The COVID-19 pandemic has highlighted a clear need to ensure rapid and equitable global access to health interventions in preparation for future coronavirus-driven pandemics. Here, we report the discovery of highly potent noncovalent inhibitors of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) with pan-coronavirus (pan-CoV) Mpro inhibition through computer-aided drug design. Virtual screening led to the identification of a noncovalent hit compound with a piperazine core. Structure-guided scaffold morphing provided a novel trisubstituted piperidine core. Free energy perturbation (FEP)-guided designs, with induced-fit of Met49/Met165 and Gln189, resulted in the identification of highly potent compound 30, which exhibits pan-CoV Mpro inhibition and cellular antiviral efficacy against the SARS-CoV-2 omicron variant. The optimized lead compound 30 was characterized by in vitro ADME/Tox assays and in vivo mouse pharmacokinetics. These findings suggest that compound 30 could be an addition to the repertoire of tools used to support future pandemic preparedness.

Selective inhibition of the RGD (Arg-Gly-Asp) integrin αvβ1 has been recently identified as an attractive therapeutic approach for the treatment of liver fibrosis given its function, target expression, and safety profile. Our identification of a non-RGD small molecule lead followed by focused, systematic changes to the core structure utilizing a crystal structure, in silico modeling, and a tractable synthetic approach resulted in the identification of a potent small molecule exhibiting a remarkable affinity for αvβ1 relative to several other integrin isoforms measured. Azabenzimidazolone 25 demonstrated antifibrotic efficacy in an in vivo rat liver fibrosis model and represents a tool compound capable of further exploring the biological consequences of selective αvβ1 inhibition.

Small-molecule antivirals that prevent the replication of the SARS-CoV-2 virus by blocking the enzymatic activity of its main protease (Mpro) are and will be a tenet of pandemic preparedness. However, the peptidic nature of such compounds often precludes the design of compounds within favorable physical property ranges, limiting cellular activity. Here we describe the discovery of peptide aldehyde Mpro inhibitors with potent enzymatic and cellular antiviral activity. This structure-activity relationship (SAR) exploration was guided by the use of calculated hydration site thermodynamic maps (WaterMap) to drive potency via displacement of waters from high-energy sites. Thousands of diverse compounds were designed to target these high-energy hydration sites and then prioritized for synthesis by physics- and structure-based Free-Energy Perturbation (FEP+) simulations, which accurately predicted biochemical potencies. This approach ultimately led to the rapid discovery of lead compounds with unique SAR that exhibited potent enzymatic and cellular activity with excellent pan-coronavirus coverage.

In 2022, 23 new small molecule chemical entities were approved as drugs by the United States FDA, European Union EMA, Japan PMDA, and China NMPA. This review describes the synthetic approach demonstrated on largest scale for each new drug based on patent or primary literature. The synthetic routes highlight practical methods to construct molecules, sometimes on the manufacturing scale, to access the new drugs. Ten additional drugs approved in 2021 and one approved in 2020 are included that were not covered in the previous year's review.

The proteinase-activated receptor (PAR) subfamily of G protein-coupled receptors (GPCRs) include four members, PAR1-PAR4, that play critical roles in hemostasis, thrombosis, embryonic development, wound healing, inflammation, and cancer progression. The PARs share a unique activation mechanism driven by proteinase cleavage at a specific site within the extracellular amino-terminus, exposing a ‘tethered ligand’ that self-activates the receptor. Subsequent activation allows PAR family members to initiate complex intracellular signaling networks via traditional G protein-mediated pathways and beta-arrestin signaling and, in this way, the PARs link extracellular protease signaling molecules to cellular functions. Despite a primary reliance on biochemical studies for understanding tethered ligand recognition, direct structural visualization of these ligand-receptor complexes has been elusive. Here, we present structural snapshots of activated PAR1 and PAR2 bound to their endogenous tethered ligands, revealing, for the first time, shallow and constricted orthosteric binding pockets and highlighting critical residues involved in ligand recognition and receptor activation. Surprisingly, comparisons with antagonist-bound structures show minimal conformational changes in the TM6 helix, a typical signature of GPCR activation, with large movements of TM7 observed upon activation. These insights lead to the identification of a common mechanism for PAR1 and PAR2 activation and provide a structural template for designing novel antagonists targeting the orthosteric binding site, potentially opening new avenues for therapeutic interventions. ### Competing Interest Statement The authors have declared no competing interest.

Each year, new drugs are introduced to the market, representingstructures that have affinity for biological targets implicated inhuman diseases and conditions. These new chemical entities (NCEs),particularly small molecules and antibody-drug conjugates,provide insight into molecular recognition and serve as potentialleads for the design of future medicines. This annual review is partof a continuing series highlighting the most likely process-scalesynthetic approaches to 35 NCEs that were first approved anywherein the world during 2021.

New drugs introduced to the market are privileged structures that have affinities for biological targets implicated in human diseases and conditions. These new chemical entities (NCEs), particularly small molecules and antibody-drug conjugates (ADCs), provide insight into molecular recognition and simultaneously function as leads for the design of future medicines. This Review is part of a continuing series presenting the most likely process-scale synthetic approaches to 44 new chemical entities approved for the first time anywhere in the world during 2020.

New drugs introduced to the market are privileged structures having affinities for biological targets implicated in human diseases and conditions. These new chemical entities (NCEs), particularly small molecules and antibody-drug conjugates, provide insight into molecular recognition and simultaneously function as leads for the design of future medicines. This review is part of a continuing series presenting the most likely process-scale synthetic approaches to 40 NCEs approved for the first time anywhere in the world in 2019.

A novel high-throughput aqueous solubility assay was developed for peptides and proteins exhibiting a high gelling propensity (in this case, antibacterial teixobactin analogues). By integrating the assessment of gel formation, as indicated by an increase in the solution viscosity, into the peptide equilibrium solubility screening assay, we were able to estimate the "free-flowing solubility", which is defined as the concentration at which the peptide solution not only is fully dissolved but also is a liquid exhibiting ideal flowing characteristics. In this workflow, peptide solutions passing the turbidity assessment were further screened by viscosity measurements based on nanobead-assisted dynamic light scattering analysis in a 96-well plate. The method is able to effectively detect the initiation of peptide gelation and facilitate compound ranking based on their aqueous solubility. The application of such an approach helped confirm that the substitution of Ser(3) in teixobactin led to desired physicochemical improvements and provided a focal point for further chemistry structure-activity relationship exploration.

The prevalence of life‐threatening, drug‐resistant microbial infections has challenged researchers to consider alternatives to currently available antibiotics. Teixobactin is a recently discovered “resistance‐proof” antimicrobial peptide that targets the bacterial cell wall precursor lipid II. In doing so, teixobactin exhibits potent antimicrobial activity against a wide range of Gram‐positive organisms. Herein we demonstrate that teixobactin and several structural analogues are capable of binding lipid II from both Gram‐positive and Gram‐negative bacteria. Furthermore, we show that when combined with known outer membrane‐disrupting peptides, teixobactin is active against Gram‐negative organisms.

A new class of potential antibacterial agents has been synthesized on a new molecular scaffold of cyclohexane carboxylate. We have tagged this new class of compounds TACCs (Trisubstituted Aryl Cyclohexanecarboxylate). These new molecules are structural analogues of an Activators of Self-Compartmentalizing Proteases 4 and 5 (ACP 4 and 5), and were synthesized to circumvent the drug-like property (drug-ability) challenges and liability noted in ACP 4 and 5. A pseudo-Robinson annulation protocol was used to furnish this new class of potential antibiotics. Structure-activity relationship (SAR) study was done to identify the pharmacophore(s) in this molecular scaffold. A selection of these compounds was used in our preliminary antibacterial inhibitory activities’ studies on Bacillus mycoides and Bacillus subtilis. These preliminary studies show that the TACCs exhibited equal, and in some cases better, antibacterial activity than ACP 4 and 5.

This laboratory experiment leverages the pedagogical value and multidisciplinary nature of biodiesel production from vegetable oil to introduce students to continuous-flow chemistry, a modern and rapidly growing approach to chemical synthesis. An interdisciplinary approach exposes students to the practical and conceptual aspects of modern continuous-flow chemistry while simultaneously reinforcing core organic chemistry techniques and investing students in issues of sustainability. Students screen reaction conditions in flow through an inquiry-guided approach and make evidence-based decisions to accomplish the sustainable conversion of waste cooking oil into biofuel. The laboratory experiment is designed to be highly modular and can be completed in two, three, five, or eight laboratory periods. By incorporating the burgeoning field of continuous-flow chemistry into the educational infrastructure, the experiments allow students to develop skills that are highly valued in the modern chemical workforce.

A series of 180 vinblastine 20' amides were prepared in three steps from commercially available starting materials, systematically exploring a typically inaccessible site in the molecule enlisting a powerful functionalization strategy. Clear structure-activity relationships and a structural model were developed in the studies which provided many such 20' amides that exhibit substantial and some even remarkable enhancements in potency, many that exhibit further improvements in activity against a Pgp overexpressing resistant cancer cell line, and an important subset of the vinblastine analogues that display little or no differential in activity against a matched pair of vinblastine sensitive and resistant (Pgp overexpressing) cell lines. The improvements in potency directly correlated with target tubulin binding affinity, and the reduction in differential functional activity against the sensitive and Pgp overexpressing resistant cell lines was found to correlate directly with an impact on Pgp-derived efflux.

Significance Vinblastine is a clinical drug used in frontline combination therapies for treatment of cancer. It acts by inhibition of mitosis through binding tubulin and disrupting microtubule formation. Because of advances in its total synthesis, we report previously inaccessible and unusual modifications to vinblastine that improve potency a remarkable 100-fold. These ultrapotent vinblastines display much higher tubulin binding affinities and likely further disrupt the tubulin head-to-tail dimer–dimer interaction by strategic placement of an added rigid, extended group along the adjacent continuing protein–protein interface. Significantly, the ultrapotent vinblastines are accessible by chemical synthesis in three steps from commercially available materials (catharanthine, $16/g; vindoline, $36/g) based on newly introduced synthetic methodology and are inaccessible by natural product derivatization, late-stage functionalization, or biosynthetic methods.

Background Cyclic acyldepsipeptides (ADEPs) are a novel class of antibacterial agents, some of which (e.g., ADEP 4) are highly active against Gram-positive bacteria. The focus of these in vivo studies is ADEP B315, a rationally designed compound that has the most potent in vitro activity of any ADEP analog reported to date. Methods In vivo efficacy experiments were performed using lethal intraperitoneal mice infection models with a methicillin-sensitive S. aureus (MSSA) and a methicillin-resistant (MRSA) strain. The infected mice were treated with ADEP B315, a des-methyl analog of ADEP 4, vancomycin, or the vehicle used for the ADEPs and their survival was assessed daily. A subset of MSSA-infected mice was sacrificed soon after inoculation and the bacterial burden was measured in their livers and spleens. The toxicity of ADEP B315 was assessed in viability assays using human whole blood cultures. Results In the MSSA experiments, all mice treated with the vehicle succumbed to the infection within 24 hours. All tested compounds were effective in prolonging survival of infected mice (p<0.001). Mice treated with ADEP B315 had a 39% survival rate by 10 days compared to 7% survival in mice treated with a des-methyl ADEP 4 analog (p = 0.017). Survival of the infected mice treated with ADEP B315 was comparable to those treated with vanocmycin (p = 0.12) at the same dose. Further, bacterial burden in the liver and spleen was significantly lower in mice treated with ADEP B315 compared to controls. In the MRSA experiments, ADEP B315 was able to significantly prolong survival compared to mice treated with either the vehicle (p = 0.001) or vancomycin (p = 0.007). ADEP B315 exhibited no significant toxicity in human whole blood cultures at concentrations up to 25 μg/ml. Conclusions ADEP B315 is safe and can cure mice that have lethal infections of methicillin-sensitive and -resistant strains of S. aureus.

Membrane protein-mediated drug efflux is a phenomenon that compromises our ability to treat both infectious diseases and cancer. Accordingly, there is much interest in the development of strategies for suppression of the mechanisms by which therapeutic agents are effluxed. Here, using resistance to the cyclic acyldepsipeptide (ADEP) antibacterial agents as a model, we demonstrate a new counter-efflux strategy wherein a fragment of an actively exported bioactive compound competitively interferes with its efflux and potentiates its activity. A fragment comprising the N-heptenoyldifluorophenylalanine side chain of the pharmacologically optimized ADEPs potentiates the antibacterial activity of the ADEPs against actinobacteria to a greater extent than reserpine, a well-known efflux inhibitor. Beyond their validation of a new approach to studying molecular recognition by drug efflux pumps, our findings have important implications for killing Mycobacterium tuberculosis with ADEPs and reclaiming the efficacies of therapeutic agents whose activity has been compromised by efflux pumps.

The cyclic acyldepsipeptide (ADEP) antibiotics act by binding the ClpP peptidase and dysregulating its activity. Their exocyclic N‐acylphenylalanine is thought to structurally mimic the ClpP‐binding, (I/L)GF tripeptide loop of the peptidase's accessory ATPases. We found that ADEP analogues with exocyclic N‐acyl tripeptides or dipeptides resembling the (I/L)GF motif were weak ClpP activators and had no bioactivity. In contrast, ADEP analogues possessing difluorophenylalanine N‐capped with methyl‐branched acyl groups—like the side chains of residues in the (I/L)GF motifs—were superior to the parent ADEP with respect to both ClpP activation and bioactivity. We contend that the ADEP's N‐acylphenylalanine moiety is not simply a stand‐in for the ATPases' (I/L)GF motif; it likely has physicochemical properties that are better suited for ClpP binding. Further, our finding that the methyl‐branching on the acyl group of the ADEPs improves activity opens new avenues for optimization.

The cyclic acyldepsipeptide (ADEP) antibiotics are a new class of antibacterial agents that kill bacteria via a mechanism that is distinct from all clinically used drugs. These molecules bind and dysregulate the activity of the ClpP peptidase. The potential of these antibiotics as antibacterial drugs has been enhanced by the elimination of pharmacological liabilities through medicinal chemistry efforts. Here, we demonstrate that the ADEP conformation observed in the ADEP-ClpP crystal structure is fortified by transannular hydrogen bonding and can be further stabilized by judicious replacement of constituent amino acids within the peptidolactone core structure with more conformationally constrained counterparts. Evidence supporting constraint of the molecule into the bioactive conformer was obtained by measurements of deuterium-exchange kinetics of hydrogens that were proposed to be engaged in transannular hydrogen bonds. We show that the rigidified ADEP analogs bind and activate ClpP at lower concentrations in vitro. Remarkably, these compounds have up to 1200-fold enhanced antibacterial activity when compared to those with the peptidolactone core structure common to two ADEP natural products. This study compellingly demonstrates how rational modulation of conformational dynamics may be used to improve the bioactivities of natural products.

This work (pp. E4551–E4559) establishes a fluorescence labeling method that can be used in living cells to derivatize specific proteins of interest with a small red fluorophore, resorufin. The method has extremely high sequence specificity and is based on a computationally designed fluorophore ligase, derived from Escherichia coli lipoic acid ligase. These results demonstrate the power of computational design to majorly reengineer enzyme specificity. Extensive screening of rationally designed enzyme mutants failed to achieve the same result.