viruses.

The Antifungal Itraconazole Is a Potent Inhibitor of Chikungunya Virus Replication.

Chikungunya virus (CHIKV) is the causative agent of chikungunya fever, a disabling disease that can cause long-term severe arthritis. Since the last large CHIKV outbreak in 2015, the reemergence of the virus represents a serious public health concern. The morbidity associated with viral infection emphasizes the need for the development of specific anti-CHIKV drugs. Herein, we describe the development and characterization of a CHIKV reporter replicon cell line and its use in replicon-based screenings. We tested 960 compounds from MMV/DNDi Open Box libraries and identified four candidates with interesting antiviral activities, which were confirmed in viral infection assays employing CHIKV-nanoluc and BHK-21 cells. The most noteworthy compound identified was itraconazole (ITZ), an orally available, safe, and cheap antifungal, that showed high selectivity indexes of >312 and >294 in both replicon-based and viral infection assays, respectively. The antiviral activity of this molecule has been described against positive-sense single stranded RNA viruses (+ssRNA) and was related to cholesterol metabolism that could affect the formation of the replication organelles. Although its precise mechanism of action against CHIKV still needs to be elucidated, our results demonstrate that ITZ is a potent inhibitor of the viral replication that could be repurposed as a broad-spectrum antiviral.

Characterization of the RNA-dependent RNA polymerase from Chikungunya virus and discovery of a novel ligand as a potential drug candidate.

Chikungunya virus (CHIKV) is the causative agent of Chikungunya fever, an acute febrile and arthritogenic illness with no effective treatments available. The development of effective therapeutic strategies could be significantly accelerated with detailed knowledge of the molecular components behind CHIKV replication. However, drug discovery is hindered by our incomplete understanding of their main components. The RNA-dependent RNA-polymerase (nsP4-CHIKV) is considered the key enzyme of the CHIKV replication complex and a suitable target for antiviral therapy. Herein, the nsP4-CHIKV was extensively characterized through experimental and computational biophysical methods. In the search for new molecules against CHIKV, a compound designated LabMol-309 was identified as a strong ligand of the nsp4-CHIKV and mapped to bind to its active site. The antiviral activity of LabMol-309 was evaluated in cellular-based assays using a CHIKV replicon system and a reporter virus. In conclusion, this study highlights the biophysical features of nsP4-CHIKV and identifies a new compound as a promising antiviral agent against CHIKV infection.

Pyronaridine Protects against SARS-CoV-2 Infection in Mouse.

There are currently relatively few small-molecule antiviral drugs that are either approved or emergency-approved for use against severe acute respiratory coronavirus 2 (SARS-CoV-2). One of these is remdesivir, which was originally repurposed from its use against Ebola. We evaluated three molecules we had previously identified computationally with antiviral activity against Ebola and Marburg and identified pyronaridine, which inhibited the SARS-CoV-2 replication in A549-ACE2 cells. The in vivo efficacy of pyronaridine has now been assessed in a K18-hACE transgenic mouse model of COVID-19. Pyronaridine treatment demonstrated a statistically significant reduction of viral load in the lungs of SARS-CoV-2-infected mice, reducing lung pathology, which was also associated with significant reduction in the levels of pro-inflammatory cytokines/chemokine and cell infiltration. Pyronaridine inhibited the viral PLpro activity in vitro (IC50 of 1.8 µM) without any effect on Mpro, indicating a possible molecular mechanism involved in its ability to inhibit SARS-CoV-2 replication. We have also generated several pyronaridine analogs to assist in understanding the structure activity relationship for PLpro inhibition. Our results indicate that pyronaridine is a potential therapeutic candidate for COVID-19.

High-throughput Antiviral Assays to Screen for Inhibitors of Zika Virus Replication.

Antiviral drug discovery requires the development of reliable biochemical and cellular assays that can be performed in high-throughput screening (HTS) formats. The flavivirus non-structural (NS) proteins are thought to co-translationally assemble on the endoplasmic reticulum (ER) membranes, forming the replication complex (RC). The NS3 and NS5 are the most studied enzymes of the RC and constitute the main targets for drug development due to their crucial roles in viral genome replication. NS3 protease domain, which requires NS2B as its cofactor, is responsible for the cleavage of the immature viral polyprotein into the mature NS proteins, whereas NS5 RdRp domain is responsible for the RNA replication. Herein, we describe in detail the protocols used in replicon-based screenings and enzymatic assays to test large compound libraries for inhibitors of the Zika virus (ZIKV) replication. Replicons are self-replicating subgenomic systems expressed in mammalian cells, in which the viral structural genes are replaced by a reporter gene. The inhibitory effects of compounds on viral RNA replication can be easily evaluated by measuring the reduction in the reporter protein activity. The replicon-based screenings were performed using a BHK-21 ZIKV replicon cell line expressing Renilla luciferase as a reporter gene. To characterize the specific targets of identified compounds, we established in-vitro fluorescence-based assays for recombinantly expressed NS3 protease and NS5 RdRp. The proteolytic activity of the viral protease was measured by using the fluorogenic peptide substrate Bz-nKRR-AMC, while the NS5 RdRp elongation activity was directly detected by the increase of the fluorescent signal of SYBR Green I during RNA elongation, using the synthetic biotinylated self-priming template 3'UTR-U30 (5'-biotin-U30-ACUGGAGAUCGAUCUCCAGU-3').

Machine Learning Models Identify Inhibitors of SARS-CoV-2.

With the rapidly evolving SARS-CoV-2 variants of concern, there is an urgent need for the discovery of further treatments for the coronavirus disease (COVID-19). Drug repurposing is one of the most rapid strategies for addressing this need, and numerous compounds have already been selected for in vitro testing by several groups. These have led to a growing database of molecules with in vitro activity against the virus. Machine learning models can assist drug discovery through prediction of the best compounds based on previously published data. Herein, we have implemented several machine learning methods to develop predictive models from recent SARS-CoV-2 in vitro inhibition data and used them to prioritize additional FDA-approved compounds for in vitro testing selected from our in-house compound library. From the compounds predicted with a Bayesian machine learning model, lumefantrine, an antimalarial was selected for testing and showed limited antiviral activity in cell-based assays while demonstrating binding (Kd 259 nM) to the spike protein using microscale thermophoresis. Several other compounds which we prioritized have since been tested by others and were also found to be active in vitro. This combined machine learning and in vitro testing approach can be expanded to virtually screen available molecules with predicted activity against SARS-CoV-2 reference WIV04 strain and circulating variants of concern. In the process of this work, we have created multiple iterations of machine learning models that can be used as a prioritization tool for SARS-CoV-2 antiviral drug discovery programs. The very latest model for SARS-CoV-2 with over 500 compounds is now freely available at www.assaycentral.org.

Non-Toxic Dimeric Peptides Derived from the Bothropstoxin-I Are Potent SARS-CoV-2 and Papain-like Protease Inhibitors.

The COVID-19 outbreak has rapidly spread on a global scale, affecting the economy and public health systems throughout the world. In recent years, peptide-based therapeutics have been widely studied and developed to treat infectious diseases, including viral infections. Herein, the antiviral effects of the lysine linked dimer des-Cys11, Lys12,Lys13-(pBthTX-I)2K ((pBthTX-I)2K)) and derivatives against SARS-CoV-2 are reported. The lead peptide (pBthTX-I)2K and derivatives showed attractive inhibitory activities against SARS-CoV-2 (EC50 = 28-65 µM) and mostly low cytotoxic effect (CC50 > 100 µM). To shed light on the mechanism of action underlying the peptides' antiviral activity, the Main Protease (Mpro) and Papain-Like protease (PLpro) inhibitory activities of the peptides were assessed. The synthetic peptides showed PLpro inhibition potencies (IC50s = 1.0-3.5 µM) and binding affinities (Kd = 0.9-7 µM) at the low micromolar range but poor inhibitory activity against Mpro (IC50 > 10 µM). The modeled binding mode of a representative peptide of the series indicated that the compound blocked the entry of the PLpro substrate toward the protease catalytic cleft. Our findings indicated that non-toxic dimeric peptides derived from the Bothropstoxin-I have attractive cellular and enzymatic inhibitory activities, thereby suggesting that they are promising prototypes for the discovery and development of new drugs against SARS-CoV-2 infection.

Development of Machine Learning Models and the Discovery of a New Antiviral Compound against Yellow Fever Virus.

Yellow fever (YF) is an acute viral hemorrhagic disease transmitted by infected mosquitoes. Large epidemics of YF occur when the virus is introduced into heavily populated areas with high mosquito density and low vaccination coverage. The lack of a specific small molecule drug treatment against YF as well as for homologous infections, such as zika and dengue, highlights the importance of these flaviviruses as a public health concern. With the advancement in computer hardware and bioactivity data availability, new tools based on machine learning methods have been introduced into drug discovery, as a means to utilize the growing high throughput screening (HTS) data generated to reduce costs and increase the speed of drug development. The use of predictive machine learning models using previously published data from HTS campaigns or data available in public databases, can enable the selection of compounds with desirable bioactivity and absorption, distribution, metabolism, and excretion profiles. In this study, we have collated cell-based assay data for yellow fever virus from the literature and public databases. The data were used to build predictive models with several machine learning methods that could prioritize compounds for in vitro testing. Five molecules were prioritized and tested in vitro from which we have identified a new pyrazolesulfonamide derivative with EC50 3.2 µM and CC50 24 µM, which represents a new scaffold suitable for hit-to-lead optimization that can expand the available drug discovery candidates for YF.

An atomic model for the human septin hexamer by cryo-EM.

In order to form functional filaments, human septins must assemble into hetero-oligomeric rod-like particles which polymerize end-to-end. The rules governing the assembly of these particles and the subsequent filaments are incompletely understood. Although crystallographic approaches have been successful in studying the separate components of the system, there has been difficulty in obtaining high resolution structures of the full particle. Here we report a first cryo-EM structure for a hexameric rod composed of human septins 2, 6 and 7 with a global resolution of ~3.6 Å and a local resolution of between ~3.0 Å and ~5.0 Å. By fitting the previously determined high-resolution crystal structures of the component subunits into the cryo-EM map, we are able to provide an essentially complete model for the particle. This exposes SEPT2 NC-interfaces at the termini of the hexamer and leaves internal cavities between the SEPT6-SEPT7 pairs. The floor of the cavity is formed by the two α0 helices including their polybasic regions. These are locked into place between the two subunits by interactions made with the α5 and α6 helices of the neighbouring monomer together with its polyacidic region. The cavity may serve to provide space allowing the subunits to move with respect to one another. The elongated particle shows a tendency to bend at its centre where two copies of SEPT7 form a homodimeric G-interface. Such bending is almost certainly related to the ability of septin filaments to recognize and even induce membrane curvature.

Reporter Replicons for Antiviral Drug Discovery against Positive Single-Stranded RNA Viruses.

Single-stranded positive RNA ((+) ssRNA) viruses include several important human pathogens. Some members are responsible for large outbreaks, such as Zika virus, West Nile virus, SARS-CoV, and SARS-CoV-2, while others are endemic, causing an enormous global health burden. Since vaccines or specific treatments are not available for most viral infections, the discovery of direct-acting antivirals (DAA) is an urgent need. Still, the low-throughput nature of and biosafety concerns related to traditional antiviral assays hinders the discovery of new inhibitors. With the advances of reverse genetics, reporter replicon systems have become an alternative tool for the screening of DAAs. Herein, we review decades of the use of (+) ssRNA viruses replicon systems for the discovery of antiviral agents. We summarize different strategies used to develop those systems, as well as highlight some of the most promising inhibitors identified by the method. Despite the genetic alterations introduced, reporter replicons have been shown to be reliable systems for screening and identification of viral replication inhibitors and, therefore, an important tool for the discovery of new DAAs.

Discovery and Characterization of Peptide Inhibitors for Calcium and Integrin Binding Protein 1.

Calcium and integrin binding protein 1 (CIB1) is an EF-hand-containing, small intracellular protein that has recently been implicated in cancer cell survival and proliferation. In particular, CIB1 depletion significantly impairs tumor growth in triple-negative breast cancer (TNBC). Thus, CIB1 is a potentially attractive target for cancer chemotherapy that has yet to be validated by a chemical probe. To produce a probe molecule to the CIB1 helix 10 (H10) pocket and demonstrate that it is a viable target for molecular intervention, we employed random peptide phage display to screen and select CIB1-binding peptides. The top peptide sequence selected, UNC10245092, was produced synthetically, and binding to CIB1 was confirmed by isothermal titration calorimetry (ITC) and a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. Both assays showed that the peptide bound to CIB1 with low nanomolar affinity. CIB1 was cocrystallized with UNC10245092, and the 2.1 Å resolution structure revealed that the peptide binds as an α-helix in the H10 pocket, displacing the CIB1 C-terminal H10 helix and causing conformational changes in H7 and H8. UNC10245092 was further derivatized with a C-terminal Tat-derived cell penetrating peptide (CPP) to demonstrate its effects on TNBC cells in culture, which are consistent with results of CIB1 depletion. These studies provide a first-in-class chemical tool for CIB1 inhibition in cell culture and validate the CIB1 H10 pocket for future probe and drug discovery efforts.

Structure and dynamics of Trichoderma harzianum Cel7B suggest molecular architecture adaptations required for a wide spectrum of activities on plant cell wall polysaccharides.

Cellulases from glycoside hydrolase family 7 (GH7) play crucial roles in plant lignocellulose deconstruction by fungi, but structural information available for GH7 fungal endoglucanases is limited when compared to the number of known sequences in the family. Here, we report the X-ray structure of the glycosylated catalytic domain (CD) of Trichoderma harzianum endoglucanase, ThCel7B, solved and refined at 2.9 Šresolution. Additionally, our extensive molecular dynamics simulations of this enzyme in complex with a variety of oligosaccharides provide a better understanding of its promiscuous hydrolytic activities on plant cell wall polysaccharides. The simulations demonstrate the importance of the hydrogen bond between substrate O2 hydroxyl in the subsite -1 and a side chain of catalytic Glu196 which renders ThCel7B capable to catalytically cleave cello and xylooligosaccharides, but not mannooligosaccharides. Moreover, detailed structural analyses and MD simulations revealed an additional binding pocket, suitable for accommodation of oligosaccharide decorations and/or substrates with mixed glycoside bonds that abuts onto the binding cleft close to subsite +2.

Structure, computational and biochemical analysis of PcCel45A endoglucanase from Phanerochaete chrysosporium and catalytic mechanisms of GH45 subfamily C members.

The glycoside hydrolase family 45 (GH45) of carbohydrate modifying enzymes is mostly comprised of ß-1,4-endoglucanases. Significant diversity between the GH45 members has prompted the division of this family into three subfamilies: A, B and C, which may differ in terms of the mechanism, general architecture, substrate binding and cleavage. Here, we use a combination of X-ray crystallography, bioinformatics, enzymatic assays, molecular dynamics simulations and site-directed mutagenesis experiments to characterize the structure, substrate binding and enzymatic specificity of the GH45 subfamily C endoglucanase from Phanerochaete chrysosporium (PcCel45A). We investigated the role played by different residues in the binding of the enzyme to cellulose oligomers of different lengths and examined the structural characteristics and dynamics of PcCel45A that make subfamily C so dissimilar to other members of the GH45 family. Due to the structural similarity shared between PcCel45A and domain I of expansins, comparative analysis of their substrate binding was also carried out. Our bioinformatics sequence analyses revealed that the hydrolysis mechanisms in GH45 subfamily C is not restricted to use of the imidic asparagine as a general base in the "Newton's cradle" catalytic mechanism recently proposed for this subfamily.

Schistosoma mansoni venom allergen-like protein 18 (SmVAL18) is a plasminogen-binding protein secreted during the early stages of mammalian-host infection.

Schistosomiasis is a neglected tropical disease caused by trematodes of the genus Schistosoma which have a complex life cycle characterized by an asexual multiplication phase in the snail intermediate host and a sexual reproduction phase in the mammalian definitive host. The initial steps of the human host infection involve the secretion of proteins contained in the acetabular glands of cercariae that promote parasite adhesion and proteolysis of the skin layers. Herein, we performed a functional analysis of SmVAL18, identified as one of the three SCP/TAPS proteins constituent of cercarial secretions. We evaluated the SmVAL18 binding to immobilized macromolecules of the extracellular matrix (ECM) and to plasma components. Recombinant protein, expressed in E. coli, was found to maintain an ordered secondary structure typical of the SCP/TAPS domain after purification. Expression of native SmVAL18 protein was verified to be restricted to cercariae and 3-h schistosomula stages; furthermore, the protein was observed in the corresponding secretions, confirming that SmVAL18 is secreted during the first 3 h of in vitro culture. rSmVAL18 was able to interact specifically with plasminogen (PLG) and enhance its conversion into plasmin in the presence of the urokinase-type plasminogen activator (uPA). Protein homology modelling suggested that the PLG-rSmVAL18 interaction was mediated by lysine residues of the protein. This was supported by in vitro data using the lysine analogue, 6-aminocaproic acid (ACA), which abolished the interaction. Finally, our results showed that both cercariae and 3-h schistosomula, as well as their corresponding secretions, exhibited the capacity to bind PLG and enhance its conversion into plasmin in vitro in the same way as observed for the recombinant protein. In conclusion, our findings show that SmVAL18 is a novel PLG-binding protein secreted during the early stages of the mammalian-host infection.

Crystal structure of Zika virus NS5 RNA-dependent RNA polymerase.

The current Zika virus (ZIKV) outbreak became a global health threat of complex epidemiology and devastating neurological impacts, therefore requiring urgent efforts towards the development of novel efficacious and safe antiviral drugs. Due to its central role in RNA viral replication, the non-structural protein 5 (NS5) RNA-dependent RNA-polymerase (RdRp) is a prime target for drug discovery. Here we describe the crystal structure of the recombinant ZIKV NS5 RdRp domain at 1.9 Å resolution as a platform for structure-based drug design strategy. The overall structure is similar to other flaviviral homologues. However, the priming loop target site, which is suitable for non-nucleoside polymerase inhibitor design, shows significant differences in comparison with the dengue virus structures, including a tighter pocket and a modified local charge distribution.

Crystal structure of a putative exo-ß-1,3-galactanase from Bifidobacterium bifidum S17.

Given the current interest in second-generation biofuels, carbohydrate-active enzymes have become the most important tool to overcome the structural recalcitrance of the plant cell wall. While some glycoside hydrolase families have been exhaustively described, others remain poorly characterized, especially with regard to structural information. The family 43 glycoside hydrolases are a diverse group of inverting enzymes; the available structure information on these enzymes is mainly from xylosidases and arabinofuranosidase. Currently, only one structure of an exo-ß-1,3-galactanase is available. Here, the production, crystallization and structure determination of a putative exo-ß-1,3-galactanase from Bifidobacterium bifidum S17 (BbGal43A) are described. BbGal43A was successfully produced and showed activity towards synthetic galactosides. BbGal43A was subsequently crystallized and data were collected to 1.4 Šresolution. The structure shows a single-domain molecule, differing from known homologues, and crystal contact analysis predicts the formation of a dimer in solution. Further biochemical studies are necessary to elucidate the differences between BbGal43A and its characterized homologues.

Recombinant Trichoderma harzianum endoglucanase I (Cel7B) is a highly acidic and promiscuous carbohydrate-active enzyme.

Trichoderma filamentous fungi have been investigated due to their ability to secrete cellulases which find various biotechnological applications such as biomass hydrolysis and cellulosic ethanol production. Previous studies demonstrated that Trichoderma harzianum IOC-3844 has a high degree of cellulolytic activity and potential for biomass hydrolysis. However, enzymatic, biochemical, and structural studies of cellulases from T. harzianum are scarce. This work reports biochemical characterization of the recombinant endoglucanase I from T. harzianum, ThCel7B, and its catalytic core domain. The constructs display optimum activity at 55 °C and a surprisingly acidic pH optimum of 3.0. The full-length enzyme is able to hydrolyze a variety of substrates, with high specific activity: 75 U/mg for ß-glucan, 46 U/mg toward xyloglucan, 39 U/mg for lichenan, 26 U/mg for carboxymethyl cellulose, 18 U/mg for 4-nitrophenyl ß-D-cellobioside, 16 U/mg for rye arabinoxylan, and 12 U/mg toward xylan. The enzyme also hydrolyzed filter paper, phosphoric acid swollen cellulose, Sigmacell 20, Avicel PH-101, and cellulose, albeit with lower efficiency. The ThCel7B catalytic domain displays similar substrate diversity. Fluorescence-based thermal shift assays showed that thermal stability is highest at pH 5.0. We determined kinetic parameters and analyzed a pattern of oligosaccharide substrates hydrolysis, revealing cellobiose as a final product of C6 degradation. Finally, we visualized effects of ThCel7B on oat spelt using scanning electron microscopy, demonstrating the morphological changes of the substrate during the hydrolysis. The acidic behavior of ThCel7B and its considerable thermostability hold a promise of its industrial applications and other biotechnological uses under extremely acidic conditions.

Molecular mechanism of peroxisome proliferator-activated receptor α activation by WY14643: a new mode of ligand recognition and receptor stabilization.

Peroxisome proliferator-activated receptors (PPARs) are members of a superfamily of nuclear transcription factors. They are involved in mediating numerous physiological effects in humans, including glucose and lipid metabolism. PPARα ligands effectively treat dyslipidemia and have significant antiinflammatory and anti-atherosclerotic activities. These effects and their ligand-dependent activity make nuclear receptors obvious targets for drug design. Here, we present the structure of the human PPARα in complex with WY14643, a member of fibrate class of drug, and a widely used PPAR activator. The crystal structure of this complex suggests that WY14643 induces activation of PPARα in an unusual bipartite mechanism involving conventional direct helix 12 stabilization and an alternative mode that involves a second ligand in the pocket. We present structural observations, molecular dynamics and activity assays that support the importance of the second site in WY14643 action. The unique binding mode of WY14643 reveals a new pattern of nuclear receptor ligand recognition and suggests a novel basis for ligand design, offering clues for improving the binding affinity and selectivity of ligand. We show that binding of WY14643 to PPARα was associated with antiinflammatory disease in a human corneal cell model, suggesting possible applications for PPARα ligands.