Macrocyclic inhibitors of 3C and 3C-like proteases of picornavirus, norovirus, and coronavirus.

The design, synthesis, and in vitro evaluation of the first macrocyclic inhibitor of 3C and 3C-like proteases of picornavirus, norovirus, and coronavirus are reported. The in vitro inhibitory activity (50% effective concentration) of the macrocyclic inhibitor toward enterovirus 3C protease (CVB3 Nancy strain), and coronavirus (SARS-CoV) and norovirus 3C-like proteases, was determined to be 1.8, 15.5 and 5.1 µM, respectively.

Potent inhibition of norovirus by dipeptidyl α-hydroxyphosphonate transition state mimics.

The design, synthesis, and evaluation of a series of dipeptidyl α-hydroxyphosphonates is reported. The synthesized compounds displayed high anti-norovirus activity in a cell-based replicon system, as well as high enzyme selectivity.

Design, synthesis and characterization of novel 1,2-benzisothiazol-3(2H)-one and 1,3,4-oxadiazole hybrid derivatives: potent inhibitors of Dengue and West Nile virus NS2B/NS3 proteases.

1,2-Benzisothiazol-3(2H)-ones and 1,3,4-oxadiazoles individually have recently attracted considerable interest in drug discovery, including as antibacterial and antifungal agents. In this study, a series of functionalized 1,2-benzisothiazol-3(2H)-one-1,3,4-oxadiazole hybrid derivatives were synthesized and subsequently screened against Dengue and West Nile virus proteases. Ten out of twenty-four compounds showed greater than 50% inhibition against DENV2 and WNV proteases ([I] = 10 µM). The IC(50) values of compound 7n against DENV2 and WNV NS2B/NS3 were found to be 3.75 ± 0.06 and 4.22 ± 0.07 µM, respectively. The kinetics data support a competitive mode of inhibition by compound 7n. Molecular modeling studies were performed to delineate the putative binding mode of this series of compounds. This study reveals that the hybrid series arising from the linking of the two scaffolds provides a suitable platform for conducting a hit-to-lead optimization campaign via iterative structure-activity relationship studies, in vitro screening and X-ray crystallography.

Bacterial Proteases as Potentially Exploitable Modulators of SARS-CoV-2 Infection: Logic from the Literature, Informatics, and Inspiration from the Dog.

(1) Background: The COVID-19 pandemic left many intriguing mysteries. Retrospective vulnerability trends tie as strongly to odd demographics as to exposure profiles, genetics, health, or prior medical history. This article documents the importance of nasal microbiome profiles in distinguishing infection rate trends among differentially affected subgroups. (2) Hypothesis: From a detailed literature survey, microbiome profiling experiments, bioinformatics, and molecular simulations, we propose that specific commensal bacterial species in the Pseudomonadales genus confer protection against SARS-CoV-2 infections by expressing proteases that may interfere with the proteolytic priming of the Spike protein. (3) Evidence: Various reports have found elevated Moraxella fractions in the nasal microbiomes of subpopulations with higher resistance to COVID-19 (e.g., adolescents, COVID-19-resistant children, people with strong dietary diversity, and omnivorous canines) and less abundant ones in vulnerable subsets (the elderly, people with narrower diets, carnivorous cats and foxes), along with bioinformatic evidence that Moraxella bacteria express proteases with notable homology to human TMPRSS2. Simulations suggest that these proteases may proteolyze the SARS-CoV-2 spike protein in a manner that interferes with TMPRSS2 priming.

Tracking of Systemic Lupus Erythematosus (SLE) Longitudinally Using Biosensor and Patient-Reported Data: A Report on the Fully Decentralized Mobile Study to Measure and Predict Lupus Disease Activity Using Digital Signals-The OASIS Study.

(1) Objective: Systemic lupus erythematosus (SLE) is a complex disease involving immune dysregulation, episodic flares, and poor quality of life (QOL). For a decentralized digital study of SLE patients, machine learning was used to assess patient-reported outcomes (PROs), QOL, and biometric data for predicting possible disease flares. (2) Methods: Participants were recruited from the LupusCorner online community. Adults self-reporting an SLE diagnosis were consented and given a mobile application to record patient profile (PP), PRO, and QOL metrics, and enlisted participants received smartwatches for digital biometric monitoring. The resulting data were profiled using feature selection and classification algorithms. (3) Results: 550 participants completed digital surveys, 144 (26%) agreed to wear smartwatches, and medical records (MRs) were obtained for 68. Mining of PP, PRO, QOL, and biometric data yielded a 26-feature model for classifying participants according to MR-identified disease flare risk. ROC curves significantly distinguished true from false positives (ten-fold cross-validation: p < 0.00023; five-fold: p < 0.00022). A 25-feature Bayesian model enabled time-variant prediction of participant-reported possible flares (P(true) > 0.85, p < 0.001; P(nonflare) > 0.83, p < 0.0001). (4) Conclusions: Regular profiling of patient well-being and biometric activity may support proactive screening for circumstances warranting clinical assessment.

Targeting of histone acetyltransferase p300 by cyclopentenone prostaglandin Δ(12)-PGJ(2) through covalent binding to Cys(1438).

Inhibitors of histone acetyltransferases (HATs) are perceived to treat diseases like cancer, neurodegeneration, and AIDS. On the basis of previous studies, we hypothesized that Cys(1438) in the substrate binding site could be targeted by Δ(12)-prostaglandin J(2) (Δ(12)-PGJ(2)), a cyclopentenone prostaglandin (CyPG) derived from PGD(2). We demonstrate here the ability of CyPGs to inhibit p300 HAT-dependent acetylation of histone H3. A cell-based assay system clearly showed that the α,ß-unsaturation in the cyclopentenone ring of Δ(12)-PGJ(2) was crucial for the inhibitory activity, while the 9,10-dihydro-15-deoxy-Δ(12,14)-PGJ(2), which lacks the electrophilic carbon (at carbon 9), was ineffective. Molecular docking studies suggested that Δ(12)-PGJ(2) places the electrophilic carbon in the cyclopentenone ring well within the vicinity of Cys(1438) of p300 to form a covalent Michael adduct. Site-directed mutagenesis of the p300 HAT domain, peptide competition assay involving p300 wild type and mutant peptides, followed by mass spectrometric analysis confirmed the covalent interaction of Δ(12)-PGJ(2) with Cys(1438). Using biotinylated derivatives of Δ(12)-PGJ(2) and 9,10-dihydro-15-deoxy-Δ(12,14)-PGJ(2), we demonstrate the covalent interaction of Δ(12)-PGJ(2) with the p300 HAT domain, but not the latter. In agreement with the in vitro filter binding assay, CyPGs were also found to inhibit H3 histone acetylation in cell-based assays. In addition, Δ(12)-PGJ(2) also inhibited the acetylation of the HIV-1 Tat by recombinant p300 in in vitro assays. This study demonstrates, for the first time, that Δ(12)-PGJ(2) inhibits p300 through Michael addition, where α,ß-unsaturated carbonyl function is absolutely required for the inhibitory activity.

Potent inhibition of norovirus 3CL protease by peptidyl α-ketoamides and α-ketoheterocycles.

A series of structurally-diverse α-ketoamides and α-ketoheterocycles was synthesized and subsequently investigated for inhibitory activity against norovirus 3CL protease in vitro, as well as anti-norovirus activity in a cell-based replicon system. The synthesized compounds were found to inhibit norovirus 3CL protease in vitro and to also exhibit potent anti-norovirus activity in a cell-based replicon system.

Inhibition of Dengue virus and West Nile virus proteases by click chemistry-derived benz[d]isothiazol-3(2H)-one derivatives.

Two click chemistry-derived focused libraries based on the benz[d]isothiazol-3(2H)-one scaffold were synthesized and screened against Dengue virus and West Nile virus NS2B-NS3 proteases. Several compounds (4l, 7j-n) displayed noteworthy inhibitory activity toward Dengue virus NS2B-NS3 protease in the absence and presence of added detergent. These compounds could potentially serve as a launching pad for a hit-to-lead optimization campaign.

Inhibitors of Dengue virus and West Nile virus proteases based on the aminobenzamide scaffold.

Dengue and West Nile viruses (WNV) are mosquito-borne members of flaviviruses that cause significant morbidity and mortality. There is no approved vaccine or antiviral drugs for human use to date. In this study, a series of functionalized meta and para aminobenzamide derivatives were synthesized and subsequently screened in vitro against Dengue virus and West Nile virus proteases. Four active compounds were identified which showed comparable activity toward the two proteases and shared in common a meta or para(phenoxy)phenyl group. The inhibition constants (K(i)) for the most potent compound 7n against Dengue and West Nile virus proteases were 8.77 and 5.55 µM, respectively. The kinetics data support a competitive mode of inhibition of both proteases by compound 7n. This conclusion is further supported by molecular modeling. This study reveals a new chemical scaffold which is amenable to further optimization to yield potent inhibitors of the viral proteases via the combined utilization of iterative medicinal chemistry/structure-activity relationship studies and in vitro screening.

Exploring chemical diversity via a modular reaction pairing strategy.

The efficient synthesis of an 80-member library of unique benzoxathiazocine 1,1-dioxides by a microwave-assisted, intermolecular nucleophilic aromatic substitution (S(N)Ar) diversification pathway is reported. Eight benzofused sultam cores were generated by means of a sulfonylation/S(N)Ar/Mitsunobu reaction pairing protocol, and subsequently diversified by intermolecular S(N)Ar with ten chiral, non-racemic amine/amino alcohol building blocks. Computational analyses were employed to explore and evaluate the chemical diversity of the library.

In Vitro, In Vivo and In Silico Assessment of the Antimicrobial and Immunomodulatory Effects of a Water Buffalo Cathelicidin (WBCATH) in Experimental Pulmonary Tuberculosis.

Tuberculosis (TB) is considered the oldest pandemic in human history. The emergence of multidrug-resistant (MDR) strains is currently considered a serious global health problem. As components of the innate immune response, antimicrobial peptides (AMPs) such as cathelicidins have been proposed to have efficacious antimicrobial activity against Mycobacterium tuberculosis (Mtb). In this work, we assessed a cathelicidin from water buffalo, Bubalus bubalis, (WBCATH), determining in vitro its antitubercular activity (MIC), cytotoxicity and the peptide effect on bacillary loads and cytokines production in infected alveolar macrophages. Our results showed that WBCATH has microbicidal activity against drug-sensitive and MDR Mtb, induces structural mycobacterial damage demonstrated by electron microscopy, improves Mtb killing and induces the production of protective cytokines by murine macrophages. Furthermore, in vivo WBCATH showed decreased bacterial loads in a model of progressive pulmonary TB in BALB/c mice infected with drug-sensitive or MDR mycobacteria. In addition, a synergistic therapeutic effect was observed when first-line antibiotics were administered with WBCATH. These results were supported by computational modeling of the potential effects of WBCATH on the cellular membrane of Mtb. Thus, this water buffalo-derived cathelicidin could be a promising adjuvant therapy for current anti-TB drugs by enhancing a protective immune response and potentially reducing antibiotic treatment duration.

The BioAssay network and its implications to future therapeutic discovery.

BACKGROUND: Despite intense investment growth and technology development, there is an observed bottleneck in drug discovery and development over the past decade. NIH started the Molecular Libraries Initiative (MLI) in 2003 to enlarge the pool for potential drug targets, especially from the "undruggable" part of human genome, and potential drug candidates from much broader types of drug-like small molecules. All results are being made publicly available in a web portal called PubChem. RESULTS: In this paper we construct a network from bioassay data in PubChem, apply network biology concepts to characterize this bioassay network, integrate information from multiple biological databases (e.g. DrugBank, OMIM, and UniHI), and systematically analyze the potential of bioassay targets being new drug targets in the context of complex biological networks. We propose a model to quantitatively prioritize this druggability of bioassay targets, and literature evidence was found to confirm our prioritization of bioassay targets at a roughly 70% accuracy. CONCLUSIONS: Our analysis provide some measures of the value of the MLI data as a resource for both basic chemical biology research and future therapeutic discovery.

Structural basis for the high-affinity inhibition of mammalian membranous adenylyl cyclase by 2',3'-o-(N-methylanthraniloyl)-inosine 5'-triphosphate.

2',3'-O-(N-Methylanthraniloyl)-ITP (MANT-ITP) is the most potent inhibitor of mammalian membranous adenylyl cyclase (mAC) 5 (AC5, K(i), 1 nM) yet discovered and surpasses the potency of MANT-GTP by 55-fold (J Pharmacol Exp Ther 329:1156-1165, 2009). AC5 inhibitors may be valuable drugs for treatment of heart failure. The aim of this study was to elucidate the structural basis for the high-affinity inhibition of mAC by MANT-ITP. MANT-ITP was a considerably more potent inhibitor of the purified catalytic domains VC1 and IIC2 of mAC than MANT-GTP (K(i), 0.7 versus 18 nM). Moreover, there was considerably more efficient fluorescence resonance energy transfer between Trp1020 of IIC2 and the MANT group of MANT-ITP compared with MANT-GTP, indicating optimal interaction of the MANT group of MANT-ITP with the hydrophobic pocket. The crystal structure of MANT-ITP in complex with the G(s)α- and forskolin-activated catalytic domains VC1:IIC2 compared with the existing MANT-GTP crystal structure revealed only subtle differences in binding mode. The higher affinity of MANT-ITP to mAC compared with MANT-GTP is probably due to fewer stereochemical constraints upon the nucleotide base in the purine binding pocket, allowing a stronger interaction with the hydrophobic regions of IIC2 domain, as assessed by fluorescence spectroscopy. Stronger interaction is also achieved in the phosphate-binding site. The triphosphate group of MANT-ITP exhibits better metal coordination than the triphosphate group of MANT-GTP, as confirmed by molecular dynamics simulations. Collectively, the subtle differences in ligand structure have profound effects on affinity for mAC.

Crystal structure of histamine dehydrogenase from Nocardioides simplex.

Histamine dehydrogenase (HADH) isolated from Nocardioides simplex catalyzes the oxidative deamination of histamine to imidazole acetaldehyde. HADH is highly specific for histamine, and we are interested in understanding the recognition mode of histamine in its active site. We describe the first crystal structure of a recombinant form of HADH (HADH) to 2.7-A resolution. HADH is a homodimer, where each 76-kDa subunit contains an iron-sulfur cluster ([4Fe-4S](2+)) and a 6-S-cysteinyl flavin mononucleotide (6-S-Cys-FMN) as redox cofactors. The overall structure of HADH is very similar to that of trimethylamine dehydrogenase (TMADH) from Methylotrophus methylophilus (bacterium W3A1). However, some distinct differences between the structure of HADH and TMADH have been found. Tyr(60), Trp(264), and Trp(355) provide the framework for the "aromatic bowl" that serves as a trimethylamine-binding site in TMADH is comprised of Gln(65), Trp(267), and Asp(358), respectively, in HADH. The surface Tyr(442) that is essential in transferring electrons to electron-transfer flavoprotein (ETF) in TMADH is not conserved in HADH. We use this structure to propose the binding mode for histamine in the active site of HADH through molecular modeling and to compare the interactions to those observed for other histamine-binding proteins whose structures are known.

The 3q29 microdeletion syndrome: report of three new unrelated patients and in silico "RNA binding" analysis of the 3q29 region.

The human 3q29 microdeletion syndrome is associated with mild facial dysmorphism, developmental delay and variable congenital malformations. We report three new unrelated patients with this syndrome. We also performed in silico RNA binding analysis in silico on the 3q29 critical region genes. Several genes within this genomic region including DLG1 and RNF168 are predicted to bind RNA. While recessive mutations in RNF168 cause RIDDLE syndrome, an immune deficiency and radiosensitivity disorder, the potential impact of heterozygous deletion of RNF168 on patients with the 3q29 deletion syndrome is still unknown.

Design, synthesis, and evaluation of inhibitors of Norwalk virus 3C protease.

The first series of peptidyl aldehyde inhibitors that incorporate in their structure a glutamine surrogate has been designed and synthesized based on the known substrate specificity of Norwalk virus 3C protease. The inhibitory activity of the compounds with the protease and with a norovirus cell-based replicon system was investigated. Members of this class of compounds exhibited noteworthy activity both in vitro and in a cell-based replicon system.

Characterizing the diversity and biological relevance of the MLPCN assay manifold and screening set.

The NIH Molecular Libraries Initiative (MLI), launched in 2004 with initial goals of identifying chemical probes for characterizing gene function and druggability, has produced PubChem, a chemical genomics knowledgebase for fostering translation of basic research into new therapeutic strategies. This paper assesses progress toward these goals by evaluating MLI target novelty and propensity for undergoing biochemically or therapeutically relevant modulations and the degree of chemical diversity and biogenic bias inherent in the MLI screening set. Our analyses suggest that while MLI target selection has not yet been fully optimized for biochemical diversity, it covers biologically interesting pathway space that complements established drug targets. We find the MLI screening set to be chemically diverse and to have greater biogenic bias than comparable collections of commercially available compounds. Biogenic enhancements such as incorporation of more metabolite-like chemotypes are suggested.

Occurrence of Antimicrobial Resistance Genes in the Oral Cavity of Cats with Chronic Gingivostomatitis.

Feline chronic gingivostomatitis (FCGS) is a severe immune-mediated inflammatory disease with concurrent oral dysbiosis (bacterial and fungal). Broad-spectrum antibiotics are used empirically in FCGS. Still, neither the occurrence of antimicrobial-resistant (AMR) bacteria nor potential patterns of co-occurrence between AMR genes and fungi have been documented in FCGS. This study explored the differential occurrence of AMR genes and the co-occurrence of AMR genes with oral fungal species. Briefly, 14 clinically healthy (CH) cats and 14 cats with FCGS were included. Using a sterile swab, oral tissue surfaces were sampled and submitted for 16S rRNA and ITS-2 next-generation DNA sequencing. Microbial DNA was analyzed using a proprietary curated database targeting AMR genes found in bacterial pathogens. The co-occurrence of AMR genes and fungi was tested using point biserial correlation. A total of 21 and 23 different AMR genes were detected in CH and FCGS cats, respectively. A comparison of AMR-gene frequencies between groups revealed statistically significant differences in the occurrence of genes conferring resistance to aminoglycosides (ant4Ib), beta-lactam (mecA), and macrolides (mphD and mphC). Two AMR genes (mecA and mphD) showed statistically significant co-occurrence with Malassezia restricta. In conclusion, resistance to clinically relevant antibiotics, such as beta-lactams and macrolides, is a significant cause for concern in the context of both feline and human medicine.

Application of kernel functions for accurate similarity search in large chemical databases.

BACKGROUND: Similarity search in chemical structure databases is an important problem with many applications in chemical genomics, drug design, and efficient chemical probe screening among others. It is widely believed that structure based methods provide an efficient way to do the query. Recently various graph kernel functions have been designed to capture the intrinsic similarity of graphs. Though successful in constructing accurate predictive and classification models, graph kernel functions can not be applied to large chemical compound database due to the high computational complexity and the difficulties in indexing similarity search for large databases. RESULTS: To bridge graph kernel function and similarity search in chemical databases, we applied a novel kernel-based similarity measurement, developed in our team, to measure similarity of graph represented chemicals. In our method, we utilize a hash table to support new graph kernel function definition, efficient storage and fast search. We have applied our method, named G-hash, to large chemical databases. Our results show that the G-hash method achieves state-of-the-art performance for k-nearest neighbor (k-NN) classification. Moreover, the similarity measurement and the index structure is scalable to large chemical databases with smaller indexing size, and faster query processing time as compared to state-of-the-art indexing methods such as Daylight fingerprints, C-tree and GraphGrep. CONCLUSIONS: Efficient similarity query processing method for large chemical databases is challenging since we need to balance running time efficiency and similarity search accuracy. Our previous similarity search method, G-hash, provides a new way to perform similarity search in chemical databases. Experimental study validates the utility of G-hash in chemical databases.

Uptake, distribution and diffusivity of reactive fluorophores in cells: implications toward target identification.

There is much recent interest in the application of copper-free click chemistry to study a wide range of biological events in vivo and in vitro. Specifically, azide-conjugated fluorescent probes can be used to identify targets which have been modified with bioorthogonal reactive groups. For intracellular applications of this chemistry, the structural and physicochemical properties of the fluorescent azide become increasingly important. Ideal fluorophores should extensively accumulate within cells, have even intracellular distribution, and be free (unbound), allowing them to efficiently participate in bimolecular reactions. We report here on the synthesis and evaluation of a set of structurally diverse fluorescent probes to examine their potential usefulness in intracellular click reactions. Total cellular uptake and intracellular distribution profiles were comparatively assessed using both quantitative and qualitative approaches. The intracellular diffusion coefficients were measured using a fluorescence recovery after photobleaching (FRAP)-based method. Many reactive fluorophores exhibited suboptimal properties for intracellular reactions. BODIPY- and TAMRA-based azides had superior cellular accumulation, whereas TAMRA-based probes had the most uniform intracellular distribution and best cytosolic diffusivity. Collectively, these results provide an unbiased comparative evaluation regarding the suitability of azide-linked fluorophores for intracellular click reactions.