SignificanceMany bacteria produce antimicrobial peptides for survival under stressful conditions. Some of these antimicrobial peptides are lasso peptides, which have a unique lasso-like topology and have generated great interest as a result of their stability in harsh conditions and amenability to functional engineering. In this study, we determined crystal structures of two lasso peptides, microcin J25 and capistruin, bound to their natural enzymatic target, the bacterial RNA polymerase (RNAP). The structures define peptide inhibitor–RNAP interactions that are important for inhibition and provide detailed insight into how the peptides inhibit RNAP function. This work provides a structural basis to guide the design of more potent lasso peptide antimicrobial approaches.

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Rifamycin antibiotics (Rifs) target bacterial RNA polymerases (RNAPs) and are widely used to treat infections including tuberculosis. The utility of these compounds is threatened by the increasing incidence of resistance (RifR). As resistance mechanisms found in clinical settings may also occur in natural environments, here we postulated that bacteria could have evolved to produce rifamycin congeners active against clinically relevant resistance phenotypes. We survey soil metagenomes and identify a tailoring enzyme-rich family of gene clusters encoding biosynthesis of rifamycin congeners (kanglemycins, Kangs) with potent in vivo and in vitro activity against the most common clinically relevant RifR mutations. Our structural and mechanistic analyses reveal the basis for Kang inhibition of RifR RNAP. Unlike Rifs, Kangs function through a mechanism that includes interfering with 5′-initiating substrate binding. Our results suggest that examining soil microbiomes for new analogues of clinically used antibiotics may uncover metabolites capable of circumventing clinically important resistance mechanisms.

Rifampicin (Rif) is a first-line therapeutic used to treat the infectious disease tuberculosis (TB), which is caused by the pathogenMycobacterium tuberculosis(Mtb). The emergence of Rif-resistant (RifR)Mtbpresents a need for new antibiotics. Rif targets the enzyme RNA polymerase (RNAP). Sorangicin A (Sor) is an unrelated inhibitor that binds in the Rif-binding pocket of RNAP. Sor inhibits a subset of RifRRNAPs, including the most prevalent clinical RifRRNAP substitution found inMtbinfected patients (S456>L of the β subunit). Here, we present structural and biochemical data demonstrating that Sor inhibits the wild-typeMtbRNAP by a similar mechanism as Rif: by preventing the translocation of very short RNAs. By contrast, Sor inhibits the RifRS456L enzyme at an earlier step, preventing the transition of a partially unwound promoter DNA intermediate to the fully opened DNA and blocking the template-strand DNA from reaching the active site in the RNAP catalytic center. By defining template-strand blocking as a mechanism for inhibition, we provide a mechanistic drug target in RNAP. Our finding that Sor inhibits the wild-type and mutant RNAPs through different mechanisms prompts future considerations for designing antibiotics against resistant targets. Also, we show that Sor has a better pharmacokinetic profile than Rif, making it a suitable starting molecule to design drugs to be used for the treatment of TB patients with comorbidities who require multiple medications.

Although the Diels-Alder reaction has long been utilized for the preparation of numerous heterocycles, opportunities to extend its power remain. Herein, we detail a simple, modular, and robust approach that combines various amines regioselectively with 4,6-dichloropyrone to create substrates which, under appropriate conditions, can directly deliver varied indolines and hydroindolines through [4+2] cycloadditions with substitution patterns difficult to access otherwise. As an initial demonstration of the power of the strategy, several different natural products have been obtained either formally or by direct total synthesis, with efforts toward one of these-the complex amaryllidaceae alkaloid gracilamine-affording the shortest route to date in terms of linear step count.