1-deoxy-D-xylulose-5-phosphate synthase from Pseudomonas aeruginosa and Klebsiella pneumoniae reveals conformational changes upon cofactor binding.

The ESKAPE bacteria are the six highly virulent and antibiotic-resistant pathogens that require the most urgent attention for the development of novel antibiotics. Detailed knowledge of target proteins specific to bacteria is essential to develop novel treatment options. The methylerythritol-phosphate (MEP) pathway, which is absent in humans, represents a potentially valuable target for the development of novel antibiotics. Within the MEP pathway, the enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXPS) catalyzes a crucial, rate-limiting first step and a branch point in the biosynthesis of the vitamins B1 and B6. We report the high-resolution crystal structures of DXPS from the important ESKAPE pathogens Pseudomonas aeruginosa and Klebsiella pneumoniae in both the co-factor-bound and the apo forms. We demonstrate that the absence of the cofactor thiamine diphosphate results in conformational changes that lead to disordered loops close to the active site that might be important for the design of potent DXPS inhibitors. Collectively, our results provide important structural details that aid in the assessment of DXPS as a potential target in the ongoing efforts to combat antibiotic resistance.

Revision of the Absolute Configurations of Chelocardin and Amidochelocardin.

Even with the aid of the available methods, the configurational assignment of natural products can be a challenging task that is prone to errors, and it sometimes needs to be corrected after total synthesis or single-crystal X-ray diffraction (XRD) analysis. Herein, the absolute configuration of amidochelocardin is revised using a combination of XRD, NMR spectroscopy, experimental ECD spectra, and time-dependent density-functional theory (TDDFT)-ECD calculations. As amidochelocardin was obtained via biosynthetic engineering of chelocardin, we propose the same absolute configuration for chelocardin based on the similar biosynthetic origins of the two compounds and result of TDDFT-ECD calculations. The evaluation of spectral data of two closely related analogues, 6-desmethyl-chelocardin and its semisynthetic derivative 1, also supports this conclusion.

Optical Modulation of Antibiotic Resistance by Photoswitchable Cystobactamids.

The rise of antibiotic resistance causes a serious health care problem, and its counterfeit demands novel, innovative concepts. The combination of photopharmacology, enabling a light-controlled reversible modulation of drug activity, with antibiotic drug design has led to first photoswitchable antibiotic compounds derived from established scaffolds. In this study, we converted cystobactamids, gyrase-inhibiting natural products with an oligoaryl scaffold and highly potent antibacterial activities, into photoswitchable agents by inserting azobenzene in the N-terminal part and/or an acylhydrazone moiety near the C-terminus, yielding twenty analogs that contain mono- as well as double-switches. Antibiotic and gyrase inhibition properties could be modulated 3.4-fold and 5-fold by light, respectively. Notably, the sensitivity of photoswitchable cystobactamids towards two known resistance factors, the peptidase AlbD and the scavenger protein AlbA, was light-dependent. While irradiation of an analog with an N-terminal azobenzene with 365 nm light led to less degradation by AlbD, the AlbA-mediated inactivation was induced. This provides a proof-of-principle that resistance towards photoswitchable antibiotics can be optically controlled.

Substrate-Inspired Fragment Merging and Growing Affords Efficacious LasB Inhibitors.

Extracellular virulence factors have emerged as attractive targets in the current antimicrobial resistance crisis. The Gram-negative pathogen Pseudomonas aeruginosa secretes the virulence factor elastase B (LasB), which plays an important role in the infection process. Here, we report a sub-micromolar, non-peptidic, fragment-like inhibitor of LasB discovered by careful visual inspection of structural data. Inspired by the natural LasB substrate, the original fragment was successfully merged and grown. The optimized inhibitor is accessible via simple chemistry and retained selectivity with a substantial improvement in activity, which can be rationalized by the crystal structure of LasB in complex with the inhibitor. We also demonstrate an improved in vivo efficacy of the optimized hit in Galleria mellonella larvae, highlighting the significance of this class of compounds as promising drug candidates.

Structure-Activity Relationship and Mode-Of-Action Studies Highlight 1-(4-Biphenylylmethyl)-1H-imidazole-Derived Small Molecules as Potent CYP121 Inhibitors.

CYP121 of Mycobacterium tuberculosis (Mtb) is an essential target for the development of novel potent drugs against tuberculosis (TB). Besides known antifungal azoles, further compounds of the azole class were recently identified as CYP121 inhibitors with antimycobacterial activity. Herein, we report the screening of a similarity-oriented library based on the former hit compound, the evaluation of affinity toward CYP121, and activity against M. bovis BCG. The results enabled a comprehensive SAR study, which was extended through the synthesis of promising compounds and led to the identification of favorable features for affinity and/or activity and hit compounds with 2.7-fold improved potency. Mode of action studies show that the hit compounds inhibit substrate conversion and highlighted CYP121 as the main antimycobacterial target of our compounds. Exemplified complex crystal structures of CYP121 with three inhibitors reveal a common binding site. Engaging in both hydrophobic interactions as well as hydrogen bonding to the sixth iron ligand, our compounds block a solvent channel leading to the active site heme. Additionally, we report the first CYP inhibitors that are able to reduce the intracellular replication of M. bovis BCG in macrophages, emphasizing their potential as future drug candidates against TB.

Tackling Pseudomonas aeruginosa Virulence by a Hydroxamic Acid-Based LasB Inhibitor.

In search of novel antibiotics to combat the challenging spread of resistant pathogens, bacterial proteases represent promising targets for pathoblocker development. A common motif for protease inhibitors is the hydroxamic acid function, yet this group has often been related to unspecific inhibition of various metalloproteases. In this work, the inhibition of LasB, a harmful zinc metalloprotease secreted by Pseudomonas aeruginosa, through a hydroxamate derivative is described. The present inhibitor was developed based on a recently reported, highly selective thiol scaffold. Using X-ray crystallography, the lack of inhibition of a range of human matrix metalloproteases could be attributed to a distinct binding mode sparing the S1' pocket. The inhibitor was shown to restore the effect of the antimicrobial peptide LL-37, decrease the formation of P. aeruginosa biofilm and, for the first time for a LasB inhibitor, reduce the release of extracellular DNA. Hence, it is capable of disrupting several important bacterial resistance mechanisms. These results highlight the potential of protease inhibitors to fight bacterial infections and point out the possibility to achieve selective inhibition even with a strong zinc anchor.

Binding Mode Characterization and Early in Vivo Evaluation of Fragment-Like Thiols as Inhibitors of the Virulence Factor LasB from Pseudomonas aeruginosa.

The increasing emergence of antibiotic resistance necessitates the development of anti-infectives with novel modes of action. Targeting bacterial virulence is considered a promising approach to develop novel antibiotics with reduced selection pressure. The extracellular collagenase elastase (LasB) plays a pivotal role in the infection process of Pseudomonas aeruginosa and therefore represents an attractive antivirulence target. Mercaptoacetamide-based thiols have been reported to inhibit LasB as well as collagenases from clostridia and bacillus species. The present work provides an insight into the structure-activity relationship (SAR) of these fragment-like LasB inhibitors, demonstrating an inverse activity profile compared to similar inhibitors of clostridial collagenase H (ColH). An X-ray cocrystal structure is presented, revealing distinct binding of two compounds to the active site of LasB, which unexpectedly maintains an open conformation. We further demonstrate in vivo efficacy in a Galleria mellonella infection model and high selectivity of the LasB inhibitors toward human matrix metalloproteinases (MMPs).

Structure and Biosynthesis of Crocagins: Polycyclic Posttranslationally Modified Ribosomal Peptides from Chondromyces crocatus.

Secondary metabolome mining efforts in the myxobacterial multiproducer of natural products, Chondromyces crocatus Cm c5, resulted in the isolation and structure elucidation of crocagins, which are novel polycyclic peptides containing a tetrahydropyrrolo[2,3-b]indole core. The gene cluster was identified through an approach combining genome analysis, targeted gene inactivation in the producer, and in vitro experiments. Based on our findings, we developed a biosynthetic scheme for crocagin biosynthesis. These natural products are formed from the three C-terminal amino acids of a precursor peptide and thus belong to a novel class of ribosomally synthesized and post-translationally modified peptides (RiPPs). We demonstrate that crocagin A binds to the carbon storage regulator protein CsrA, thereby inhibiting the ability of CsrA to bind to its cognate RNA target.