Optimized small-molecule 3C-like protease inhibitors show potency against human coronaviruses in both cell culture and a mouse model.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-related CoVs encode 3 tandem macrodomains within nonstructural protein (nsp3). The first macrodomain, Mac1, is conserved throughout binds to hydrolyzes mono-ADP-ribose (MAR) from target proteins. Mac1 likely counters host-mediated antiviral ADP-ribosylation, a posttranslational modification that part of the host response viral infections. essential for pathogenesis in multiple animal models CoV infection, implicating...

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to be a serious global public health threat. The 3C-like protease (3CLpro) is virus encoded by SARS-CoV-2, which essential for replication. We have previously reported series of small-molecule 3CLpro inhibitors effective inhibiting replication human coronaviruses including SARS-CoV-2 in cell culture and animal models. Here we generated deuterated variants inhibitor, GC376, evaluated the antiviral effect against...

A series of nondeuterated and deuterated dipeptidyl aldehyde masked inhibitors that incorporate in their structure a conformationally constrained cyclohexane moiety was synthesized found to potently inhibit severe acute respiratory syndrome coronavirus-2 3CL protease biochemical cell-based assays. Several the were also be nanomolar Middle East coronavirus protease. The corresponding latent bisulfite adducts equipotent precursor aldehydes. High-resolution cocrystal structures confirmed...

The worldwide impact of the ongoing COVID-19 pandemic on public health has made imperative discovery and development direct-acting antivirals aimed at targeting viral and/or host targets. SARS-CoV-2 3C-like protease (3CLpro) emerged as a validated target for therapeutics because pivotal role it plays in replication. We describe herein structure-guided design highly potent inhibitors 3CLpro that incorporate their structure novel spirocyclic elements optimizing potency by accessing new...

The COVID-19 pandemic is having a major impact on public health worldwide, and there an urgent need for the creation of armamentarium effective therapeutics, including vaccines, biologics, small-molecule to combat SARS-CoV-2 emerging variants. Inspection virus life cycle reveals multiple viral- host-based choke points that can be exploited virus. 3C-like protease (3CLpro), enzyme essential viral replication, attractive target therapeutic intervention, design inhibitors may lead emergence...

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-like-CoVs encode 3 tandem macrodomains within non-structural protein (nsp3). The first macrodomain, Mac1, is conserved throughout CoVs, binds to hydrolyzes mono-ADP-ribose (MAR) from target proteins. Mac1 likely counters host-mediated anti-viral ADP-ribosylation, a posttranslational modification that part of the host response viral infections. essential for pathogenesis in multiple animal models CoV...

Abstract Inhibition of the proteolytic processing hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP) is an attractive approach for drug discovery novel anticancer therapeutics which prevent tumor progression metastasis. Here, we utilized improved expanded version positional scanning substrate combinatorial libraries (PS‐SCL) technique called HyCoSuL to optimize peptidomimetic inhibitors HGF/MSP activating serine proteases, HGFA, matriptase, hepsin. These have...

RNA macromolecules, like proteins, fold to assume shapes that are intimately connected their broadly recognized biological functions; however, because of high charge and dynamic nature, structures far more challenging determine. We introduce an approach exploits the brilliance x-ray free-electron laser sources reveal formation ready identification angstrom-scale features in structured unstructured RNAs. Previously unrecognized structural signatures secondary tertiary identified through...

The advent of SARS-CoV-2, the causative agent COVID-19, and its worldwide impact on global health, have provided impetus for development effective countermeasures that can be deployed against virus, including vaccines, monoclonal antibodies, direct-acting antivirals (DAAs). Despite these efforts, current paucity DAAs has created an urgent need creation enhanced diversified portfolio broadly acting agents with different mechanisms action effectively abrogate viral infection. SARS-CoV-2...

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to be a serious global public health threat. The 3C-like protease (3CLpro) is virus encoded by SARS-CoV-2, which essential for replication. We have previously reported series of small molecule 3CLpro inhibitors effective inhibiting replication human coronaviruses including SARS-CoV-2 in cell culture and animal models. Here we generated deuterated variants inhibitor, GC376, evaluated the antiviral effect...

We report the structural, biochemical, and functional characterization of product gene PA0962 from Pseudomonas aeruginosa PAO1. The protein, termed Pa Dps, adopts Dps subunit fold oligomerizes into a nearly spherical 12-mer quaternary structure at pH 6.0 or in presence divalent cations neutral above. 12-Mer contains two di-iron centers interface each dimer, coordinated by conserved His, Glu, Asp residues. In vitro, catalyze oxidation Fe2+ utilizing H2O2 (not O2) as an oxidant, suggesting...

Protease inhibitor drug discovery is challenged by the lack of cellular and oral permeability, selectivity, metabolic stability, rapid clearance peptides. Here, we describe rational design, synthesis, evaluation peptidomimetic side-chain-cyclized macrocycles which converted into covalent serine protease inhibitors with addition an electrophilic ketone warhead. We have identified potent selective TMPRSS2, matriptase, hepsin, HGFA demonstrated their improved pharmacokinetic (PK) properties....

ABSTRACT In diderm bacteria, the Lol pathway canonically mediates periplasmic transport of lipoproteins from inner membrane (IM) to outer (OM) and therefore plays an essential role in bacterial envelope homeostasis. After extrusion modified IM via LolCDE complex, chaperone LolA carries through periplasm transfers them OM lipoprotein insertase LolB, itself a with LolA-like fold. Yet, LolB homologs appear restricted ψ-proteobacteria are missing spirochetes like tick-borne Lyme disease pathogen...

Acute gastroenteritis caused by noroviruses has a major impact on public health worldwide in terms of morbidity, mortality, and economic burden. The disease impacts most severely immunocompromised patients, the elderly, children. current lack approved vaccines small-molecule therapeutics for treatment prophylaxis norovirus infections underscores need development norovirus-specific drugs. studies described herein entail use gem-dimethyl moiety as means improving pharmacological activity...

Protonation of key histidine residues has been long implicated in the acid-mediated cellular action diphtheria toxin translocation (T-) domain, responsible for delivery catalytic domain into cell. Here, we use a combination computational (constant-pH Molecular Dynamics simulations) and experimental (NMR, circular dichroism, fluorescence spectroscopy along with X-ray crystallography) approaches to characterize initial stages conformational change happening solution wild-type T-domain...

Abstract Transferrins function in iron sequestration and transport by binding tightly reversibly. Vertebrate transferrins coordinate through interactions with two tyrosines, an aspartate, a histidine, carbonate anion, conformational changes that occur upon release have been described. Much less is known about the structure functions of insect transferrin‐1 (Tsf1), which present hemolymph influences homeostasis mostly unknown mechanisms. Amino acid sequence biochemical analyses suggested...

Mutations in two different domains of the ubiquitously expressed TRIM32 protein give rise to clinically separate diseases, one which is Limb-girdle muscular dystrophy type 2H (LGMD2H). Uncovering muscle-specific role LGMD2H pathogenesis has proven difficult, as neurogenic phenotypes, independent pathology, are present KO mice. We previously established a platform study using Drosophila melanogaster model. Here we show that disease-causing mutations NHL domain molecularly and structurally...

Musashi-2 (MSI2) belongs to Musashi family of RNA binding proteins (RBP). Like Musashi-1 (MSI1), it is overexpressed in a variety cancers and promising therapeutic target. Both MSI contain two N-terminal recognition motifs play roles posttranscriptional regulation target mRNAs. Previously, we have identified several inhibitors MSI1, all which bind MSI2 as well. In order design MSI2-specific compare the differences mode inhibitors, set out solve structure MSI2-RRM1, key motif that responsible...

Diphtheria toxin, an exotoxin secreted by Corynebacterium that causes disease in humans inhibiting protein synthesis, enters the cell via receptor-mediated endocytosis. The subsequent endosomal acidification triggers a series of conformational changes, resulting refolding and membrane insertion translocation (T-)domain ultimately leading to catalytic domain into cytoplasm. Here, we use X-ray crystallography along with circular dichroism fluorescence spectroscopy gain insight mechanism early...

Catalysis of human phosphoglycerate mutase is dependent on a 2,3-bisphosphoglycerate cofactor (dPGM), whereas the nonhomologous isozyme in many parasitic species independent (iPGM). This mechanistic and phylogenetic diversity offers an opportunity for selective pharmacologic targeting glycolysis disease-causing organisms. We previously discovered ipglycermide, potent inhibitor iPGM, from large combinatorial cyclic peptide library. To fully delineate ipglycermide pharmacophore, herein we...

Abstract NADH cytochrome b 5 oxidoreductase (Ncb5or) is a cytosolic ferric reductase implicated in diabetes and neurological conditions. Ncb5or comprises ( ) R) domains separated by CHORD‐Sgt1 (CS) linker domain. redox activity depends on proper inter‐domain interactions to mediate electron transfer from or NADPH via FAD heme. While full‐length human has proven resistant crystallization, we have succeeded obtaining high‐resolution atomic structures of the domain construct containing CS R...

Herein the crystal structure of VapBC-1 complex from nontypeable Haemophilus influenzae (NTHi) is described. Our results show that some mutations in PIN domain VapC-1 toxin were associated with decreased toxicity E. coli , but mutants retained ability to homodimerize and heterodimerize wild-type cognate antitoxin, VapB-1. A new system was designed constructed quantify effects these on NTHi survival during infections primary human tissues ex vivo . Any mutation a conserved amino acid...

Abstract Gram-positive bacteria utilize a Fatty Acid Kinase (FAK) complex to harvest fatty acids from the environment. The complex, consisting of acid kinase, FakA, and an acyl carrier protein, FakB, is known impact virulence disease outcomes. However, FAK’s structure enzymatic mechanism remain poorly understood. Here, we used combination modeling, biochemical, cell-based approaches establish critical details FAK activity. Solved structures apo ligand-bound FakA kinase domain captured...