Toxoplasma gondii is a globally important intracellular parasite, and treatment regimens are limited by the failure of drugs to target latent tissue cysts. Developing new candidates for treatment also needs to address the potential for resistance to arise. Herein, we developed a minimum inoculum for resistance assay as a semiquantitative metric for evaluating inhibitors of T. gondii. The resistance assay, adapted from malaria, measures the frequency of pre-existing resistance alleles by exposing different-sized parasite populations to drug pressure. We profiled a series of bicyclic pyrrolidone analogues that inhibit phenylalanine tRNA synthetase. We demonstrate that these inhibitors require higher inocula to lead to parasite resistance (up to >10(8) parasites) in comparison with an inhibitor of DNA synthesis and that resistance values vary across inhibitors with closely related chemical structures. Clonal analysis of resistant parasites emerging from resistance assays revealed both new and previously identified resistance-conferring mutations in T. gondii phenylalanine tRNA synthetase, and structural modeling revealed their potential impact on the enzyme active site. The minimum inoculum for resistance assay provides a functional benchmark to compare new and existing inhibitors, allowing for rational prioritization of lead compounds with a high genetic barrier to resistance.

Dengue virus (DENV) is a mosquito-transmitted flavivirus that circulates globally as four distinct serotypes and poses a substantial threat to public health. There are an estimated ~96 million symptomatic infections yearly, including severe cases of dengue fever, underscoring the urgency of identifying effective therapeutics targeting all four serotypes. Nucleoside analogs, which mimic endogenous nucleosides to inhibit viral RNA replication, offer a promising strategy for broad-spectrum antiviral development. Here, we conducted a high-throughput screen of 1,101 nucleoside analogs against DENV serotype 2 (DENV2) in a panel of human cell models, including human epithelial cells, hepatocytes, and fibroblasts. Candidates that were active against DENV2 were screened against all four serotypes. Since flaviviruses including West Nile virus and Zika virus are also important human pathogens, we screened these compounds for activity and identified compounds that were broadly active in these cellular and viral models. We further evaluated antivirals in primary human keratinocytes and fibroblasts, which are early targets of mosquito-transmitted DENV infection. From this screen, we identified 23 nucleoside analogs with broad antiviral activity against DENV and focused on two purine analogs UPGNUC255 and UPGNUC558, that demonstrated potent pan-flaviviral activity achieving >10-fold viral load reduction across all four DENV serotypes and other flaviviruses across cell models. Mechanistic studies revealed that both compounds target the viral RNA-dependent RNA polymerase (RdRp) domain of NS5. Resistance to UPGNUC558 was associated with a conserved S604T substitution, conferring cross-resistance to other 2'C-substituted nucleoside analogs. Resistance to UPGNUC255 was linked to a previously unknown R355Q mutation, located near the catalytic GDD motif of RdRp. These findings highlight UPGNUC255 and UPGNUC558 as promising leads for the development of broad-spectrum antiviral agents against flaviviruses.

Vector-borne diseases represent a significant global health concern, and effective vector control in animals often involves using orally administered drugs that kill arthropod vectors of human pathogens. Isoxazoline ectoparasiticides may have promise in humans if they can be optimized for safe use due to their selectivity for invertebrate over mammalian ion channels. Yet, isoxazolines can cause neurological side effects due to their ability to cross the blood brain barrier, and thus, we synthesized novel isoxazolines with improved physiochemical properties to reduce brain exposure without reducing toxicity to arthropod pests. Our medicinal chemistry campaigns led to the discovery of lead compound mCMV280 that is 3× more toxic to ticks and equitoxic to mosquitoes, with an ∼5× reduction in mammalian brain exposure and an ∼8× lower brain-to-plasma ratio compared to fluralaner. These findings highlight the promise of new isoxazoline scaffolds for safer and more effective drug-based vector control strategies in humans.

Developing safe and effective treatments against emerging RNA viruses is an important goal in pandemic preparedness efforts. 4'-fluorouridine (4'-FlU) is a broad-spectrum antiviral that was shown to inhibit viral RNA-dependent RNA polymerases (RdRps). Given its notable range of antiviral activity, this class of nucleoside analogs warrants further investigation. Here, we studied the antiviral activity and underlying mechanism of inhibition of 4'-fluoroadenosine (4'-FlA). Like 4'-FlU, 4'-FlA demonstrates a broad-spectrum of antiviral activity against eight prototypic viruses representing diverse families. Enzyme kinetics shows that the triphosphate (4'-fluoroadenosine triphosphate) is efficiently incorporated by viral RdRps. A cryo-EM structure of the RdRp of severe acute respiratory syndrome coronavirus 2 in complex with double-stranded RNA and the incorporated monophosphate (4'-fluoroadenosine monophosphate) characterizes interactions at the active site. The incorporated analog elicits heterogeneous inhibition patterns in primer extension reactions. In contrast, templates with embedded 4'-fluoroadenosine monophosphate inhibit incorporation of complementary uridine triphosphate (UTP) across the viral RdRps. However, incorporation of 4'-fluoroadenosine triphosphate is not limited to viral polymerases and likewise includes human mitochondrial RNA polymerase. These results demonstrate the general potential for 4'-fluorinated nucleotides as antiviral drugs and guide the development of more selective derivatives for medical use in appropriate settings.

Inhibition of phenylalanine tRNA synthetase (PheRS) by bicyclic pyrrolidines provides a potent and specific inhibition of parasite growth. Herein, we describe novel bicyclic pyrrolidines designed to explore structure-activity relationships with Toxoplasma gondii vs human PheRS. Modification of the biaryl alkyne extension, which fits into the phenylalanine-binding site, showed a strong preference for ortho hydroxyl addition over meta and para. Further addition of N to both the proximal and distal phenyl rings of the biaryl alkyne and to the methoxyphenyl urea moiety, which fits into a unique auxiliary site present in the parasite enzyme, identified compounds with reduced plasma protein binding and lower hERG activity. Finally, we identified a potent lead with improved pharmacokinetics, extended plasma exposure, central nervous system penetration, and low-dose cure of acute infection in mouse. Collectively, these findings advance new candidates for the treatment of toxoplasmosis based on selective and potent inhibitors of parasite PheRS.

The coronavirus main protease (Mpro, 3CLpro) remains an attractive target for the development of antiviral therapeutics due to its essential role in viral replication and high sequence conservation across coronaviruses. The oxamide functionality represents a versatile medicinal chemistry motif capable of acting as both a hydrogen-bond donor and acceptor while imparting conformational constraint to bioactive molecules. Building on an oxamide-based hit compound, we designed, synthesized, and evaluated an expanded series of Nterminal oxamide capped peptidomimetics to optimize antiviral potency and developability. Structure-activity relationship studies explored variations in the oxamide P2-cap as a function of proline and non-proline modifications at the P2 motif. Evaluation against SARS-CoV-2 identified multiple analogs with sub-100 nM antiviral activity while maintaining favorable metabolic stability in microsomes. Among these, mCMV911 emerged as a lead candidate, exhibiting broad-spectrum antiviral activity (e.g., ALI-HBEC SARS-CoV-2 EC90 = 19 nM, HCoV-OC43 EC90 = 186 nM, HCoV-229E EC90 = 59 nM), while maintaining favorable aqueous solubility (0.4-0.7 mg/mL), moderate microsomal stability, and oral bioavailability (FPO = 22% in dogs). Furthermore, we discuss the pharmacokinetic properties of mCMV911, and proof-of-concept efficacy data from a coronavirus mouse model.

The use of covalent warheads targeting the catalytic cysteine has been a cornerstone in the coronavirus main protease (Mpro) inhibitor development. Various electrophilic motifs have been explored, including aldehydes, nitriles, ketoamides, and hydroxymethyl ketones (HMKs). Recent efforts have mostly centered around nitrile warheads, given the success of Nirmatrelvir in the clinic. However, it is essential to identify and develop alternative chemotypes with distinct chemical and pharmacological profiles to prepare for future pandemics. Among such alternatives, HMKs are of particular interest because they balance reduced intrinsic electrophilicity with an excellent selectivity profile. Nevertheless, early HMK-based compounds, such as the clinical-stage Mpro inhibitor PF-00835231, suffered from poor oral bioavailability and therefore required intravenous administration, with or without prodrug derivatization of the hydroxyl group. In this work, we describe our efforts to advance the HMK field by discovering mCMX110, a lead compound that exhibits superior potency, increased unbound exposure in vivo, and favorable oral bioavailability in preclinical studies.

ABSTRACT The use of covalent warheads targeting the catalytic cysteine has been a cornerstone in coronavirus main protease (M pro ) inhibitor development, where various electrophilic motifs have been used including aldehydes, nitriles, ketoamides, and hydroxymethyl ketones (HMKs). Recent efforts have been mostly centered around nitrile warheads, given the success of compounds like Nirmatrelvir and Ensitrelvir in the clinic. However, finding and advancing alternative chemotypes with differentiating chemical and pharmacological profiles is essential for future pandemic preparedness. Among such alternatives, HMKs hold special interest because they balance reduced intrinsic electrophilicity with an excellent selectivity profile. Nevertheless, early HMK-based compounds, such as the clinical-stage M pro inhibitor PF-00835231, suffered from poor oral bioavailability and therefore required intravenous administration, with or without prodrug derivatization of the hydroxyl group. Here, we describe our efforts in advancing the HMK field via the discovery of mCMX110 , a lead that has superior potency, increased unbound exposure in vivo , and favorable oral bioavailability in preclinical studies. Graphical Abstract

Mycobacterium tuberculosis (Mtb) remains the world's deadliest bacterial pathogen1. There is an urgent medical need to develop new drugs that shorten the treatment duration to combat widespread multi-drug-resistant and extensive-drug-resistant Mtb. Here, we present a preclinical covalent compound, CMX410, that contains an aryl fluorosulfate (SuFEx)2 warhead and uniquely targets the acyltransferase domain of Pks13, an essential enzyme in cell-wall biosynthesis. CMX410 is equipotent against drug-sensitive and drug-resistant strains of Mtb and efficacious in multiple mouse models of infection. Inhibition by CMX410 is irreversible through a previously undescribed mechanism: CMX410 reacts with the catalytic serine of the AT domain of Pks13, rapidly and irreversibly disabling the active site by forming a β-lactam. CMX410 is highly selective for its target and thus demonstrates excellent pharmacological and safety profiles, including no adverse effects in a 14-day rat toxicity study up to 1,000 mg kg-1 per day. The distinctive mode of action from current drugs, high potency across all tested clinical isolates, oral bioavailability, favourable performance in drug combination testing and superior pharmacological and safety characteristics make CMX410 a promising first-in-class candidate to replace outdated cell-wall biosynthesis inhibitors, such as isoniazid and ethambutol, in tuberculosis regimens.

Toxoplasma gondii causes widespread chronic infections that are not cured by current treatments due to the inability to affect semidormant bradyzoite stages within tissue cysts. To identify compounds to eliminate chronic infection, we developed an HTS using a recently characterized strain of T. gondii that undergoes efficient conversion to bradyzoites in vitro. Stage-specific expression of luciferase was used to selectively monitor the growth inhibition of bradyzoites by the Library of Pharmacological Active Compounds, consisting of 1280 drug-like compounds. We identified 44 compounds with >50% inhibitory effects against bradyzoites, including new highly potent compounds, several of which have precedent for antimicrobial activity. Subsequent characterization of the compound sanguinarine sulfate revealed potent and rapid killing against in vitro-produced bradyzoites and bradyzoites harvested from chronically infected mice, including potent activity against intact cysts. These findings provide a platform for expanded screening and identify promising compounds for further preclinical development against T. gondii bradyzoites that are responsible for chronic infection.

Radical cure of Plasmodium vivax malaria must include elimination of quiescent ‘hypnozoite’ forms in the liver; however, the only FDA-approved treatments are contraindicated in many vulnerable populations. To identify new drugs and drug targets for hypnozoites, we screened the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library and a collection of epigenetic inhibitors against P. vivax liver stages. From both libraries, we identified inhibitors targeting epigenetics pathways as selectively active against P. vivax and P. cynomolgi hypnozoites. These include DNA methyltransferase inhibitors as well as several inhibitors targeting histone post-translational modifications. Immunofluorescence staining of Plasmodium liver forms showed strong nuclear 5-methylcystosine signal, indicating liver stage parasite DNA is methylated. Using bisulfite sequencing, we mapped genomic DNA methylation in sporozoites, revealing DNA methylation signals in most coding genes. We also demonstrated that methylation level in proximal promoter regions as well as in the first exon of the genes may affect, at least partially, gene expression in P. vivax . The importance of selective inhibitors targeting epigenetic features on hypnozoites was validated using MMV019721, an acetyl-CoA synthetase inhibitor that affects histone acetylation and was previously reported as active against P. falciparum blood stages. In summary, our data indicate that several epigenetic mechanisms are likely modulating hypnozoite formation or persistence and provide an avenue for the discovery and development of improved radical cure antimalarials.

Screening of the ChemDiv molecular library in cholesterol media against Mycobacterium tuberculosis (Mtb) H37Rv strain identified a novel isoxazole thiophene hit as a putative Rv1625c/Cya activator with a promising in vitro activity and good pharmacokinetic properties. Twenty-nine analogs were synthesized to assess the structure-activity relationships (SAR) to further improve potency. The most notable analog was P15, which showed an intramacrophage EC50 = 1.96 mu M and exhibited 58.0 % oral bioavailability when it was dosed orally at 20 mg/kg in a mouse pharmacokinetic (PK) study. The overall medicinal chemistry campaign revealed limited SAR that did not support further investigation into this series.

The papain-like protease (PLpro) is a highly conserved domain encoded by the coronavirus (CoV) genome and it plays an essential role in the replication and maturation of the virus in addition to weakening host immune response. Due to the virus's reliance on PLpro for survival and propagation, small-molecule inhibitors of PLpro serve as an attractive model for direct-acting antiviral therapeutic agents against SARS-CoV-2. Building upon existing work aimed at designing covalent inhibitors against PLpro, we report the synthesis and structure-activity relationship of analogs based on the known covalent inhibitor 1 (Sanders, et al.2023). To evaluate the efficacy of synthesized derivatives, we conducted enzymatic inhibition assays, SARS-CoV-2/HeLa-ACE2 cellular potency and toxicity assays, and profiled the most promising analogs via in vitro ADME and in vivo pharmacokinetic studies. Additionally, we describe computational docking of profiled compounds bound to PLpro to elucidate the structure-activity relationship of compounds based on 1 and offer suggestions for optimizing the potency and selectivity of the electrophilic warhead and improving ADME and PK properties for this chemotype. Relative to the parent compound, new designs demonstrate comparable potency and target selectivity for PLpro. The accomplished SAR campaign provides novel insight for future development of antivirals against SARS-CoV-2.

The SARS-CoV-2 frameshift stimulation element (FSE) is a critical RNA structure that is essential for viral replication and represents a promising target for antiviral intervention. Here, Chemical Cross-Linking and Isolation by Pull-down (Chem-CLIP) covalent target validation and binding site mapping was applied, to identify small-molecule binding pockets within the FSE and ultimately develop a ligandability map. These studies employed ∼ 190 Chem-CLIP fragments, including the fluoroquinolone merafloxacin, previously shown to interact with this element. Covalent mapping defined merafloxacin's binding pocket at a nucleotide-level resolution and revealed interactions that, along with structure-based design, efficient one-pot on-plate synthesis and competitive displacement assays, enabled the development of bioactive compounds with antiviral activity. Complementary chemical probing with dimethyl sulfate (DMS) in the presence of a bioactive ligand, coupled to Deconvolution of RNA Alternative Conformations (DRACO), revealed that compound binding increased the reactivity of specific nucleotides with DMS, indicative of changes in local RNA folding. These results highlight the importance of combining Chem-CLIP and DMS profiling to differentiate direct ligand binding from ligand-induced changes in RNA structure. In addition, in silico pocket analysis of FSE structures derived from cryogenic-electron microscopy (cryo-EM) studies identified four recurring cavities, including the experimentally determined merafloxacin and Chem-CLIP fragments binding pockets. Altogether, the findings advance our understanding of RNA-ligand interactions and support a strategy to design and discover small molecules that bind RNA structures.

Drug resistance is a widespread problem across therapeutic areas including malaria, but what accounts for resistance propensity remains poorly understood. Here, we reveal that two HSP90 inhibitors targeting the identical ATP-binding site exhibit dramatically different resistance profiles in P. falciparum. Geldanamycin readily selected 10 distinct resistance mutations conferring up to 22-fold resistance, while AUY-922 required 44 weeks to yield a single A41S mutation with only 2-fold resistance to AUY-922 but not to geldanamycin. Resistance mapping in parasites and yeast revealed geldanamycin resistance mutations distributed throughout the binding pocket whereas AUY-922 resistance mutations localized close to the ATP-binding site. Unexpectedly, the A41S mutation enhanced AUY-922 binding affinity without changing geldanamycin binding. In silico analysis suggested this enhancement occurs through additional hydrogen bonding, yet stronger binding correlated with resistance. In yeast, A41S had opposite effects, hypersensitizing cells to all HSP90 inhibitors tested. Additionally, conditional HSP90 knockdown increased geldanamycin sensitivity but left AUY-922 activity unaffected, indicating different target dependencies despite shared binding sites. Based on these data, we propose a multi-target hypothesis where AUY-922's lower resistance risk stems from engaging multiple HSP90 family members. Our findings reveal how enhanced drug-target binding can paradoxically correlate with resistance and demonstrate that resistance risk cannot be predicted from binding site identity alone, providing insights for developing more durable drugs across therapeutic areas.

The functionally and evolutionarily conserved Hippo-YAP signaling pathway plays a critical role in regulating cellular proliferation, organ size control, and regeneration. Accordingly, activators of YAP-driven transcription hold therapeutic promise for treating disease states driven by insufficient proliferative repair, yet only a handful of pharmacological mechanisms exist for augmenting YAP activity. Here we report the discovery of sCMF231, a small molecule activator of YAP discovered from high throughput screening that acts by targeting the poorly characterized ubiquitin-like protein FUBI. Using canonical ubiquitin conjugation machinery—UBA1, UBE2C, and APC/C—FUBI covalently modifies the Hippo pathway protein Annexin A2, reinforcing its YAP suppressive role at the plasma membrane. Binding of sCMF231 to FUBI discourages its conjugation to Annexin A2, resulting in the membrane delocalization of Annexin A2 and a liberated, transcriptionally active form of YAP. This work provides the first definitive evidence of covalent modification of proteins by FUBI, a post-translational modification termed fubylation, and defines how fubylation regulates the activity of a central growth pathway. ### Competing Interest Statement The authors have declared no competing interest. National Institute of General Medical Sciences, https://ror.org/04q48ey07, GM146865 California Institute for Regenerative Medicine, EDUC4-12811

A focused small-molecule library was screened against extracellular Mycobacterium tuberculosis (Mtb) across four distinct carbon sources that mimic different metabolic states of the pathogen. This screen identified a novel tetrazol-2-yl-acetamide compound, sALT629 (P1), with potent intramacrophage activity (EC50 = 1.5 μM). sALT629 showed broad-spectrum activities across all carbon sources, equipotent efficacy against drug-resistant Mtb, and activity against both slow-replicating and nonreplicating Mtb. Structure-activity relationship (SAR) studies optimized the potency and drug-like properties, leading to analogue P39 with improved intramacrophage activity (EC50 = 0.68 μM) and pharmacokinetics (PK) properties. In mice, P39 achieved a plasma exposure of 58,754 ng/mL and maintained plasma concentrations above EC50 for 16 h after a 20 mg/kg oral dose. Additionally, sALT629 showed good exposure and tolerability after repeated dosing for 4 days at 200 mg/kg once daily (QD) or 100 mg/kg twice daily (BID), indicating low toxicity liability and the potential for further development as an anti-tuberculosis (TB) drug candidate.

Type 2 alveolar epithelial cells (AEC2s) are stem cells in the adult lung that contribute to lower airway repair. Agents that promote the selective expansion of these cells might stimulate regeneration of the compromised alveolar epithelium, an etiology-defining event in several pulmonary diseases. From a high-content imaging screen of the drug repurposing library ReFRAME, we identified that dipeptidyl peptidase 4 (DPP4) inhibitors, widely used type 2 diabetes medications, selectively expand AEC2s and are broadly efficacious in several mouse models of lung damage. Mechanism of action studies revealed that the protease DPP4, in addition to processing incretin hormones, degrades IGF-1 and IL-6, essential regulators of AEC2 expansion whose levels are increased in the luminal compartment of the lung in response to drug treatment. To selectively target DPP4 in the lung with sufficient drug exposure, we developed NZ-97, a locally delivered, lung persistent DPP4 inhibitor that broadly promotes efficacy in mouse lung damage models with minimal peripheral exposure and good tolerability. This work reveals DPP4 as a central regulator of AEC2 expansion and affords a promising therapeutic approach to broadly stimulate regenerative repair in pulmonary disease.

Academic and other non-profit institutions have a long-term vision to improve human health where commercial interests can be limited for profit organizations. Medicinal chemistry to these diseases with no commercial benefit needs is well suited in the academic environment and this chapter outlines some work conducted at Calibr-Skaggs around antibiotic drug development that has led to initiation of multiple clinical trials over the last decade.

There remains a need to develop novel SARS-CoV-2 therapeutic options that improve upon existing therapies by an increased robustness of response, fewer safety liabilities, and global-ready accessibility. Functionally critical viral main protease (Mpro, 3CLpro) of SARS-CoV-2 is an attractive target due to its homology within the coronaviral family, and lack thereof toward human proteases. In this disclosure, we outline the advent of a novel SARS-CoV-2 3CLpro inhibitor, CMX990, bearing an unprecedented trifluoromethoxymethyl ketone warhead. Compared with the marketed drug nirmatrelvir (combination with ritonavir = Paxlovid), CMX990 has distinctly differentiated potency (∼5× more potent in primary cells) and human in vitro clearance (>4× better microsomal clearance and >10× better hepatocyte clearance), with good in vitro-to-in vivo correlation. Based on its compelling preclinical profile and projected once or twice a day dosing supporting unboosted oral therapy in humans, CMX990 advanced to a Phase 1 clinical trial as an oral drug candidate for SARS-CoV-2.