The hydroxyl functionality and a rigid proximal N are required for forming a novel non-covalent quinine-heme complex

Quinoline antimalarial drugs bind both monomeric and dimeric forms of free heme, with distinct preferences depending on the chemical environment. Under biological conditions, chloroquine (CQ) appears to prefer to bind to μ-oxo dimeric heme, while quinine (QN) preferentially binds monomer. To further explore this important distinction, we study three newly synthesized and several commercially available QN analogues lacking various functional groups. We find that removal of the QN hydroxyl lowers heme affinity, hemozoin (Hz) inhibition efficiency, and antiplasmodial activity. Elimination of the rigid quinuclidyl ring has similar effects, but elimination of either the vinyl or methoxy group does not. Replacing the quinuclidyl N with a less rigid tertiary aliphatic N only partially restores activity. To further study these trends, we probe drug-heme interactions via NMR studies with both Fe and Zn protoporphyrin IX (FPIX, ZnPIX) for QN, dehydroxyQN (DHQN), dequinuclidylQN (DQQN), and deamino-dequinuclidylQN (DADQQN). Magnetic susceptibility measurements in the presence of FPIX demonstrate that these compounds differentially perturb FPIX monomer-dimer equilibrium. We also isolate the QN-FPIX complex formed under mild aqueous conditions and analyze it by mass spectrometry, as well as fluorescence, vibrational, and solid state NMR spectroscopies. The data elucidate key features of QN pharmacology and allow us to propose a refined model for the preferred binding of QN to monomeric FPIX under biologically relevant conditions. With this model in hand, we also propose how QN, CQ, and amodiaquine (AQ) differ in their ability to inhibit Hz formation

Comparison of proteomic responses as global approach to antibiotic mechanism of action elucidation

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. New antibiotics are urgently needed to address the mounting resistance challenge. In early drug discovery, one of the bottlenecks is the elucidation of targets and mechanisms. To accelerate antibiotic research, we provide a proteomic approach for the rapid classification of compounds into those with precedented and unprecedented modes of action. We established a proteomic response library of Bacillus subtilis covering 91 antibiotics and comparator compounds, and a mathematical approach was developed to aid data analysis. Comparison of proteomic responses (CoPR) allows the rapid identification of antibiotics with dual mechanisms of action as shown for atypical tetracyclines. It also aids in generating hypotheses on mechanisms of action as presented for salvarsan (arsphenamine) and the antirheumatic agent auranofin, which is under consideration for repurposing. Proteomic profiling also provides insights into the impact of antibiotics on bacterial physiology through analysis of marker proteins indicative of the impairment of cellular processes and structures. As demonstrated for trans-translation, a promising target not yet exploited clinically, proteomic profiling supports chemical biology approaches to investigating bacterial physiology

Antibiotic that inhibits trans-translation blocks binding of EF-Tu to tmRNA but not to tRNA

ABSTRACT trans-Translation is conserved throughout bacteria and is essential in many species. High-throughput screening identified a tetrazole-based trans-translation inhibitor, KKL-55, that has broad-spectrum antibiotic activity. A biotinylated version of KKL-55 pulled down elongation factor thermo-unstable (EF-Tu) from bacterial lysates. Purified EF-Tu bound KKL-55 in vitro with a K d = 2 µM, confirming a high-affinity interaction. An X-ray crystal structure showed that KKL-55 binds in domain 3 of EF-Tu, and mutation of residues in the binding pocket abolished KKL-55 binding. RNA-binding assays in vitro showed that KKL-55 inhibits binding between EF-Tu and transfer-messenger RNA (tmRNA) but not between EF-Tu and tRNA. These data demonstrate a new mechanism for the inhibition of EF-Tu function and suggest that this specific inhibition of EF-Tu•tmRNA binding is a viable target for antibiotic development. IMPORTANCE Elongation factor thermo-unstable (EF-Tu) is a universally conserved translation factor that mediates productive interactions between tRNAs and the ribosome. In bacteria, EF-Tu also delivers transfer-messenger RNA (tmRNA)-SmpB to the ribosome during trans-translation. We report the first small molecule, KKL-55, that specifically inhibits EF-Tu activity in trans-translation without affecting its activity in normal translation. KKL-55 has broad-spectrum antibiotic activity, suggesting that compounds targeted to the tmRNA-binding interface of EF-Tu could be developed into new antibiotics to treat drug-resistant infections

Pathogen-Specific De Novo Antimicrobials Engineered Through Membrane Porin Biomimicry

Precision antimicrobials that can kill pathogens without damaging host commensals hold potential to cure disease without antibiotic-associated dysbiosis. Here we report the de novo design of host defense peptides that have been rationally engineered to precisely target specific pathogens by mimicking key molecular features of the target microbe’s unique channel-forming membrane proteins, or porins. This biomimetic strategy exploits physical and structural motifs of the pathogen envelope, rather than targeting resistance-susceptible protein biochemical pathways, to construct fast-acting precision bacteriolytics. Utilizing this approach, we design an antitubercular sequence that undergoes instructed, tryptophan-zippered assembly within the mycolic-acid rich outer membrane of Mycobacterium tuberculosis (Mtb) to specifically kill the pathogen without collateral toxicity towards lung commensals or host tissue. These mycomembrane-templated mechanisms are rapid and synergistically enhance the potency of antibiotics that otherwise poorly diffuse across the rigid Mtb envelope, particularly those that exploit porins for antimycobacterial activity. This new porin-mimetic paradigm may serve as a conceptual basis for the directed design of new narrow-spectrum antimicrobial scaffolds.</p

Ribosome Rescue Inhibitors Kill Actively Growing and Nonreplicating Persister <i>Mycobacterium tuberculosis</i> Cells

The emergence of <i>Mycobacterium tuberculosis</i> (MTB) strains that are resistant to most or all available antibiotics has created a severe problem for treating tuberculosis and has spurred a quest for new antibiotic targets. Here, we demonstrate that <i>trans</i>-translation is essential for growth of MTB and is a viable target for development of antituberculosis drugs. We also show that an inhibitor of <i>trans</i>-translation, KKL-35, is bactericidal against MTB under both aerobic and anoxic conditions. Biochemical experiments show that this compound targets helix 89 of the 23S rRNA. <i>In silico</i> molecular docking predicts a binding pocket for KKL-35 adjacent to the peptidyl-transfer center in a region not targeted by conventional antibiotics. Computational solvent mapping suggests that this pocket is a druggable hot spot for small molecule binding. Collectively, our findings reveal a new target for antituberculosis drug development and provide critical insight on the mechanism of antibacterial action for KKL-35 and related 1,3,4-oxadiazole benzamides

Antiplasmodial and Antiproliferative Pseudoguaianolides of Athroisma proteiforme from the Madagascar Dry Forest

Investigation of extracts from the plant Athroisma proteiforme (Humbert) Mattf. (Asteraceae) for antimalarial activity led to the isolation of the five new sesquiterpene lactones <b>1</b>–<b>5</b> together with centaureidin (<b>6</b>). The structures of the new compounds were deduced from analyses of physical and spectroscopic data, and the absolute configuration of compound <b>1</b> was confirmed by an X-ray crystallographic study. Athrolides C (<b>3</b>) and D (<b>4</b>) both showed antiplasmodial activities with IC₅₀ values of 6.6 (<b>3</b>) and 7.2 μM (<b>4</b>) against the HB3 strain and 5.5 (<b>3</b>) and 4.2 μM (<b>4</b>) against the Dd2 strain of the malarial parasite Plasmodium falciparum. The isolates <b>1</b>–<b>6</b> also showed antiproliferative activity against A2780 human ovarian cancer cells, with IC₅₀ values ranging from 0.4 to 2.5 μM