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Although not currently in the infectious disease spotlight, there is still a pressing need for new agents to treat tuberculosis caused by Mycobacterium tuberculosis . As there is an ever-increasing amount of clinical resistance to the current drugs, ideally new drugs would be found against novel targets to circumvent pre-existing resistance. A phenotypic growth screen identified a novel singleton, 1 , as an inhibitor of M. tuberculosis growth. Mechanism-of-action studies determined that 1 targeted Pks13, an essential enzyme in cell wall biosynthesis that, as of yet, has not been targeted by agents in the clinic. The reactive nature of the pentafluorophenyl warhead meant that the molecule was inherently metabolically unstable. A medicinal chemistry optimization program is described that resulted in the identification of a compound that was reactive enough to still inhibit Pks13 and M. tuberculosis growth while being metabolically stable enough to explore in vivo.

While treatment options for human African trypanosomiasis (HAT) have improved significantly, there is still a need for new drugs with eradication now a realistic possibility. Here, we report the development of 2,4-diaminothiazoles that demonstrate significant potency against Trypanosoma brucei , the causative agent of HAT. Using phenotypic screening to guide structure-activity relationships, potent drug-like inhibitors were developed. Proof of concept was established in an animal model of the hemolymphatic stage of HAT. To treat the meningoencephalitic stage of infection, compounds were optimized for pharmacokinetic properties, including blood-brain barrier penetration. However, in vivo efficacy was not achieved, in part due to compounds evolving from a cytocidal to a cytostatic mechanism of action. Subsequent studies identified a nonessential kinase involved in the inositol biosynthesis pathway as the molecular target of these cytostatic compounds. These studies highlight the need for cytocidal drugs for the treatment of HAT and the importance of static-cidal screening of analogues.

Enzymes involved in rescuing stalled ribosomes and recycling translation machinery are ubiquitous in bacteria and required for growth. Peptidyl tRNA drop-off is a type of abortive translation that results in the release of a truncated peptide that is still bound to tRNA (peptidyl tRNA) into the cytoplasm. Peptidyl tRNA hydrolase (Pth) recycles the released tRNA by cleaving off the unfinished peptide and is essential in most bacteria. We developed a sequencing-based strategy called copper sulfate-based tRNA sequencing (Cu-tRNAseq) to study the physiological role of Pth in Mycobacterium tuberculosis (Mtb). While most peptidyl tRNA species accumulated in a strain with impaired Pth expression, peptidyl prolyl-tRNA was particularly enriched, suggesting that Pth is required for robust peptidyl prolyl-tRNA turnover. Reducing Pth levels increased Mtb's susceptibility to tRNA synthetase inhibitors that are in development to treat tuberculosis (TB) and rendered this pathogen highly susceptible to macrolides, drugs that are ordinarily ineffective against Mtb. Collectively, our findings reveal the potency of Cu-tRNAseq for profiling peptidyl tRNAs and suggest that targeting Pth would open new therapeutic approaches for TB. IMPORTANCE Peptidyl tRNA hydrolase (Pth) is an enzyme that cuts unfinished peptides off tRNA that has been prematurely released from a stalled ribosome. Pth is essential in nearly all bacteria, including the pathogen Mycobacterium tuberculosis (Mtb), but it has not been clear why. We have used genetic and novel biochemical approaches to show that when Pth levels decline in Mtb, peptidyl tRNA accumulates to such an extent that usable tRNA pools drop. Thus, Pth is needed to maintain normal tRNA levels, most strikingly for prolyl-tRNAs. Many antibiotics act on protein synthesis and could be affected by altering the availability of tRNA. This is certainly true for tRNA synthetase inhibitors, several of which are drug candidates for tuberculosis. We find that their action is potentiated by Pth depletion. Furthermore, Pth depletion results in hypersensitivity to macrolides, drugs that are not active enough under ordinary circumstances to be useful for tuberculosis.

Tuberculosis is a major global cause of both mortality and financial burden mainly in low and middle-income countries. Given the significant and ongoing rise of drug-resistant strains of Mycobacterium tuberculosis within the clinical setting, there is an urgent need for the development of new, safe and effective treatments. Here the development of a drug-like series based on a fused dihydropyrrolidino-pyrimidine scaffold is described. The series has been developed against M. tuberculosis lysyl-tRNA synthetase (LysRS) and cellular studies support this mechanism of action. DDD02049209, the lead compound, is efficacious in mouse models of acute and chronic tuberculosis and has suitable physicochemical, pharmacokinetic properties and an in vitro safety profile that supports further development. Importantly, preliminary analysis using clinical resistant strains shows no pre-existing clinical resistance towards this scaffold., (© 2022. The Author(s).)

With the emergence of multi-drug-resistant strains of Mycobacterium tuberculosis, there is a pressing need for new oral drugs with novel mechanisms of action. A number of scaffolds with potent anti-tubercular in vitro activity have been identified from phenotypic screening that appear to target MmpL3. However, the scaffolds are typically lipophilic, which facilitates partitioning into hydrophobic membranes, and several contain basic amine groups. Highly lipophilic basic amines are typically cytotoxic against mammalian cell lines and have associated off-target risks, such as inhibition of human ether-à-go-go related gene (hERG) and IKr potassium current modulation. The spirocycle compound 3 was reported to target MmpL3 and displayed promising efficacy in a murine model of acute tuberculosis (TB) infection. However, this highly lipophilic monobasic amine was cytotoxic and inhibited the hERG ion channel. Herein, the related spirocycles ( 1-2 ) are described, which were identified following phenotypic screening of the Eli Lilly corporate library against M. tuberculosis . The novel N-alkylated pyrazole portion offered improved physicochemical properties, and optimization led to identification of a zwitterion series, exemplified by lead 29 , with decreased HepG2 cytotoxicity as well as limited hERG ion channel inhibition. Strains with mutations in MmpL3 were resistant to 29 , and under replicating conditions, 29 demonstrated bactericidal activity against M. tuberculosis . Unfortunately, compound 29 had no efficacy in an acute model of TB infection; this was most likely due to the in vivo exposure remaining above the minimal inhibitory concentration for only a limited time., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

The leishmaniases are diseases that affect millions of people across the world, in particular visceral leishmaniasis (VL) which is fatal unless treated. Current standard of care for VL suffers from multiple issues and there is a limited pipeline of new candidate drugs. As such, there is a clear unmet medical need to identify new treatments. This paper describes the optimization of a phenotypic hit against Leishmania donovani, the major causative organism of VL. The key challenges were to balance solubility and metabolic stability while maintaining potency. Herein, strategies to address these shortcomings and enhance efficacy are discussed, culminating in the discovery of preclinical development candidate GSK3186899/DDD853651 (1) for VL.

With the emergence of multidrug-resistant strains of Mycobacterium tuberculosis there is a pressing need for new oral drugs with novel mechanisms of action. Herein, we describe the identification of a novel morpholino-thiophenes (MOT) series following phenotypic screening of the Eli Lilly corporate library against M. tuberculosis strain H37Rv. The design, synthesis, and structure-activity relationships of a range of analogues around the confirmed actives are described. Optimized leads with potent whole cell activity against H37Rv, no cytotoxicity flags, and in vivo efficacy in an acute murine model of infection are described. Mode-of-action studies suggest that the novel scaffold targets QcrB, a subunit of the menaquinol cytochrome c oxidoreductase, part of the bc1-aa3-type cytochrome c oxidase complex that is responsible for driving oxygen-dependent respiration.