Exploration of the P1 residue in 3CL protease inhibitors leading to the discovery of a 2-tetrahydrofuran P1 replacement.

The virally encoded 3C-like protease (3CLpro) is a well-validated drug target for the inhibition of coronaviruses including Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Most inhibitors of 3CLpro are peptidomimetic, with a γ-lactam in place of Gln at the P1 position of the pseudopeptide chain. An effort was pursued to identify a viable alternative to the γ-lactam P1 mimetic which would improve physicochemical properties while retaining affinity for the target. Discovery of a 2-tetrahydrofuran as a suitable P1 replacement that is a potent enzymatic inhibitor of 3CLpro in SARS-CoV-2 virus is described herein.

Cyclic AMP-Mediated Inhibition of Cholesterol Catabolism in Mycobacterium tuberculosis by the Novel Drug Candidate GSK2556286.

Despite the deployment of combination tuberculosis (TB) chemotherapy, efforts to identify shorter, nonrelapsing treatments have resulted in limited success. Recent evidence indicates that GSK2556286 (GSK286), which acts via Rv1625c, a membrane-bound adenylyl cyclase in Mycobacterium tuberculosis, shortens treatment in rodents relative to standard of care drugs. Moreover, GSK286 can replace linezolid in the three-drug, Nix-TB regimen. Given its therapeutic potential, we sought to better understand the mechanism of action of GSK286. The compound blocked growth of M. tuberculosis in cholesterol media and increased intracellular cAMP levels ~50-fold. GSK286 did not inhibit growth of an rv1625c transposon mutant in cholesterol media and did not induce cyclic AMP (cAMP) production in this mutant, suggesting that the compound acts on this adenylyl cyclase. GSK286 also induced cAMP production in Rhodococcus jostii RHA1, a cholesterol-catabolizing actinobacterium, when Rv1625c was heterologously expressed. However, these elevated levels of cAMP did not inhibit growth of R. jostii RHA1 in cholesterol medium. Mutations in rv1625c conferred cross-resistance to GSK286 and the known Rv1625c agonist, mCLB073. Metabolic profiling of M. tuberculosis cells revealed that elevated cAMP levels, induced using either an agonist or a genetic tool, did not significantly affect pools of steroid metabolites in cholesterol-incubated cells. Finally, the inhibitory effect of agonists was not dependent on the N-acetyltransferase MtPat. Together, these data establish that GSK286 is an Rv1625c agonist and sheds light on how cAMP signaling can be manipulated as a novel antibiotic strategy to shorten TB treatments. Nevertheless, the detailed mechanism of action of these compounds remains to be elucidated.

GSK2556286 Is a Novel Antitubercular Drug Candidate Effective In Vivo with the Potential To Shorten Tuberculosis Treatment.

As a result of a high-throughput compound screening campaign using Mycobacterium tuberculosis-infected macrophages, a new drug candidate for the treatment of tuberculosis has been identified. GSK2556286 inhibits growth within human macrophages (50% inhibitory concentration [IC50] = 0.07 µM), is active against extracellular bacteria in cholesterol-containing culture medium, and exhibits no cross-resistance with known antitubercular drugs. In addition, it has shown efficacy in different mouse models of tuberculosis (TB) and has an adequate safety profile in two preclinical species. These features indicate a compound with a novel mode of action, although still not fully defined, that is effective against both multidrug-resistant (MDR) or extensively drug-resistant (XDR) and drug-sensitive (DS) M. tuberculosis with the potential to shorten the duration of treatment in novel combination drug regimens. (This study has been registered at ClinicalTrials.gov under identifier NCT04472897).

1,3-Diarylpyrazolyl-acylsulfonamides Target HadAB/BC Complex in Mycobacterium tuberculosis.

Alternative mode-of-inhibition of clinically validated targets is an effective strategy for circumventing existing clinical drug resistance. Herein, we report 1,3-diarylpyrazolyl-acylsulfonamides as potent inhibitors of HadAB/BC, a 3-hydroxyl-ACP dehydratase complex required to iteratively elongate the meromycolate chain of mycolic acids in Mycobacterium tuberculosis (Mtb). Mutations in compound 1-resistant Mtb mutants mapped to HadC (Rv0637; K157R), while chemoproteomics confirmed the compound's binding to HadA (Rv0635), HadB (Rv0636), and HadC. The compounds effectively inhibited the HadAB and HadBC enzyme activities and affected mycolic acid biosynthesis in Mtb, in a concentration-dependent manner. Unlike known 3-hydroxyl-ACP dehydratase complex inhibitors of clinical significance, isoxyl and thioacetazone, 1,3-diarylpyrazolyl-acylsulfonamides did not require activation by EthA and thus are not liable to EthA-mediated resistance. Further, the crystal structure of a key compound in a complex with Mtb HadAB revealed unique binding interactions within the active site of HadAB, providing a useful tool for further structure-based optimization of the series.

The multi-target aspect of an MmpL3 inhibitor: The BM212 series of compounds bind EthR2, a transcriptional regulator of ethionamide activation.

The emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb) ensures that drug discovery efforts remain at the forefront of TB research. There are multiple different experimental approaches that can be employed in the discovery of anti-TB agents. Notably, inhibitors of MmpL3 are numerous and structurally diverse in Mtb and have been discovered through the generation of spontaneous resistant mutants and subsequent whole genome sequencing studies. However, this approach is not always reliable and can lead to incorrect target assignment and requires orthogonal confirmatory approaches. In fact, many of these inhibitors have also been shown to act as multi-target agents, with secondary targets in Mtb, as well as in other non-MmpL3-containing pathogens. Herein, we have investigated further the cellular targets of the MmpL3-inhibitor BM212 and a number of BM212 analogues. To determine the alternative targets of BM212, which may have been masked by MmpL3 mutations, we have applied a combination of chemo-proteomic profiling using bead-immobilised BM212 derivatives and protein extracts, along with whole-cell and biochemical assays. The study identified EthR2 (Rv0078) as a protein that binds BM212 analogues. We further demonstrated binding of BM212 to EthR2 through an in vitro tryptophan fluorescence assay, which showed significant quenching of tryptophan fluorescence upon addition of BM212. Our studies have demonstrated the value of revisiting drugs with ambiguous targets, such as MmpL3, in an attempt to find alternative targets and the study of off-target effects to understand more precisely target engagement of new hits emerging from drug screening campaigns.

Ca2+ signals critical for egress and gametogenesis in malaria parasites depend on a multipass membrane protein that interacts with PKG.

Calcium signaling regulated by the cGMP-dependent protein kinase (PKG) controls key life cycle transitions in the malaria parasite. However, how calcium is mobilized from intracellular stores in the absence of canonical calcium channels in Plasmodium is unknown. Here, we identify a multipass membrane protein, ICM1, with homology to transporters and calcium channels that is tightly associated with PKG in both asexual blood stages and transmission stages. Phosphoproteomic analyses reveal multiple ICM1 phosphorylation events dependent on PKG activity. Stage-specific depletion of Plasmodium berghei ICM1 prevents gametogenesis due to a block in intracellular calcium mobilization, while conditional loss of Plasmodium falciparum ICM1 is detrimental for the parasite resulting in severely reduced calcium mobilization, defective egress, and lack of invasion. Our findings suggest that ICM1 is a key missing link in transducing PKG-dependent signals and provide previously unknown insights into atypical calcium homeostasis in malaria parasites essential for pathology and disease transmission.

Inhibition of Resistance-Refractory P. falciparum Kinase PKG Delivers Prophylactic, Blood Stage, and Transmission-Blocking Antiplasmodial Activity.

The search for antimalarial chemotypes with modes of action unrelated to existing drugs has intensified with the recent failure of first-line therapies across Southeast Asia. Here, we show that the trisubstituted imidazole MMV030084 potently inhibits hepatocyte invasion by Plasmodium sporozoites, merozoite egress from asexual blood stage schizonts, and male gamete exflagellation. Metabolomic, phosphoproteomic, and chemoproteomic studies, validated with conditional knockdown parasites, molecular docking, and recombinant kinase assays, identified cGMP-dependent protein kinase (PKG) as the primary target of MMV030084. PKG is known to play essential roles in Plasmodium invasion of and egress from host cells, matching MMV030084's activity profile. Resistance selections and gene editing identified tyrosine kinase-like protein 3 as a low-level resistance mediator for PKG inhibitors, while PKG itself never mutated under pressure. These studies highlight PKG as a resistance-refractory antimalarial target throughout the Plasmodium life cycle and promote MMV030084 as a promising Plasmodium PKG-targeting chemotype.

Novel Antitubercular 6-Dialkylaminopyrimidine Carboxamides from Phenotypic Whole-Cell High Throughput Screening of a SoftFocus Library: Structure-Activity Relationship and Target Identification Studies.

A BioFocus DPI SoftFocus library of ∼35 000 compounds was screened against Mycobacterium tuberculosis (Mtb) in order to identify novel hits with antitubercular activity. The hits were evaluated in biology triage assays to exclude compounds suggested to function via frequently encountered promiscuous mechanisms of action including inhibition of the QcrB subunit of the cytochrome bc1 complex, disruption of cell-wall homeostasis, and DNA damage. Among the hits that passed this screening cascade, a 6-dialkylaminopyrimidine carboxamide series was prioritized for hit to lead optimization. Compounds from this series were active against clinical Mtb strains, while no cross-resistance to conventional antituberculosis drugs was observed. This suggested a novel mechanism of action, which was confirmed by chemoproteomic analysis leading to the identification of BCG_3193 and BCG_3827 as putative targets of the series with unknown function. Initial structure-activity relationship studies have resulted in compounds with moderate to potent antitubercular activity and improved physicochemical properties.

Essential but Not Vulnerable: Indazole Sulfonamides Targeting Inosine Monophosphate Dehydrogenase as Potential Leads against Mycobacterium tuberculosis.

A potent, noncytotoxic indazole sulfonamide was identified by high-throughput screening of >100,000 synthetic compounds for activity against Mycobacterium tuberculosis (Mtb). This noncytotoxic compound did not directly inhibit cell wall biogenesis but triggered a slow lysis of Mtb cells as measured by release of intracellular green fluorescent protein (GFP). Isolation of resistant mutants followed by whole-genome sequencing showed an unusual gene amplification of a 40 gene region spanning from Rv3371 to Rv3411c and in one case a potential promoter mutation upstream of guaB2 (Rv3411c) encoding inosine monophosphate dehydrogenase (IMPDH). Subsequent biochemical validation confirmed direct inhibition of IMPDH by an uncompetitive mode of inhibition, and growth inhibition could be rescued by supplementation with guanine, a bypass mechanism for the IMPDH pathway. Beads containing immobilized indazole sulfonamides specifically interacted with IMPDH in cell lysates. X-ray crystallography of the IMPDH-IMP-inhibitor complex revealed that the primary interactions of these compounds with IMPDH were direct pi-pi interactions with the IMP substrate. Advanced lead compounds in this series with acceptable pharmacokinetic properties failed to show efficacy in acute or chronic murine models of tuberculosis (TB). Time-kill experiments in vitro suggest that sustained exposure to drug concentrations above the minimum inhibitory concentration (MIC) for 24 h were required for a cidal effect, levels that have been difficult to achieve in vivo. Direct measurement of guanine levels in resected lung tissue from tuberculosis-infected animals and patients revealed 0.5-2 mM concentrations in caseum and normal lung tissue. The high lesional levels of guanine and the slow lytic, growth-rate-dependent effect of IMPDH inhibition pose challenges to developing drugs against this target for use in treating TB.