αβ,α'β'-Diepoxyketones are mechanism-based inhibitors of nucleophilic cysteine enzymes

Epoxides are an established class of electrophilic alkylating agents that react with nucleophilic protein residues. We report αβ,α′β′-diepoxyketones (DEKs) as a new type of mechanism-based inhibitors of nucleophilic cysteine enzymes. Studies with the L,D-transpeptidase LdtMt2 from Mycobacterium tuberculosis and the main protease from SARS-CoV-2 (Mpro) reveal that following epoxide ring opening by a nucleophilic cysteine, further reactions can occur, leading to irreversible alkylation

5‑Substituted Pyridine-2,4-dicarboxylate Derivatives Have Potential for Selective Inhibition of Human Jumonji‑C Domain-Containing Protein 5

Jumonji-C domain-containing protein 5 (JMJD5) is a 2-oxoglutarate (2OG)-dependent oxygenase that plays important roles in development, circadian rhythm, and cancer through unclear mechanisms. JMJD5 has been reported to have activity as a histone protease, as an Nε-methyl lysine demethylase, and as an arginine residue hydroxylase. Small-molecule JMJD5-selective inhibitors will be useful for investigating its (patho)physiological roles. Following the observation that the broad-spectrum 2OG oxygenase inhibitor pyridine-2,4-dicarboxylic acid (2,4-PDCA) is a 2OG-competing JMJD5 inhibitor, we report that 5-aminoalkyl-substituted 2,4-PDCA derivatives are potent JMJD5 inhibitors manifesting selectivity for JMJD5 over other human 2OG oxygenases. Crystallographic analyses with five inhibitors imply induced fit binding and reveal that the 2,4-PDCA C5 substituent orients into the JMJD5 substrate-binding pocket. Cellular studies indicate that the lead compounds display similar phenotypes as reported for clinically observed JMJD5 variants, which have a reduced catalytic activity compared to wild-type JMJD5

Cyclic β^2,3-amino acids improve the serum stability of macrocyclic peptide inhibitors targeting the SARS-CoV-2 main protease

Due to their constrained conformations, cyclic β2,3-amino acids (cβAA) are key building blocks that can fold peptides into compact and rigid structures, improving peptidase resistance and binding affinity to target proteins, due to their constrained conformations. Although the translation efficiency of cβAAs is generally low, our engineered tRNA, referred to as tRNAPro1E2, enabled efficient incorporation of cβAAs into peptide libraries using the flexible in vitro translation (FIT) system. Here we report on the design and application of a macrocyclic peptide library incorporating three kinds of cβAAs: (1R,2S)-2-aminocyclopentane carboxylic acid (β1), (1S,2S)-2-aminocyclohexane carboxylic acid (β2), and (1R,2R)-2-aminocyclopentane carboxylic acid. This library was applied to an in vitro selection against the SARS-CoV-2 main protease (Mpro). The resultant peptides, BM3 and BM7, bearing one β2 and two β1, exhibited potent inhibitory activities with IC50 values of 40 nM and 20 nM, respectively. BM3 and BM7 also showed remarkable serum stability with half-lives of 48 h and >168 h, respectively. Notably, BM3A and BM7A, wherein the cβAAs were substituted with alanine, lost their inhibitory activities against Mpro and displayed substantially shorter serum half-lives. This observation underscores the significant contribution of cβAA to the activity and stability of peptides. Overall, our results highlight the potential of cβAA in generating potent and highly stable macrocyclic peptides with drug-like properties

Open science discovery of potent noncovalent SARS-CoV-2 main protease inhibitors

INTRODUCTION COVID-19 became a global pandemic partially as a result of the lack of easily deployable, broad-spectrum oral antivirals, which complicated its containment. Even endemically, and with effective vaccinations, it will continue to cause acute disease, death, and long-term sequelae globally unless there are accessible treatments. COVID-19 is not an isolated event but instead is the latest example of a viral pandemic threat to human health. Therefore, antiviral discovery and development should be a key pillar of pandemic preparedness efforts. RATIONALE One route to accelerate antiviral drug discovery is the establishment of open knowledge bases, the development of effective technology infrastructures, and the discovery of multiple potent antivirals suitable as starting points for the development of therapeutics. In this work, we report the results of the COVID Moonshot—a fully open science, crowdsourced, and structure-enabled drug discovery campaign—against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro). This collaboration may serve as a roadmap for the potential development of future antivirals. RESULTS On the basis of the results of a crystallographic fragment screen, we crowdsourced design ideas to progress from fragment to lead compounds. The crowdsourcing strategy yielded several key compounds along the optimization trajectory, including the starting compound of what became the primary lead series. Three additional chemically distinct lead series were also explored, spanning a diversity of chemotypes. The collaborative and highly automated nature of the COVID Moonshot Consortium resulted in >18,000 compound designs, >2400 synthesized compounds, >490 ligand-bound x-ray structures, >22,000 alchemical free-energy calculations, and >10,000 biochemical measurements—all of which were made publicly available in real time. The recently approved antiviral ensitrelvir was identified in part based on crystallographic data from the COVID Moonshot Consortium. This campaign led to the discovery of a potent [median inhibitory concentration (IC50) = 37 ± 2 nM] and differentiated (noncovalent and nonpeptidic) lead compound that also exhibited potent cellular activity, with a median effective concentration (EC50) of 64 nM in A549-ACE2-TMPRSS2 cells and 126 nM in HeLa-ACE2 cells without measurable cytotoxicity. Although the pharmacokinetics of the reported compound is not yet optimal for therapeutic development, it is a promising starting point for further antiviral discovery and development. CONCLUSION The success of the COVID Moonshot project in producing potent antivirals, building open knowledge bases, accelerating external discovery efforts, and functioning as a useful information-exchange hub is an example of the potential effectiveness of open science antiviral discovery programs. The open science, patent-free nature of the project enabled a large number of collaborators to provide in-kind support, including synthesis, assays, and in vitro and in vivo experiments. By making all data immediately available and ensuring that all compounds are purchasable from Enamine without the need for materials transfer agreements, we aim to accelerate research globally along parallel tracks. In the process, we generated a detailed map of the structural plasticity of Mpro, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data to spur further research into antivirals and discovery methodologies. We hope that this can serve as an alternative model for antiviral discovery and future pandemic preparedness. Further, the project also showcases the role of machine learning, computational chemistry, and high-throughput structural biology as force multipliers in drug design. Artificial intelligence and machine learning algorithms help accelerate chemical synthesis while balancing multiple competing molecular properties. The design-make-test-analyze cycle was accelerated by these algorithms combined with planetary-scale biomolecular simulations of protein-ligand interactions and rapid structure determination

Epithelial-immune cell interplay in primary Sjogren syndrome salivary gland pathogenesis

In primary Sjogren syndrome (pSS), the function of the salivary glands is often considerably reduced. Multiple innate immune pathways are likely dysregulated in the salivary gland epithelium in pSS, including the nuclear factor-kappa B pathway, the inflammasome and interferon signalling. The ductal cells of the salivary gland in pSS are characteristically surrounded by a CD4(+) T cell-rich and B cell-rich infiltrate, implying a degree of communication between epithelial cells and immune cells. B cell infiltrates within the ducts can initiate the development of lymphoepithelial lesions, including basal ductal cell hyperplasia. Vice versa, the epithelium provides chronic activation signals to the glandular B cell fraction. This continuous stimulation might ultimately drive the development of mucosa-associated lymphoid tissue lymphoma. This Review discusses changes in the cells of the salivary gland epithelium in pSS (including acinar, ductal and progenitor cells), and the proposed interplay of these cells with environmental stimuli and the immune system. Current therapeutic options are insufficient to address both lymphocytic infiltration and salivary gland dysfunction. Successful rescue of salivary gland function in pSS will probably demand a multimodal therapeutic approach and an appreciation of the complicity of the salivary gland epithelium in the development of pSS. Salivary gland dysfunction is an important characteristic of primary Sjogren syndrome (pSS). In this Review, the authors discuss various epithelial abnormalities in pSS and the mechanisms by which epithelial cell-immune cell interactions contribute to disease development and progression

Fluorinated derivatives of pyridine-2,4-dicarboxylate are potent inhibitors of human 2-oxoglutarate dependent oxygenases

2-Oxoglutarate (2OG) oxygenases have important roles in human biology and are validated medicinal chemistry targets. Improving the selectivity profile of broad-spectrum 2OG oxygenase inhibitors may help enable the identification of selective inhibitors for use in functional assignment work. We report the synthesis of F- and CF3-substituted derivatives of the broad-spectrum 2OG oxygenase inhibitor pyridine-2,4-dicarboxylate (2,4-PDCA). Their inhibition selectivity profile against selected functionally distinct human 2OG oxygenases was determined using mass spectrometry-based assays. F-substituted 2,4-PDCA derivatives efficiently inhibit the 2OG oxygenases aspartate/asparagine-β-hydroxylase (AspH) and the JmjC lysine-specific Nε-demethylase 4E (KDM4E); The F- and CF3-substituted 2,4-PDCA derivatives were all less efficient inhibitors of the tested 2OG oxygenases than 2,4-PDCA itself, except for the C5 F-substituted 2,4-PDCA derivative which inhibited AspH with a similar efficiency as 2,4-PDCA. Notably, the introduction of a F- or CF3-substituent at the C5 position of 2,4-PDCA results in a substantial increase in selectivity for AspH over KDM4E compared to 2,4-PDCA. Crystallographic studies inform on the structural basis of our observations, which exemplifies how a small change on a 2OG analogue can make a substantial difference in the potency of 2OG oxygenase inhibition

2-Oxoglutarate derivatives can selectively enhance or inhibit the activity of human oxygenases

2-Oxoglutarate (2OG) oxygenases are validated agrochemical and human drug targets. The potential for modulating their activity with 2OG derivatives has not been explored, possibly due to concerns regarding selectivity. We report proof-of-principle studies demonstrating selective enhancement or inhibition of 2OG oxygenase activity by 2-oxo acids. The human 2OG oxygenases studied, factor inhibiting hypoxia-inducible transcription factor HIF-α (FIH) and aspartate/asparagine-β-hydroxylase (AspH), catalyze C3 hydroxylations of Asp/Asn-residues. Of 35 tested 2OG derivatives, 10 enhance and 17 inhibit FIH activity. Comparison with results for AspH reveals that 2OG derivatives selectively enhance or inhibit FIH or AspH. Comparison of FIH structures complexed with 2OG derivatives to those for AspH provides insight into the basis of the observed selectivity. 2-Oxo acid derivatives have potential as drugs, for use in biomimetic catalysis, and in functional studies. The results suggest that the in vivo activity of 2OG oxygenases may be regulated by natural 2-oxo acids other than 2OG

Synthesis of novel pyridine-carboxylates as small-molecule inhibitors of human aspartate/asparagine-β-hydroxylase

The human 2-oxoglutarate (2OG)-dependent oxygenase aspartate/asparagine-β-hydroxylase (AspH) is a potential medicinal chemistry target for anti-cancer therapy. AspH is overexpressed on the cell surface of invasive cancer cells and accepts epidermal growth factor-like domain (EGFDs) substrates with a non-canonical ( i.e. Cys 1-2, 3-4, 5-6) disulfide pattern. We report a concise synthesis of C-3 substituted derivatives of pyridine-2,4-dicarboxylic acid (2,4-PDCA) as 2OG competitors for use in SAR studies on AspH inhibition. AspH inhibition was assayed using a mass spectrometry based assay employing a stable thioether-analogue of a natural EGFD AspH substrate. Certain C-3 substituted 2,4-PDCA derivatives were potent AspH inhibitors, manifesting selectivity over some, but not all, other tested human 2OG oxygenases. The results raise questions about the use of pyridine-carboxylate related 2OG analogues as selective functional probes for specific 2OG oxygenases, and should aid the development of AspH inhibitors suitable for in vivo use

Structure-activity studies reveal scope for optimisation of Ebselen-type inhibition of SARS-COV-2 main protease

The reactive organoselenium compound ebselen is being investigated for treatment of coronavirus disease 2019 (COVID-19) and other diseases. We report structure-activity studies on sulfur analogues of ebselen with the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro), employing turnover and protein-observed mass spectrometry-based assays. The results reveal scope for optimisation of ebselen/ebselen derivative- mediated inhibition of Mpro, particularly with respect to improved selectivity.</p