Schistosomiasis, caused by a parasitic blood fluke of the genus Schistosoma, is a global health problem for which new chemotherapeutic options are needed. We explored the scaffold of gallinamide A, a natural peptidic metabolite of marine cyanobacteria that has previously been shown to inhibit cathepsin L-type proteases. We screened a library of 19 synthetic gallinamide A analogs and identified nanomolar inhibitors of the cathepsin B-type protease SmCB1, which is a drug target for the treatment of schistosomiasis mansoni. Against cultured S. mansoni schistosomula and adult worms, many of the gallinamides generated a range of deleterious phenotypic responses. Imaging with a fluorescent-activity-based probe derived from gallinamide A demonstrated that SmCB1 is the primary target for gallinamides in the parasite. Furthermore, we solved the high-resolution crystal structures of SmCB1 in complex with gallinamide A and its two analogs and describe the acrylamide covalent warhead and binding mode in the active site. Quantum chemical calculations evaluated the contribution of individual positions in the peptidomimetic scaffold to the inhibition of the target and demonstrated the importance of the P1' and P2 positions. Our study introduces gallinamides as a powerful chemotype that can be exploited for the development of novel antischistosomal chemotherapeutics.

• Keywords
• Schistosoma mansoni, acrylamide inhibitor, cathepsin B, cysteine protease, drug target, parasite,
• MeSH
• Cathepsin B * antagonists & inhibitors   metabolism   MeSH
• Crystallography, X-Ray MeSH
• Models, Molecular MeSH
• Schistosoma mansoni * enzymology   drug effects   MeSH
• Schistosomicides pharmacology   chemistry   MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cathepsin B * MeSH
• Schistosomicides MeSH

A collaborative, open-science team undertook discovery of novel small molecule inhibitors of the SARS-CoV-2 nsp16-nsp10 2'-O-methyltransferase using a high throughput screening approach with the potential to reveal new inhibition strategies. This screen yielded compound 5a, a ligand possessing an electron-deficient double bond, as an inhibitor of SARS-CoV-2 nsp16 activity. Surprisingly, X-ray crystal structures revealed that 5a covalently binds within a previously unrecognized cryptic pocket near the S-adenosylmethionine binding cleft in a manner that prevents occupation by S-adenosylmethionine. Using a multidisciplinary approach, we examined the mechanism of binding of compound 5a to the nsp16 cryptic pocket and developed 5a derivatives that inhibited nsp16 activity and murine hepatitis virus replication in rat lung epithelial cells but proved cytotoxic to cell lines canonically used to examine SARS-CoV-2 infection. Our study reveals the druggability of this newly discovered SARS-CoV-2 nsp16 cryptic pocket, provides novel tool compounds to explore the site, and suggests a new approach for discovery of nsp16 inhibition-based pan-coronavirus therapeutics through structure-guided drug design.

• Keywords
• antiviral, coronavirus, covalent inhibitors, nsp16 methyltransferase, structural biology,
• MeSH
• COVID-19 * MeSH
• Rats MeSH
• Methyltransferases MeSH
• Mice MeSH
• S-Adenosylmethionine chemistry   metabolism   MeSH
• SARS-CoV-2 * metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Methyltransferases MeSH
• S-Adenosylmethionine MeSH

Schistosomiasis, a parasitic disease caused by blood flukes of the genus Schistosoma, is a global health problem with over 200 million people infected. Treatment relies on just one drug, and new chemotherapies are needed. Schistosoma mansoni cathepsin B1 (SmCB1) is a critical peptidase for the digestion of host blood proteins and a validated drug target. We screened a library of peptidomimetic vinyl sulfones against SmCB1 and identified the most potent SmCB1 inhibitors reported to date that are active in the subnanomolar range with second order rate constants (k2nd) of ∼2 × 105 M-1 s-1. High resolution crystal structures of the two best inhibitors in complex with SmCB1 were determined. Quantum chemical calculations of their respective binding modes identified critical hot spot interactions in the S1' and S2 subsites. The most potent inhibitor targets the S1' subsite with an N-hydroxysulfonic amide moiety and displays favorable functional properties, including bioactivity against the pathogen, selectivity for SmCB1 over human cathepsin B, and reasonable metabolic stability. Our results provide structural insights for the rational design of next-generation SmCB1 inhibitors as potential drugs to treat schistosomiasis.

• Keywords
• Schistosoma mansoni, cathepsin B, cysteine peptidase, drug target, parasite, vinyl sulfone inhibitor,
• MeSH
• Cathepsin B * MeSH
• Humans MeSH
• Schistosoma mansoni MeSH
• Schistosomiasis * drug therapy   MeSH
• Sulfones pharmacology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• divinyl sulfone MeSH Browser
• Cathepsin B * MeSH
• Sulfones MeSH

Azapeptide nitriles are postulated to reversibly covalently react with the active-site cysteine residue of cysteine proteases and form isothiosemicarbazide adducts. We investigated the interaction of azadipeptide nitriles with the cathepsin B1 drug target (SmCB1) from Schistosoma mansoni, a pathogen that causes the global neglected disease schistosomiasis. Azadipeptide nitriles were superior inhibitors of SmCB1 over their parent carba analogs. We determined the crystal structure of SmCB1 in complex with an azadipeptide nitrile and analyzed the reaction mechanism using quantum chemical calculations. The data demonstrate that azadipeptide nitriles, in contrast to their carba counterparts, undergo a change from E- to Z-configuration upon binding, which gives rise to a highly favorable energy profile of noncovalent and covalent complex formation. Finally, azadipeptide nitriles were considerably more lethal than their carba analogs against the schistosome pathogen in culture, supporting the further development of this chemotype as a treatment for schistosomiasis.

• Keywords
• azapeptide inhibitors, cysteine proteases, protein structures, schistosomiasis, structure−activity relationships,
• MeSH
• Cathepsin B MeSH
• Peptide Hydrolases * MeSH
• Schistosoma mansoni * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Cathepsin B MeSH
• Peptide Hydrolases * MeSH

BACKGROUND: Serine proteases are important virulence factors for many pathogens. Recently, we discovered a group of trypsin-like serine proteases with domain organization unique to flatworm parasites and containing a thrombospondin type 1 repeat (TSR-1). These proteases are recognized as antigens during host infection and may prove useful as anthelminthic vaccines, however their molecular characteristics are under-studied. Here, we characterize the structural and proteolytic attributes of serine protease 2 (SmSP2) from Schistosoma mansoni, one of the major species responsible for the tropical infectious disease, schistosomiasis. METHODOLOGY/PRINCIPAL FINDINGS: SmSP2 comprises three domains: a histidine stretch, TSR-1 and a serine protease domain. The cleavage specificity of recombinant SmSP2 was determined using positional scanning and multiplex combinatorial libraries and the determinants of specificity were identified with 3D homology models, demonstrating a trypsin-like endopeptidase mode of action. SmSP2 displayed restricted proteolysis on protein substrates. It activated tissue plasminogen activator and plasminogen as key components of the fibrinolytic system, and released the vasoregulatory peptide, kinin, from kininogen. SmSP2 was detected in the surface tegument, esophageal glands and reproductive organs of the adult parasite by immunofluorescence microscopy, and in the excretory/secretory products by immunoblotting. CONCLUSIONS/SIGNIFICANCE: The data suggest that SmSP2 is secreted, functions at the host-parasite interface and contributes to the survival of the parasite by manipulating host vasodilatation and fibrinolysis. SmSP2 may be, therefore, a potential target for anti-schistosomal therapy.

• MeSH
• Fibrinolysis drug effects   MeSH
• Blood Coagulation drug effects   MeSH
• Hemostatics antagonists & inhibitors   MeSH
• Blood Pressure drug effects   MeSH
• Models, Molecular MeSH
• Plasminogen drug effects   MeSH
• Protein Domains MeSH
• Helminth Proteins chemistry   genetics   pharmacology   MeSH
• Proteolysis drug effects   MeSH
• Recombinant Proteins MeSH
• Schistosoma mansoni enzymology   MeSH
• Schistosomiasis mansoni parasitology   MeSH
• Amino Acid Sequence MeSH
• Serine Endopeptidases chemistry   genetics   pharmacology   MeSH
• Tissue Plasminogen Activator drug effects   MeSH
• Vasodilation drug effects   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Hemostatics MeSH
• Plasminogen MeSH
• Helminth Proteins MeSH
• Recombinant Proteins MeSH
• Serine Endopeptidases MeSH
• Tissue Plasminogen Activator MeSH
• trypsin-like serine protease MeSH Browser

General and reliable description of structures and energetics in protein-ligand (PL) binding using the docking/scoring methodology has until now been elusive. We address this urgent deficiency of scoring functions (SFs) by the systematic development of corrected semiempirical quantum mechanical (SQM) methods, which correctly describe all types of noncovalent interactions and are fast enough to treat systems of thousands of atoms. Two most accurate SQM methods, PM6-D3H4X and SCC-DFTB3-D3H4X, are coupled with the conductor-like screening model (COSMO) implicit solvation model in so-called "SQM/COSMO" SFs and have shown unique recognition of native ligand poses in cognate docking in four challenging PL systems, including metalloprotein. Here, we apply the two SQM/COSMO SFs to 17 diverse PL complexes and compare their performance with four widely used classical SFs (Glide XP, AutoDock4, AutoDock Vina, and UCSF Dock). We observe superior performance of the SQM/COSMO SFs and identify challenging systems. This method, due to its generality, comparability across the chemical space, and lack of need for any system-specific parameters, gives promise of becoming, after comprehensive large-scale testing in the near future, a useful computational tool in structure-based drug design and serving as a reference method for the development of other SFs.

• Publication type
• Journal Article MeSH

BACKGROUND: Blood flukes of the genus Schistosoma cause schistosomiasis, a parasitic disease that infects over 240 million people worldwide, and for which there is a need to identify new targets for chemotherapeutic interventions. Our research is focused on Schistosoma mansoni prolyl oligopeptidase (SmPOP) from the serine peptidase family S9, which has not been investigated in detail in trematodes. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that SmPOP is expressed in adult worms and schistosomula in an enzymatically active form. By immunofluorescence microscopy, SmPOP is localized in the tegument and parenchyma of both developmental stages. Recombinant SmPOP was produced in Escherichia coli and its active site specificity investigated using synthetic substrate and inhibitor libraries, and by homology modeling. SmPOP is a true oligopeptidase that hydrolyzes peptide (but not protein) substrates with a strict specificity for Pro at P1. The inhibition profile is analogous to those for mammalian POPs. Both the recombinant enzyme and live worms cleave host vasoregulatory, proline-containing hormones such as angiotensin I and bradykinin. Finally, we designed nanomolar inhibitors of SmPOP that induce deleterious phenotypes in cultured schistosomes. CONCLUSIONS/SIGNIFICANCE: We provide the first localization and functional analysis of SmPOP together with chemical tools for measuring its activity. We briefly discuss the notion that SmPOP, operating at the host-parasite interface to cleave host bioactive peptides, may contribute to the survival of the parasite. If substantiated, SmPOP could be a new target for the development of anti-schistosomal drugs.

• MeSH
• DNA Primers genetics   MeSH
• Escherichia coli MeSH
• Microscopy, Fluorescence MeSH
• Hydrolysis MeSH
• Immunoblotting MeSH
• Catalytic Domain genetics   MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Models, Molecular * MeSH
• Prolyl Oligopeptidases MeSH
• Gene Expression Regulation, Enzymologic physiology   MeSH
• Recombinant Proteins genetics   metabolism   MeSH
• Schistosoma mansoni enzymology   MeSH
• Serine Endopeptidases genetics   metabolism   MeSH
• Gene Expression Profiling MeSH
• Substrate Specificity MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Primers MeSH
• Prolyl Oligopeptidases MeSH
• Recombinant Proteins MeSH
• Serine Endopeptidases MeSH

Carborane-based compounds are promising lead structures for development of inhibitors of carbonic anhydrases (CAs). Here, we report structural and computational analysis applicable to structure-based design of carborane compounds with selectivity toward the cancer-specific CAIX isoenzyme. We determined the crystal structure of CAII in complex with 1-methylenesulfamide-1,2-dicarba-closo-dodecaborane at 1.0 Å resolution and used this structure to model the 1-methylenesulfamide-1,2-dicarba-closo-dodecaborane interactions with CAIX. A virtual glycine scan revealed the contributions of individual residues to the energy of binding of 1-methylenesulfamide-1,2-dicarba-closo-dodecaborane to CAII and CAIX, respectively.

• MeSH
• Glycine chemistry   MeSH
• Carbonic Anhydrase Inhibitors chemistry   pharmacology   MeSH
• Carbonic Anhydrases chemistry   MeSH
• Catalytic Domain MeSH
• Crystallography, X-Ray MeSH
• Quantum Theory * MeSH
• Humans MeSH
• Models, Molecular * MeSH
• Boron Compounds chemistry   pharmacology   MeSH
• Substrate Specificity drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• dodecaborate MeSH Browser
• Glycine MeSH
• Carbonic Anhydrase Inhibitors MeSH
• Carbonic Anhydrases MeSH
• Boron Compounds MeSH