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Pathogenic bacteria sense and respond to environmental fluctuations, a capability essential for establishing successful infections. The YmoA/Hha protein family are conserved transcription regulators in Enterobacteriaceae, playing a critical role in these responses. Specifically, YmoA in Yersinia adjusts the expression of virulence-associated traits upon temperature shift. Still, the molecular mechanisms transducing environmental signals through YmoA remain elusive. Our study employs nuclear magnetic resonance spectroscopy, biological assays and RNA-seq analysis to elucidate these mechanisms. We demonstrate that YmoA undergoes structural fluctuations and conformational dynamics in response to temperature and osmolarity changes, correlating with changes in plasmid copy number, bacterial fitness and virulence function. Notably, chemical shift analysis identifies key roles of a few specific residues and of the C-terminus region in sensing both temperature and salt-driven switch. These findings demonstrate that YmoA acts as a central stress sensor in Yersinia, fine-tuning virulence gene expression and balancing metabolic trade-offs.

Type II NADH dehydrogenase (NDH-2) is an essential component of electron transfer in many microbial pathogens but has remained largely unexplored as a potential drug target. Previously, quinolinyl pyrimidines were shown to inhibit Mycobacterium tuberculosis NDH-2, as well as the growth of the bacteria [Shirude, P. S.; ACS Med. Chem. Lett. 2012, 3, 736−740]. Here, we synthesized a number of novel quinolinyl pyrimidines and investigated their properties. In terms of inhibition of the NDH-2 enzymes from M. tuberculosis and Mycobacterium smegmatis, the best compounds were of similar potency to previously reported inhibitors of the same class (half-maximal inhibitory concentration (IC50) values in the low-μM range). However, a number of the compounds had much better activity against Gram-negative pathogens, with minimum inhibitory concentrations (MICs) as low as 2 μg/mL. Multivariate analyses (partial least-squares (PLS) and principle component analysis (PCA)) showed that overall ligand charge was one of the most important factors in determining antibacterial activity, with patterns that varied depending on the particular bacterial species. In some cases (e.g., mycobacteria), there was a clear correlation between the IC50 values and the observed MICs, while in other instances, no such correlation was evident. When tested against a panel of protozoan parasites, the compounds failed to show activity that was not linked to cytotoxicity. Further, a strong correlation between hydrophobicity (estimated as clog P) and cytotoxicity was revealed; more hydrophobic analogues were more cytotoxic. By contrast, antibacterial MIC values and cytotoxicity were not well correlated, suggesting that the quinolinyl pyrimidines can be optimized further as antimicrobial agents.

Oligopeptide boronates with a lipophilic tail are known to inhibit the type I signal peptidase in E. coli, which is a promising drug target for developing novel antibiotics. Antibacterial activity depends on these oligopeptides having a cationic modification to increase their permeation. Unfortunately, this modification is associated with cytotoxicity, motivating the need for novel approaches. The sulfonimidamide functionality has recently gained much interest in drug design and discovery, as a means of introducing chirality and an imine-handle, thus allowing for the incorporation of additional substituents. This in turn can tune the chemical and biological properties, which are here explored. We show that introducing the sulfonimidamide between the lipophilic tail and the peptide in a series of signal peptidase inhibitors resulted in antibacterial activity, while the sulfonamide isostere and previously known non-cationic analogs were inactive. Additionally, we show that replacing the sulfonamide with a sulfonimidamide resulted in decreased cytotoxicity, and similar results were seen by adding a cationic sidechain to the sulfonimidamide motif. This is the first report of incorporation of the sulfonimidamide functional group into bioactive peptides, more specifically into antibacterial oligopeptides, and evaluation of its biological effects.

As antibiotic resistance of various pathogens emerged globally, the need for new effective drugs with novel modes of action became urgent. In this thesis, we focus on infectious diseases, e.g. tuberculosis, malaria, and nosocomial infections, and the corresponding causative pathogens, Mycobacterium tuberculosis, Plasmodium falciparum, and the Gram-negative ESKAPE pathogens that underlie so many healthcare-acquired diseases. Following the same-target-other-pathogen (STOP) strategy, we attempted to comprehensively explore the properties of three promising drug targets.

Signal peptidase I (SPase I), existing both in Gram-negative and Gram-positive bacteria, as well as in parasites, is vital for cell viability, due to its critical role in signal peptide cleavage, thus, protein maturation, and secreted protein transport. Three factors, comprising essentiality, a unique mode of action, and easy accessibility, make it an attractive drug target. We have established a platform, investigating the protein purification, enzymatic kinetics, and inhibition. A full-length SPase I from E. coli, including two transmembrane segments, was produced and purified in the presence of 0.5 % Triton X-100. In the in vitro biochemical assay, it exhibits proteolytic activity on antigen 85A from M. tuberculosis, with a K_m of 20 µM and a k_cat of 135 s^-1_­. A series of macrocyclic oligopeptides that have been proven inhibitory to E. coli SPase I also showed potency against a panel of Gram-negative bacteria. 

1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is responsible for the production of methylerythritol phosphate (MEP) in the non-mevalonate pathway of isoprenoid biosynthesis, and is thus essential for cell growth. DXRs from M. tuberculosis and P. falciparum have been under investigation in our lab for years. I addressed structural and biochemical characterizations of PfDXR with analogs of 3-(N-formyl-N-hydroxyamino)propyl- phosphonate (fosmidomycin) and 3-(N-acetyl-N-hydroxyamino)propyl- phosphonate (FR-9000098), two natural products showing potency against P. falciparum. Chemical modifications, methylation at Cg, and double bond formation between Ca and Cb, were investigated to increase the pathogenicidal activity. Crystallographic complex structures of PfDXR and four novel compounds inhibitory to PfDXR in a dose-dependent manner were solved, and ligand binding will be discussed in detail.

Type II NADH dehydrogenase (NDH-2) is an essential component in the respiratory chain, playing an important role in electron transfer. Biomembrane-bound NDH-2 from M. tuberculosis was over-expressed in E. coli, as well as the homolog from M. smegmatis. The purified NDH-2s were kinetically characterized, and showed a similar affinity to previously reported NDH-2s expressed M. smegmatis. A collection of novel inhibitors in the scaffold of quinolinyl pyrimidines were synthesized and tested for inhibition in a biochemical assay.

Type I signal peptidase, with its vital role in bacterial viability, is a promising but underexploited antibacterial drug target. In the light of steadily increasing rates of antimicrobial resistance, we have developed novel macrocyclic lipopeptides, linking P2 and P1' by a boronic ester warhead, capable of inhibiting Escherichia coli type I signal peptidase (EcLepB) and exhibiting good antibacterial activity. Structural modifications of the macrocyclic ring, the peptide sequence and the lipophilic tail led us to 14 novel macrocyclic boronic esters. It could be shown that macrocyclization is well tolerated in terms of EcLepB inhibition and antibacterial activity. Among the synthesized macrocycles, potent enzyme inhibitors in the low nanomolar range (e.g. compound 42f, EcLepB IC₅₀ = 29 nM) were identified also showing good antimicrobial activity (e.g. compound 42b, E. coli WT MIC = 16 μg/mL). The unique macrocyclic boronic esters described here were based on previously published linear lipopeptidic EcLepB inhibitors in an attempt to address cytotoxicity and hemolysis. We show herein that structural changes to the macrocyclic ring influence both the cytotoxicity and hemolytic activity suggesting that the P2 to P1' linker provide means for optimizing off-target effects. However, for the present set of compounds we were not able to separate the antibacterial activity and cytotoxic effect.

Type I signal peptidases are potential targets for the development of new antibacterial agents. Here we report finding potent inhibitors of E. coli type I signal peptidase (LepB), by optimizing a previously reported hit compound, decanoyl-PTANA-CHO, through modifications at the N- and C-termini. Good improvements of inhibitory potency were obtained, with IC50s in the low nanomolar range. The best inhibitors also showed good antimicrobial activity, with MICs in the low μg/mL range for several bacterial species. The selection of resistant mutants provided strong support for LepB as the target of these compounds. The cytotoxicity and hemolytic profiles of these compounds are not optimal but the finding that minor structural changes cause the large effects on these properties suggests that there is potential for optimization in future studies.

Fosmidomycin and FR-9000098 have been confirmed to show parasiticidal activity against Plasmodium falciparum, targeting DXR involved in the MEP pathway. We designed a construct of PfDXR that has successfully been overexpressed in E. coli BL21(DE3) C43, and purified by IMAC and SEC, with  the final yield of 1.2 mg/ 8 L culture. PfDXR was concentrated to 20 mg/ml, and co-crystallized with previously tested inhibitors in the FR-9000098 scaffold in the presence of Mn2+. Three FR-9000098 analogues with double-bonded Ca-Cband/or a phenyl ring with various lengths to N1, showed inhibitory activities with IC50s roughly 50 nM. Three crystals were in triclinic P1space group, with similar dimensions in the unit cell (51Å, 56Å, 86Å, 103°, 103°, 101°). All four complex structures have been crystallographically determined at resolutions in the range 1.86 Å, 2.45 Å, 2.13Å, 2.05 Å. Given the high similarity in structures, the initial phases were determined by rigid body refinement with search model PfDXR-FN3 complex, followed by restrained refinement in refmac5. Subsequently, the ligands and surrounding amino acid residues were manually rebuilt with theqdstools in O. the Ca-Cbbonds of the three ligands were altered from a single to double bond based on the structure of FR9000098. In addition, two ligands were extended at the Cdwith a phenyl group, and with the benzyl group connected by two carbons. N-terminal NADPH binding domains from four complexes undergo minor rigid body movement, and more details of conformational changes in the flap region are discussed.