ChemMedChem

The main protease (Mpro) of SARS-CoV-2 is a validated antiviral drug target. Several Mpro inhibitors have been reported with potent enzymatic inhibition and cellular antiviral activity, including GC376, boceprevir, calpain inhibitors II and XII, each containing a reactive warhead that covalently modifies the catalytic Cys145. In this study, we report an expedited drug discovery approach by coupling structure-based design and Ugi four-component (Ugi-4CR) reaction methodology to the design of non-covalent Mpro inhibitors. The most potent compound 23R had cellular antiviral activity similar to covalent inhibitors such as GC376. Our designs were guided by overlaying the structure of SARS-CoV Mpro + ML188 (R), a non-covalent inhibitor derived from Ug-4CR, with the X-ray crystal structures of SARS-CoV-2 Mpro + calpain inhibitor XII/GC376/UAWJ247. Binding site analysis suggests a strategy of extending the P2 and P3 substitutions in ML188 (R) to achieve optimal shape complementary with SARS-CoV-2 Mpro. Lead optimization led to the discovery of 23R, which inhibits SARS-CoV-2 Mpro and SARS-CoV-2 viral replication with an IC50 of 0.31 M and EC50 of 1.27 M, respectively. The binding and specificity of 23R to SARS-CoV-2 Mpro were confirmed in a thermal shift assay and native mass spectrometry assay. The co-crystal structure of SARS-CoV-2 Mpro with 23R revealed the P2 biphenyl fits snuggly into the S2 pocket and the benzyl group in the -methylbenzyl faces towards the core of the enzyme, occupying a previously unexplored binding site located in between the S2 and S4 pockets. Overall, this study revealed the most potent non-covalent SARS-CoV-2 Mpro inhibitors reported to date and a novel binding pocket that can be explored for Mpro inhibitor design.

Small molecule agonists of TLR7/8, such as imidazoquinolines, are validated agonists for the treatment of cancer and for use in vaccine adjuvants. Imidazoquinolines have been extensively modified to understand the structure-activity relationship (SAR) at the N1- and C2-positions resulting in the clinical drug imiquimod, resiquimod, and several other highly potent analogues. However, the SAR of the aryl ring has not been fully elucidated in the literature. This initial study examines the SAR of C7-substituted imidazoquinolines. These compounds not only demonstrated that TLR7/8 tolerate changes at the C7 position but can increase potency and change their cytokine profiles. The most notable TLR7/8 agonists developed from this study 5, 8, and 14 which are up to 4-fold and 2-fold more active than resiquimod for TLR8 and/or TLR7, respectively, and up to 100-fold more active than the FDA approved imiquimod for TLR7.

Emerging RNA viruses including SARS-CoV-2 continue to be a major threat around the globe. The cell entry of SARS-CoV-2 particles via the endosomal pathway involves the cysteine protease cathepsin L (CatL) among other proteases. CatL is rendered as a promising drug target in the context of different viral and lysosome-related diseases. Hence, drug discovery and structure-based optimization of inhibitors is of high pharmaceutical interest. We herein verified and compared the anti-SARS-CoV-2 activity of a set of carbonyl and succinyl-epoxide-based inhibitors, which have previously been identified as cathepsin inhibitors. Calpain inhibitor XII (CI-XII), MG-101 and CatL inhibitor IV (CLI-IV) possess antiviral activity in the very low nanomolar IC50 range in Vero E6 cells. Experimental structural data on how these and related compounds bind to CatL are however notably lacking, despite their therapeutic potential. Consequently, we present and compare crystal structures of CatL in complex with 14 compounds, namely BOCA (N-BOC-2-aminoacetaldehyde), CLI-IV, CI-III, CI-VI, CI-XII, the main protease -ketoamide inhibitor 13b, MG-101, MG-132 as well as E-64d (aloxistatin), E-64, CLIK148, CAA0225, TC-I (CID 16725315) and TPCK at resolutions better than 2 [A]. Overall, the presented data comprise a broad and solid basis for structure-guided understanding and optimization of CatL inhibitors towards protease drug development.

Alphaviruses are mosquito-borne RNA viruses that pose a significant public health threat, with no FDA-approved antiviral therapeutics available. The non-structural protein 2 helicase (nsP2hel) is an enzyme involved in unwinding dsRNA essential for alphavirus replication. This study reports the discovery and optimization of first-in-class oxaspiropiperidine inhibitors targeting nsP2hel. Structure-activity relationship (SAR) studies identified potent cyclic sulfonamide analogs with nanomolar antiviral activity against chikungunya virus (CHIKV). Biochemical analyses of nsP2hel ATPase and RNA unwindase activities showed these compounds act by a non-competitive mode suggesting that they are allosteric inhibitors. Viral resistance mutations mapped to nsP2hel and a fluorine-labeled analog exhibited direct binding to the protein by 19F NMR. The lead inhibitor, 2o, demonstrated broad-spectrum antialphaviral activity, reducing titers of CHIKV, Mayaro virus (MAYV), and Venezuelan equine encephalitis virus (VEEV). These findings support nsP2hel as a viable target for development of broad-spectrum direct-acting antialphaviral drugs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/641060v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@856df5org.highwire.dtl.DTLVardef@1f6225borg.highwire.dtl.DTLVardef@4997d5org.highwire.dtl.DTLVardef@18f42f2_HPS_FORMAT_FIGEXP M_FIG C_FIG

The emergence and spread of artemisinin-resistant, malaria-causing P. falciparum provide the impetus for the development of novel antimalarials. Pantothenamides are potent inhibitors of malaria parasite proliferation, however their clinical use is hindered by pantetheinase-mediated degradation in human serum. Here we report the synthesis and biological activity of a series of pantothenamide-mimics in which the labile amide bond is replaced by a thiazole ring with various orientations. Out of 23 novel compounds generated and tested in the presence of pantetheinase, several display sub-micromolar antiplasmodial activity in vitro. A selection of compounds was studied in more detail and CoA biosynthesis and/or utilisation pathways were confirmed to be the target. Toxicity to human cells was not observed. Kinetic studies identified the selected compounds as substrates of the HsPanK3 enzyme, but with much lower affinity compared to that of the natural substrate pantothenate. The most potent thiazole-bearing antiplasmodial compound was found to bind to PfPanK with a 120-fold higher affinity compared to HsPanK, highlighting excellent selectivity, not only against the key first enzyme in the CoA biosynthesis pathway, but also at the whole-cell level. In conclusion, thiazole substitution of the labile amide bond represents a promising avenue for the development of antimalarial pantothenamide-mimics.

The increasing resistance of influenza A viruses to adamantane-based antivirals underscores the need for new inhibitors targeting both wild-type (WT) and mutant M2 ion channels. Here, we report the synthesis and biological evaluation of polycyclic cage amines designed to replace the adamantane scaffold as M2 inhibitors. These include ring-contracted and ring-expanded analogues, evaluated both as primary amines and as aryl-/heteroaryl-substituted derivatives. Most of the polycyclic amines inhibited the WT M2 channel as demonstrated by electrophysiological assays. Among them, compound 10, a 3,4,8,9-tetramethyltetracyclo[4.4.0.03..0.]decan-1-amine, emerged as a triple blocker active against M2 WT, M2 L27F, and M2 V27A channels. In contrast, compound 6c, a noradamantane-isoxazole derivative, showed selective inhibition of the S31N mutant. Although no antiviral activity was observed against influenza A virus in infected cell assays, both compounds 6c and 10 displayed significant antiviral activity against human coronavirus 229E. Furthermore, compound 10 demonstrated favourable pharmacokinetic properties. MD simulations show that noradamantane 6c binds inside the M2 S31N pore, with its ammonium forming H-bonds to Asn31 and the isoxazole positioned near Val27, restricting water entry. In contrast, larger polycyclic amines likely cannot access the pore due to steric hindrance.

The discovery of new targets for treatment of malaria advanced with the demonstration that orally administered inhibitors of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) could clear infection in a murine model. This enthusiasm was tempered by unsatisfactory safety and/or pharmacokinetic issues found with these chemotypes. To address the urgent need for new scaffolds, we recently reported the discovery and optimization of novel, potent isoxazole-based PfPKG inhibitors that lacked any obvious safety warnings. This manuscript presents representative in vitro ADME, hERG characterization and cell-based antiparasitic activity of these PfPKG inhibitors. We also report the discovery and structure-activity relationships of a new series with good potency, low hERG activity and cell-based anti-parasitic activity comparable to a literature standard.

The SARS CoV-2 Papain-Like protease has multiple roles in the viral replication cycle, related to both its polypeptide cleavage function and its capacity to antagonize host immune response. Targeting PLpro function is recognized as a promising mechanism to modulate viral replication whilst supporting host immune responses. However, development of PLpro specific inhibitors remains challenging. Upcoming studies revealed the limitation of reported inhibitors by profiling them through a pipeline of enzymatic, binding and cellular activity assays showing unspecific activity. GRL-0617 remained the only validated molecule with demonstrated anti-viral activity in cells. In this study we refer to the pitfalls of redox-sensitivity of PLpro. Using a screening-based approach to identify inhibitors of PLpro proteolytic activity, we made extensive efforts to validate the active compounds over a range of conditions and readouts, emphasising the need for comprehensive orthogonal data when profiling putative PLpro inhibitors. The remaining active compound CPI-169, showed to compete with GRL-0617 in NMR-based experiments, suggesting to share a similar binding mode, opening novel design opportunities for further developments as antiviral agents. Author summaryThe increasing knowledge about SARS-CoV-2 allowed the development of multiple strategies to contain the spread of COVID-19 infection. Nevertheless, effective antiviral pharmacological treatments are still rare and viral evolution allowed a fast adaptation and escape from available containment methods. The papain like protease (PLpro) has now become the next most promising SARS-CoV-2 therapeutic due to its multiple functions in virus replication cycle and antagonization of host immune response. However, due to inherent flexibility and sensitivity of this enzyme specific inhibitors are rare. Here we report on a screening strategy using repurposing of known drugs that takes into account PLpro characteristics to identify new inhibitors, showing the success of the approach by identifying CPI-169 that competitive targets the well described GRL-0617 inhibitor binding pocket of PLpro and helping to design further antiviral agents.

Human Neutrophil Elastase (HNE) plays a vital role in several inflammatory diseases, however its role in the tumour microenvironment and the potential in cancer treatment is still unrevealed. Considering the potential of {beta}-lactams as HNE inhibitors, the present work describes the development of a synthetic strategy to obtain two different types (Type I and Type II) of quenched activity-based probes (qABPs), using a {beta}-lactam ring as a warhead and BODIPY-FL as a fluorophore. The two types differ in mechanism and relative position between the fluorophore and the quencher moiety. The qABPs synthesized presented IC50 values against HNE lower than 0.5 {micro}M, and high selectivity compared with homologous serine hydrolases. Type II qABPs showed a more efficient turn-on mechanism, and selectively targeted HNE in different cell lysates. The qABP 22 was internalized in U937 cells and in human neutrophils and successfully targeted HNE in both.

Non-polio enteroviruses (NPEV) such as enterovirus D68 (EV-D68) that are highly infectious and associated with polio-like neurological complications have caused out-breaks, globally, in recent years. While some clinical and preclinical compounds have shown efficacy against NPEV in-vitro, liabilities that caused historical compounds such as pleconaril to fall short of FDA approval still remain. We present herein SAR and SPR studies of analogues of clinical compounds such as pleconaril and vapendavir against EV-D68 as a representative NPEV. Numerous structurally differentiated analogues with EV-D68 antiviral activity and useful ADME properties were discovered, which could serve as starting points for future EV drug discovery campaigns. Screening against a panel of enteroviruses revealed moderately broad-spectrum anti-EV activity of compound 26.

The emergence of antibiotic resistance necessitates the discovery of new scaffolds with improved efficacy and drug-like properties. In this work, a new series of acid-functionalized carbazole derivatives was designed, synthesized, and comprehensively evaluated for their antibacterial potential. The incorporation of acidic functionalities into the carbazole framework enhanced physicochemical properties and biological interactions, yielding distinct strain-specific antibacterial activities. Minimum inhibitory concentration (MIC) studies demonstrated that 3-methyl-1,4-dioxo-4,9-dihydro-1H-carbazole-6-carboxylic acid (2) exhibited broad-spectrum potency, particularly against S. aureus and E. coli, while 6-methyl-9H-carbazole-3-carboxylic acid (3) showed selectivity against B. cereus and (E)-3-methyl-1-(2-tosylhydrazono)-2,3,4,9-tetrahydro-1H-carbazole-6-carboxylic acid (1) displayed strong activity toward S. typhimurium. Molecular docking studies revealed favourable binding affinities of all derivatives toward bacterial dihydrofolate reductase (DHFR), with compound 1 showing the highest docking scores. Molecular dynamics simulations further confirmed the broad conformational adaptability of compound 1, the target-specific stability of compound 3, and protein-dependent binding behaviour of compound 2. Complementary ADMET predictions indicated that all compounds adhered to Lipinskis rules, with compound 3 displaying the most favourable pharmacokinetic profile, including high oral bioavailability and low toxicity risk. Together, these experimental and computational findings establish acid-functionalized carbazole scaffolds as promising antibacterial candidates.

New drugs targeting multiple stages of the malaria-causing parasite, Plasmodium, are needed to reduce and eliminate malaria worldwide. N-Myristoyltransferase (NMT) is an essential eukaryotic enzyme, and a validated chemically tractable drug target for malaria. Previous efforts have failed to target NMT owing to the low selectivity for the Plasmodium enzyme compared with human NMTs. Herein, we applied a structure-guided approach using previously reported NMT inhibitors as scaffolds to develop a new generation of Plasmodium vivax NMT (PvNMT) targeting compounds. We report a series of compounds with IC50 values in the nM range and an order of magnitude improved selectivity to Plasmodium NMT over human NMT (HsNMT). X-ray co-crystallization of PvNMT with a representative lead compound, 12b, supported the prevailing hypothesis that a conformational difference in a key tyrosine residue of PvNMT and HsNMT drives the selectivity between these enzymes. The compounds were triaged based on their selectivity for PvNMT. They significantly decreased P. falciparum blood-stage parasite load, with IC50 values ranging from 0.36 M to 1.25 M. The compounds exhibited a dose-dependent inhibition of P. vivax liver stage schizont and hypnozoite infection, consistently, in three different P. vivax isolates with IC50 values ranging from 2.2 M to 6 M and from 1.2 M to 12 M. Our data provide evidence that NMT inhibitors could be multistage antimalarials, targeting both dormant and developing liver stage parasites, which is essential for malaria elimination. One Sentence SummaryPotent and selective N-myristoyltransferase inhibitors of Plasmodium vivax hypnozoites and schizonts were synthesized and tested.

The development of effective broad-spectrum antivirals forms an important part of preparing for future pandemics. One cause for concern is the currently emerging pathogen Enterovirus D68 (EV-D68) which primarily spreads through respiratory routes causing mostly mild to severe respiratory illness but, in severe cases, acute flaccid myelitis. The 3C protease of EV-D68 (3Cpro) is a potential target for the development of antiviral drugs due to its essential role in the viral life cycle and high sequence conservation amongst family members. In this study, we describe the identification of fragments which bind to3Cpro using crystallographic screening and the expansion of these into more lead-like compounds. The hits revealed interesting directions for hit-to-lead progression, specifically the importance of the pocket occupied by the conserved glutamine sidechain of the substrates and the interactions formed. Additionally, two pockets could be joined by not following the backbone of the native substrates, thus circumventing the screening issues arising from the flexibility of the catalytic triad. These observations of the novel binding modes of the chemical matter found by this screen can help shape future drug design campaigns against 3C proteases.

Fungal pathogens are an escalating global public health concern, particularly in the context of invasive and opportunistic infections. Cryptococcosis, primarily caused by Cryptococcus neoformans var. grubii, can manifest as acute, subacute, or chronic disease, affecting multiple organs and frequently leading to life-threatening meningitis in immunocompromised individuals. Given the limited antifungal therapeutic strategies and the emergence of resistance and toxicity-related constraints, the development of novel anti-cryptococcal agents remains an urgent priority. In this study, a library of innovative hybrids (5a-f) based on the 3-hydroxypyridin-4(1H)-one scaffold was developed. Their antimicrobial activity was evaluated towards a panel of clinically relevant Gram-positive (methicillin-resistant Staphylococcus aureus - MRSA) and Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii), as well as fungal species Candida albicans and Cryptococcus neoformans var. grubbi. Cytotoxicity was assessed in HEK293 and HepG2 cell lines, and haemolytic profile was determined to evaluate safety. In addition, iron-chelating capacity and lipophilic properties were also investigated. All compounds formed stable complexes with iron(III) and were non-toxic at concentrations up to 25 M. Lipophilicity studies showed that compounds in series 1 (5a-c) exhibited lower lipophilicity than those in Series 2 (5d-f), mainly due to the regioisomeric position of the hydroxyl group on the 2-methyl-4-pyridone scaffold; specifically, the C3-substitution pattern in Series 2 that enhances the hydrophobic character compared to the C5-substitution in Series 1. Fluorination further increased lipophilicity in both series. Notably, compounds 5c-5f emerged as potent, selective, and non-toxic antifungal agents against Cryptococcus neoformans var. grubii (MIC < 16 {micro}g/mL; CC50 > 32 {micro}g/mL; HC10 > 32 {micro}g/mL). Their distinct structural features appear to play a key role in antifungal selectivity, supporting the potential of these 3-hydroxypyridin-4(1H)-one-based hybrids as promising approach for the development of novel therapeutics for cryptococcal meningitis.

The family of dopamine D2-like receptors represent an interesting target for a variety of neurological diseases, e.g. Parkinsons disease (PD), addiction or schizophrenia. In this study we describe the synthesis of a new set of fluorescent ligands as tools for visualization of dopamine D2-like receptors. Pharmacological characterization in radioligand binding studies identified UR-MN212 (20) as a high-affinity ligand for D2-like receptors (pKi (D2longR) = 8.24, pKi (D3R) = 8.58, pKi (D4R) = 7.78) with decent selectivity towards D1-like receptors. Compound 20 is a neutral antagonist in a Go1 activation assay at the D2longR, D3R and D4R, which is an important feature for studies using whole cells. The neutral antagonist 20, equipped with a 5-TAMRA dye, displayed rapid association to the D2longR in binding studies using confocal microscopy demonstrating its suitability for fluorescence microscopy. Furthermore, in molecular brightness studies, the ligands binding affinity could be determined in a single-digit nanomolar range that was in good agreement with radioligand binding data. Therefore, the fluorescent compound can be used for quantitative characterization of native D2-like receptors in a broad variety of experimental setups.

The discovery of new targets for treatment of malaria and in particular those aimed at the pre-erythrocytic stage in the life cycle, advanced with the demonstration that orally administered inhibitors of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) could clear infection in a murine model. This enthusiasm was tempered by unsatisfactory safety and/or pharmacokinetic issues found with these chemotypes. To address the urgent need for new scaffolds, this manuscript presents initial structure-activity relationships in an imidazole scaffold at four positions, representative in vitro ADME, hERG characterization and cell-based anti-parasitic activity. This series of PfPKG inhibitors has good in vitro PfPKG potency, low hERG activity and cell-based anti-parasitic activity against multiple Plasmodium species that appears to correlate with in vitro potency.

The antibacterial activity of 22 thiolato-bridged dinuclear ruthenium(II)-arene compounds was assessed in vitro against Escherichia coli, Streptococcus pneumoniae and Staphylococcus aureus. None of the compounds efficiently inhibited the growth of the three E. coli strains tested and only compound 5 exhibited a medium activity against this bacterium (MIC (minimum inhibitory concentration) of 25 M). However, a significant antibacterial activity was observed against S. pneumoniae, with MIC values ranging from 1.3 to 2.6 M for compounds 1-3, 5 and 6. Similarly, compounds 2, 5-7 and 20-22 had MIC values ranging from 2.5 to 5 M against S. aureus. The tested diruthenium compounds have a bactericidal effect significantly faster than that of penicillin. Fluorescence microscopy assays performed on S. aureus using the BODIPY-tagged diruthenium complex 15 showed that this type of metal compound enter the bacteria and do not accumulate in the cell wall of gram-positive bacteria. Cellular internalization was further confirmed by inductively coupled plasma mass spectrometry (ICP-MS) experiments. The nature of the substituents anchored on the bridging thiols and the compounds molecular weight appear to significantly influence the antibacterial activity. Thus, if overall a decrease of the bactericidal effect with the increase of compounds molecular weight is observed, however the complexes bearing larger benzo-fused lactam substituents had low MIC values. This first antibacterial activity screening demonstrated that the thiolato-diruthenium compounds exhibit promising activity against S. aureus and S. pneumoniae and deserve to be considered for further studies.

This scientific study explores the binding mechanisms of saccharine derivatives with human carbonic anhydrase IX (hCA IX), an antitumor drug target, with the aim of facilitating the design of potent and selective inhibitors. Through the use of crystallographic analysis, we investigate the structures of hCA IX - saccharine derivative complexes, unveiling their unique binding modes that exhibit both similarities to sulfonamides and distinct orientations of the ligand tail. Our comprehensive structural insights provide information regarding the crucial interactions between the ligands and the protein, shedding light on interactions that dictate inhibitor binding and selectivity. Through a comparative analysis of the binding modes observed in hCA II and hCA IX, isoform-specific interactions are identified, offering promising strategies for the development of isoform-selective inhibitors that specifically target tumor-associated hCA IX. The findings of this study significantly deepen our understanding of the binding mechanisms of hCA inhibitors, laying a solid foundation for the rational design of more effective inhibitors.

The 2020 SARS-CoV-2 coronavirus pandemic highlighted the urgent need for novel small molecule antiviral drugs. (S)-x38 DNDI-6510 is a non-covalent SARS-CoV-2 main protease inhibitor developed by the open science collaboration COVID Moonshot. Here, we report on the metabolic and toxicologic optimization of the lead series previously disclosed by the COVID Moonshot Initiative, leading up to the selection of (S)-x38 DNDI-6510 as the preclinical candidate. We describe the thorough profiling of the series, identifying key risks such as formation of genotoxic metabolites and high clearance, which were successfully addressed during lead optimization. In addition, we disclose the in vitro and in vivo evaluation of (S)-x38 DNDI-6510 in pharmacokinetic and pharmacodynamic models, exploring multiple approaches to ameliorate rodent-specific metabolic clearance, and show that both co-dosing of (S)-x38 DNDI-6510 with an ABT inhibitor and utilizing a metabolically humanized mouse model (8HUM) achieve significant improvements in exposure. Through comparisons of ABT co-dosing and humanized mouse models in efficacy experiments, we demonstrate that continuous exposure over cellular EC90 is required for SARS-CoV-2 antiviral efficacy in vivo in an antiviral model using a mouse-adapted SARS-CoV-2 strain. Finally, (S)-x38 DNDI-6510 was assessed in maximum tolerated dose experiments in two species, demonstrating significant in vivo PXR-linked auto-induction of metabolism, leading to the discontinuation of this compound. In summary, we report the successful effort to overcome series-specific AMES liabilities in a lead development program. Downstream optimization of existing series will require in-depth optimization of rodent-specific liabilities and metabolic induction profile.

Protein-based therapeutics such as monoclonal antibodies and cytokines are important therapies in various pathophysiological conditions such as oncology, auto-immune disorders, and viral infections. However, the wide application of such protein therapeutics is often hindered by dose-limiting toxicities and adverse effects, namely cytokine storm syndrome, organ failure and others. Therefore, spatiotemporal control of the activities of these proteins is crucial to further expand their application. Here, we report the design and application of small molecule-controlled switchable protein therapeutics by taking advantage of a previously engineered OFF-switch system. We used Rosetta modeling suite to computationally optimize the affinity between B-cell lymphoma 2 (Bcl-2) protein and a previously developed computationally designed protein partner (LD3) to obtain a fast and efficient heterodimer disruption upon addition of a competing drug (Venetoclax). The incorporation of the engineered OFF-switch system into CTLA4, anti-HER2 antibodies or an Fc-fused IL-15 cytokine demonstrated an efficient disruption in vitro, as well as fast clearance in vivo upon addition of the competing drug Venetoclax. These results provide a proof-of-concept for the rational design of controllable biologics by introducing a drug-induced OFF-switch into existing protein-based therapeutics.