Antibiotic class with potent in vivo activity targeting lipopolysaccharide synthesis in Gram-negative bacteria.

Here, we describe the identification of an antibiotic class acting via LpxH, a clinically unexploited target in lipopolysaccharide synthesis. The lipopolysaccharide synthesis pathway is essential in most Gram-negative bacteria and there is no analogous pathway in humans. Based on a series of phenotypic screens, we identified a hit targeting this pathway that had activity on efflux-defective strains of Escherichia coli. We recognized common structural elements between this hit and a previously published inhibitor, also with activity against efflux-deficient bacteria. With the help of X-ray structures, this information was used to design inhibitors with activity on efflux-proficient, wild-type strains. Optimization of properties such as solubility, metabolic stability and serum protein binding resulted in compounds having potent in vivo efficacy against bloodstream infections caused by the critical Gram-negative pathogens E. coli and Klebsiella pneumoniae. Other favorable properties of the series include a lack of pre-existing resistance in clinical isolates, and no loss of activity against strains expressing extended-spectrum-ß-lactamase, metallo-ß-lactamase, or carbapenemase-resistance genes. Further development of this class of antibiotics could make an important contribution to the ongoing struggle against antibiotic resistance.

Synthesis and kinetic evaluation of phosphomimetic inhibitors targeting type B ribose-5-phosphate isomerase from Mycobacterium tuberculosis.

Because tuberculosis is still a major health threat worldwide, identification of new drug targets is urgently needed. In this study, we considered type B ribose-5-phosphate isomerase from Mycobacterium tuberculosis as a potential target, and addressed known problems of previous inhibitors in terms of their sensitivity to hydrolysis catalyzed by phosphatase enzymes, which impaired their potential use as drugs. To this end, we synthesized six novel phosphomimetic compounds designed to be hydrolytically stable analogs of the substrate ribose 5-phosphate and the best known inhibitor 5-phospho-d-ribonate. The phosphate function was replaced by phosphonomethyl, sulfate, sulfonomethyl, or malonate groups. Inhibition was evaluated on type A and type B ribose-5-phosphate isomerases, and stability towards hydrolysis using alkaline phosphatase and veal serum was assessed. One of the phosphomimetic analogs, 5-deoxy-5-phosphonomethyl-d-ribonate, emerged as the first strong and specific inhibitor of the M. tuberculosis enzyme that is resistant to hydrolysis.

Bacterial type I signal peptidase inhibitors - Optimized hits from nature.

The ever-increasing number of bacteria resistant to the currently available antibacterial agents is a great medical problem today, and new antibiotics with novel mechanisms of action are urgently needed. Among the validated antibacterial drug targets against which new classes of antibiotics might be directed is bacterial type I signal peptidase (SPase I), an essential part of the Tat and Sec secretory systems. SPase I is responsible for the hydrolysis of the N-terminal signal peptides from proteins secreted across the cytoplasmic membrane and plays a key role in bacterial viability and virulence. This review focuses on the antibacterial activity of natural and synthetic SPase I inhibitors reported to date, namely ß-lactams, lipopeptides, and arylomycins, but also an example of SPase I activator was presented.

Synthesis and In Vitro Biological Evaluation of Quinolinyl Pyrimidines Targeting Type II NADH-Dehydrogenase (NDH-2).

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.

Antibacterial sulfonimidamide-based oligopeptides as type I signal peptidase inhibitors: Synthesis and biological evaluation.

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.

Boronic ester-linked macrocyclic lipopeptides as serine protease inhibitors targeting Escherichia coli type I signal peptidase.

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.

Design, synthesis and in vitro biological evaluation of oligopeptides targeting E. coli type I signal peptidase (LepB).

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.

Targeting an Aromatic Hotspot in Plasmodium falciparum 1-Deoxy-d-xylulose-5-phosphate Reductoisomerase with ß-Arylpropyl Analogues of Fosmidomycin.

Blocking the 2-C-methyl-d-erythrithol-4-phosphate pathway for isoprenoid biosynthesis offers new ways to inhibit the growth of Plasmodium spp. Fosmidomycin [(3-(N-hydroxyformamido)propyl)phosphonic acid, 1] and its acetyl homologue FR-900098 [(3-(N-hydroxyacetamido)propyl)phosphonic acid, 2] potently inhibit 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr), a key enzyme in this biosynthetic pathway. Arylpropyl substituents were introduced at the ß-position of the hydroxamate analogue of 2 to study changes in lipophilicity, as well as electronic and steric properties. The potency of several new compounds on the P. falciparum enzyme approaches that of 1 and 2. Activities against the enzyme and parasite correlate well, supporting the mode of action. Seven X-ray structures show that all of the new arylpropyl substituents displace a key tryptophan residue of the active-site flap, which had made favorable interactions with 1 and 2. Plasticity of the flap allows substituents to be accommodated in many ways; in most cases, the flap is largely disordered. Compounds can be separated into two classes based on whether the substituent on the aromatic ring is at the meta or para position. Generally, meta-substituted compounds are better inhibitors, and in both classes, smaller size is linked to better potency.

Expanding the Catalytic Triad in Epoxide Hydrolases and Related Enzymes.

Potato epoxide hydrolase 1 exhibits rich enantio- and regioselectivity in the hydrolysis of a broad range of substrates. The enzyme can be engineered to increase the yield of optically pure products as a result of changes in both enantio- and regioselectivity. It is thus highly attractive in biocatalysis, particularly for the generation of enantiopure fine chemicals and pharmaceuticals. The present work aims to establish the principles underlying the activity and selectivity of the enzyme through a combined computational, structural, and kinetic study using the substrate trans-stilbene oxide as a model system. Extensive empirical valence bond simulations have been performed on the wild-type enzyme together with several experimentally characterized mutants. We are able to computationally reproduce the differences between the activities of different stereoisomers of the substrate and the effects of mutations of several active-site residues. In addition, our results indicate the involvement of a previously neglected residue, H104, which is electrostatically linked to the general base H300. We find that this residue, which is highly conserved in epoxide hydrolases and related hydrolytic enzymes, needs to be in its protonated form in order to provide charge balance in an otherwise negatively charged active site. Our data show that unless the active-site charge balance is correctly treated in simulations, it is not possible to generate a physically meaningful model for the enzyme that can accurately reproduce activity and selectivity trends. We also expand our understanding of other catalytic residues, demonstrating in particular the role of a noncanonical residue, E35, as a "backup base" in the absence of H300. Our results provide a detailed view of the main factors driving catalysis and regioselectivity in this enzyme and identify targets for subsequent enzyme design efforts.

Optimization and Evaluation of 5-Styryl-Oxathiazol-2-one Mycobacterium tuberculosis Proteasome Inhibitors as Potential Antitubercular Agents.

This is the first report of 5-styryl-oxathiazol-2-ones as inhibitors of the Mycobacterium tuberculosis (Mtb) proteasome. As part of the study, the structure-activity relationship of oxathiazolones as Mtb proteasome inhibitors has been investigated. Furthermore, the prepared compounds displayed a good selectivity profile for Mtb compared to the human proteasome. The 5-styryl-oxathiazol-2-one inhibitors identified showed little activity against replicating Mtb, but were rapidly bactericidal against nonreplicating bacteria. (E)-5-(4-Chlorostyryl)-1,3,4-oxathiazol-2-one) was most effective, reducing the colony-forming units (CFU)/mL below the detection limit in only seven days at all concentrations tested. The results suggest that this new class of Mtb proteasome inhibitors has the potential to be further developed into novel antitubercular agents for synergistic combination therapies with existing drugs.

Synthesis and bioactivity of ß-substituted fosmidomycin analogues targeting 1-deoxy-D-xylulose-5-phosphate reductoisomerase.

Blocking the 2-C-methyl-d-erythrithol-4-phosphate (MEP) pathway for isoprenoid biosynthesis offers interesting prospects for inhibiting Plasmodium or Mycobacterium spp. growth. Fosmidomycin (1) and its homologue FR900098 (2) potently inhibit 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr), a key enzyme in this pathway. Here we introduced aryl or aralkyl substituents at the ß-position of the hydroxamate analogue of 2. While direct addition of a ß-aryl moiety resulted in poor inhibition, longer linkers between the carbon backbone and the phenyl ring were generally associated with better binding to the enzymes. X-ray structures of the parasite Dxr-inhibitor complexes show that the "longer" compounds generate a substantially different flap structure, in which a key tryptophan residue is displaced, and the aromatic group of the ligand lies between the tryptophan and the hydroxamate's methyl group. Although the most promising new Dxr inhibitors lack activity against Escherichia coli and Mycobacterium smegmatis, they proved to be highly potent inhibitors of Plasmodium falciparum in vitro growth.

Arabidopsis cytosolic acyl-CoA-binding proteins ACBP4, ACBP5 and ACBP6 have overlapping but distinct roles in seed development.

Eukaryotic cytosolic ACBPs (acyl-CoA-binding proteins) bind acyl-CoA esters and maintain a cytosolic acyl-CoA pool, but the thermodynamics of their protein-lipid interactions and physiological relevance in plants are not well understood. Arabidopsis has three cytosolic ACBPs which have been identified as AtACBP4, AtACBP5 and AtACBP6, and microarray data indicated that all of them are expressed in seeds; AtACBP4 is expressed in early embryogenesis, whereas AtACBP5 is expressed later. ITC (isothermal titration calorimetry) in combination with transgenic Arabidopsis lines were used to investigate the roles of these three ACBPs from Arabidopsis thaliana. The dissociation constants, stoichiometry and enthalpy change of AtACBP interactions with various acyl-CoA esters were determined using ITC. Strong binding of recombinant (r) AtACBP6 with long-chain acyl-CoA (C16- to C18-CoA) esters was observed with dissociation constants in the nanomolar range. However, the affinity of rAtACBP4 and rAtACBP5 to these acyl-CoA esters was much weaker (dissociation constants in the micromolar range), suggesting that they interact with acyl-CoA esters differently from rAtACBP6. When transgenic Arabidopsis expressing AtACBP6pro::GUS was generated, strong GUS (ß-glucuronidase) expression in cotyledonary-staged embryos and seedlings prompted us to measure the acyl-CoA contents of the acbp6 mutant. This mutant accumulated higher levels of C18:1-CoA and C18:1- and C18:2-CoAs in cotyledonary-staged embryos and seedlings, respectively, in comparison with the wild type. The acbp4acbp5acbp6 mutant showed the lightest seed weight and highest sensitivity to abscisic acid during germination, suggesting their physiological functions in seeds.

Inhibition of glutamine synthetase: a potential drug target in Mycobacterium tuberculosis.

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. Globally, tuberculosis is second only to AIDS in mortality and the disease is responsible for over 1.3 million deaths each year. The impractically long treatment schedules (generally 6-9 months) and unpleasant side effects of the current drugs often lead to poor patient compliance, which in turn has resulted in the emergence of multi-, extensively- and totally-drug resistant strains. The development of new classes of anti-tuberculosis drugs and new drug targets is of global importance, since attacking the bacterium using multiple strategies provides the best means to prevent resistance. This review presents an overview of the various strategies and compounds utilized to inhibit glutamine synthetase, a promising target for the development of drugs for TB therapy.

DXR inhibition by potent mono- and disubstituted fosmidomycin analogues.

The antimalarial compound fosmidomycin targets DXR, the enzyme that catalyzes the first committed step in the MEP pathway, producing the essential isoprenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate. The MEP pathway is used by a number of pathogens, including Mycobacterium tuberculosis and apicomplexan parasites, and differs from the classical mevalonate pathway that is essential in humans. Using a structure-based approach, we designed a number of analogues of fosmidomycin, including a series that are substituted in both the Cα and the hydroxamate positions. The latter proved to be a stable framework for the design of inhibitors that extend from the polar and cramped (and so not easily druggable) substrate-binding site and can, for the first time, bridge the substrate and cofactor binding sites. A number of these compounds are more potent than fosmidomycin in terms of killing Plasmodium falciparum in an in vitro assay; the best has an IC50 of 40 nM.

Trisubstituted imidazoles as Mycobacterium tuberculosis glutamine synthetase inhibitors.

Mycobacterium tuberculosis glutamine synthetase (MtGS) is a promising target for antituberculosis drug discovery. In a recent high-throughput screening study we identified several classes of MtGS inhibitors targeting the ATP-binding site. We now explore one of these classes, the 2-tert-butyl-4,5-diarylimidazoles, and present the design, synthesis, and X-ray crystallographic studies leading to the identification of MtGS inhibitors with submicromolar IC(50) values and promising antituberculosis MIC values.

Structural studies on Mycobacterium tuberculosis DXR in complex with the antibiotic FR-900098.

A number of pathogens, including the causative agents of tuberculosis and malaria, synthesize the essential isoprenoid precursor isopentenyl diphosphate via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway rather than the classical mevalonate pathway that is found in humans. As part of a structure-based drug-discovery program against tuberculosis, DXR, the enzyme that carries out the second step in the MEP pathway, has been investigated. This enzyme is the target for the antibiotic fosmidomycin and its active acetyl derivative FR-900098. The structure of DXR from Mycobacterium tuberculosis in complex with FR-900098, manganese and the NADPH cofactor has been solved and refined. This is a new crystal form that diffracts to a higher resolution than any other DXR complex reported to date. Comparisons with other ternary complexes show that the conformation is that of the enzyme in an active state: the active-site flap is well defined and the cofactor-binding domain has a conformation that brings the NADPH into the active site in a manner suitable for catalysis. The substrate-binding site is highly conserved in a number of pathogens that use this pathway, so any new inhibitor that is designed for the M. tuberculosis enzyme is likely to exhibit broad-spectrum activity.

Synthesis of functionalized cinnamaldehyde derivatives by an oxidative Heck reaction and their use as starting materials for preparation of Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate reductoisomerase inhibitors.

Cinnamaldehyde derivatives were synthesized in good to excellent yields in one step by a mild and selective, base-free palladium(II)-catalyzed oxidative Heck reaction starting from acrolein and various arylboronic acids. Prepared α,ß-unsaturated aldehydes were used for synthesis of novel α-aryl substituted fosmidomycin analogues, which were evaluated for their inhibition of Mycobacterium tuberculosis 1-deoxy-D-xylulose 5-phosphate reductoisomerase. IC(50) values between 0.8 and 27.3 µM were measured. The best compound showed activity comparable to that of the most potent previously reported α-aryl substituted fosmidomycin-class inhibitor.

Design, synthesis, and X-ray crystallographic studies of α-aryl substituted fosmidomycin analogues as inhibitors of Mycobacterium tuberculosis 1-deoxy-D-xylulose 5-phosphate reductoisomerase.

The natural antibiotic fosmidomycin acts via inhibition of 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), an essential enzyme in the non-mevalonate pathway of isoprenoid biosynthesis. Fosmidomycin is active on Mycobacterium tuberculosis DXR (MtDXR), but it lacks antibacterial activity probably because of poor uptake. α-Aryl substituted fosmidomycin analogues have more favorable physicochemical properties and are also more active in inhibiting malaria parasite growth. We have solved crystal structures of MtDXR in complex with 3,4-dichlorophenyl substituted fosmidomycin analogues; these show important differences compared to our previously described forsmidomycin-DXR complex. Our best inhibitor has an IC(50) = 0.15 µM on MtDXR but still lacked activity in a mycobacterial growth assay (MIC > 32 µg/mL). The combined results, however, provide insights into how DXR accommodates the new inhibitors and serve as an excellent starting point for the design of other novel and more potent inhibitors, particularly against pathogens where uptake is less of a problem, such as the malaria parasite.

Structural and functional studies of mycobacterial IspD enzymes.

A number of pathogens, including the causative agents of tuberculosis and malaria, synthesize isopentenyl diphosphate via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway rather than the classical mevalonate pathway found in humans. As part of a structure-based drug-discovery program against tuberculosis, IspD, the enzyme that carries out the third step in the MEP pathway, was targeted. Constructs of both the Mycobacterium smegmatis and the Mycobacterium tuberculosis enzymes that were suitable for structural and inhibitor-screening studies were engineered. Two crystal structures of the M. smegmatis enzyme were produced, one in complex with CTP and the other in complex with CMP. In addition, the M. tuberculosis enzyme was crystallized in complex with CTP. Here, the structure determination and crystallographic refinement of these crystal forms and the enzymatic characterization of the M. tuberculosis enzyme construct are reported. A comparison with known IspD structures allowed the definition of the structurally conserved core of the enzyme. It indicates potential flexibility in the enzyme and in particular in areas close to the active site. These well behaved constructs provide tools for future target-based screening of potential inhibitors. The conserved nature of the extended active site suggests that any new inhibitor will potentially exhibit broad-spectrum activity.

Expression and ß-glucan binding properties of Scots pine (Pinus sylvestris L.) antimicrobial protein (Sp-AMP).

Scots pine (Pinus sylvestris) secretes a number of small, highly-related, disulfide-rich proteins (Sp-AMPs) in response to challenges with fungal pathogens such as Heterobasidion annosum, although their biological role has been unknown. Here, we examined the expression patterns of these genes, as well as the structure and function of the encoded proteins. Northern blots and quantitative real time PCR showed increased levels of expression that are sustained during the interactions of host trees with pathogens, but not non-pathogens, consistent with a function in conifer tree defenses. Furthermore, the genes were up-regulated after treatment with salicylic acid and an ethylene precursor, 1-aminocyclopropane-1-carboxylic-acid, but neither methyl jasmonate nor H(2)O(2) induced expression, indicating that Sp-AMP gene expression is independent of the jasmonic acid signaling pathways. The cDNA encoding one of the proteins was cloned and expressed in Pichia pastoris. The purified protein had antifungal activity against H. annosum, and caused morphological changes in its hyphae and spores. It was directly shown to bind soluble and insoluble ß-(1,3)-glucans, specifically and with high affinity. Furthermore, addition of exogenous glucan is linked to higher levels of Sp-AMP expression in the conifer. Homology modeling and sequence comparisons suggest that a conserved patch on the surface of the globular Sp-AMP is a carbohydrate-binding site that can accommodate approximately four sugar units. We conclude that these proteins belong to a new family of antimicrobial proteins (PR-19) that are likely to act by binding the glucans that are a major component of fungal cell walls.