SummaryWidespread resistance to first-line TB drugs is a major problem that will likely only be resolved through the development of new drugs with novel mechanisms of action. We have used structure-guided methods to develop a lead molecule that targets the thioesterase activity of polyketide synthase Pks13, an essential enzyme that forms mycolic acids, required for the cell wall of Mycobacterium tuberculosis. Our lead, TAM16, is a benzofuran class inhibitor of Pks13 with highly potent in vitro bactericidal activity against drug-susceptible and drug-resistant clinical isolates of M. tuberculosis. In multiple mouse models of TB infection, TAM16 showed in vivo efficacy equal to the first-line TB drug isoniazid, both as a monotherapy and in combination therapy with rifampicin. TAM16 has excellent pharmacological and safety profiles, and the frequency of resistance for TAM16 is ∼100-fold lower than INH, suggesting that it can be developed as a new antitubercular aimed at the acute infection.PaperClip

New antibiotics are needed to combat rising resistance, with new Mycobacterium tuberculosis (Mtb) drugs of highest priority. Conventional whole-cell and biochemical antibiotic screens have failed. We developed a novel strategy termed PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets) in which we screen compounds against pools of strains depleted for essential bacterial targets. We engineered strains targeting 474 Mtb essential genes and screened pools of 100-150 strains against activity-enriched and unbiased compounds libraries, measuring > 8.5-million chemical-genetic interactions. Primary screens identified > 10-fold more hits than screening wild-type Mtb alone, with chemical-genetic interactions providing immediate, direct target insight. We identified > 40 novel compounds targeting DNA gyrase, cell wall, tryptophan, folate biosynthesis, and RNA polymerase, as well as inhibitors of a novel target EfpA. Chemical optimization yielded EfpA inhibitors with potent wild-type activity, thus demonstrating PROSPECT's ability to yield inhibitors against novel targets which would have eluded conventional drug discovery.

Itaconate is an immunometabolite with both anti-inflammatory and bactericidal effects. Its coenzyme A (CoA) derivative, itaconyl-CoA, inhibits B12-dependent methylmalonyl-CoA mutase (MCM) by an unknown mechanism. We demonstrate that itaconyl-CoA is a suicide inactivator of human and Mycobacterium tuberculosis MCM, which forms a markedly air-stable biradical adduct with the 5′-deoxyadenosyl moiety of the B12 coenzyme. Termination of the catalytic cycle in this way impairs communication between MCM and its auxiliary repair proteins. Crystallography and spectroscopy of the inhibited enzyme are consistent with a metal-centered cobalt radical ~6 angstroms away from the tertiary carbon-centered radical and suggest a means of controlling radical trajectories during MCM catalysis. Mycobacterial MCM thus joins enzymes in the glyoxylate shunt and the methylcitrate cycle as targets of itaconate in pathogen propionate metabolism.

ycobacterium tuberculosis kills more humans than any other pathogen 1 . Whereas most bacterial pathogens cause acute disease, Mtb usually undergoes a years-long infection cycle. Mtb persists in humans in part through parasitism of macrophage phagosomes. Survival in this intracellular niche is accomplished by slowing phagosomal maturation and reducing intracellular killing mechanisms 2-4 , while offering partial cloaking from immune cells and access to lipids and other host nutrients 5,6 . As Mtb interactions with the host play out over years and at diverse anatomical sites, pinpointing specific events that determine tuberculosis (TB) disease outcome is challenging. However, a successful approach has been the comparative profiling of mycobacteria of varying virulence to discover factors selectively present in highly virulent species. Mycobacterium species naturally differ in their potential to infect, persist and cause TB, and transmit among hosts. With an estimated 1.7 billion infections worldwide 1 , only Mtb has broadly colonized the human species, and humans represent its only natural host. These observations highlight the need to identify factors selectively expressed in Mtb but not in other mycobacterial species.Comparative genomics and transcriptomics of Mtb and Bacille Calmette-Guèrin (BCG) have isolated factors selectively present in Mtb, such as the ESX-1 transporter 7 . Whereas genetic techniques are widely used, comparative chemical biology screens are uncommon in mycobacteria. An HPLC-mass spectrometry (MS)-based lipidomics platform was developed for analysis of all chloroform/methanol-extractable mycobacterial lipids 8,9 . Comparative lipidomics of Mtb and BCG identified a previously unknown, Mtb-specific lipid missed by genomics approaches: 1-tuberculosinyladenosine (1-TbAd, 1) 10 . Cyclization of geranylgeranyl pyrophosphate into tuberculosinyl pyrophosphate occurs via the enzyme, Rv3377c, and tuberculosinyl transferase (Rv3378c) generates 1-TbAd, which can chemically rearrange to N 6 -TbAd (2) [10][11][12] . So far 1-TbAd has been detected only in Mtb 12 , so its expression correlates with evolved virulence. However, 1-TbAd has been studied only in laboratoryadapted strains 12,13 , and the extent to which it is produced by patientderived Mtb strains remains unknown.Furthermore, 1-TbAd's function remains unknown. Transposon inactivation of Rv3377c or Rv3378c reduced Mtb uptake, phagosomal acidification and killing of Mtb in mouse macrophages 14 . Therefore, 1-TbAd might influence some aspects of these processes in host cells. However, any host receptor, receptor-independent mechanism or other target of 1-TbAd in host cells remains unknown. Commonly used bioinformatic predictors were not helpful for understanding 1-TbAd function, because it was not possible to identify orthologous biosynthetic genes or similar 1-linked purines in other species. Therefore, diverse candidate mechanisms