Antibacterial Design Research Articles

Synthesis and antibacterial activity of peptide deformylase inhibitors.

Peptide deformylase catalyzes the removal of the N-terminal formyl group from newly synthesized polypeptides in eubacteria. Its essential character in bacterial cells makes it an attractive target for antibacterial drug design. In this work, we have rationally designed and synthesized a series of peptide thiols that act as potent, reversible inhibitors of purified recombinant peptide deformylase from Escherichia coli and Bacillus subtilis. The most potent inhibitor has a K(I) value of 11 nM toward the B. subtilis enzyme. These inhibitors showed antibacterial activity against both Gram-positive and Gram-negative bacteria, with minimal inhibitory concentrations (MIC) as low as 5 microM ( approximately 2 microg/mL). The PDF inhibitors induce bacterial cell lysis and are bactericidal toward all four bacterial strains that have been tested, B. subtilis, Staphylococcus epidermidis, Enterococcus faecalis, and E. coli. Resistance evaluation of one of the inhibitors (1b) against B. subtilis showed that no resistant clone could be found from >1 x 10(9) cells. Quantitative analysis using a set of inhibitors designed to possess varying potencies against the deformylase enzyme revealed a linear correlation between the MIC values and the K(I) values. These results suggest that peptide deformylase is the likely molecular target responsible for the antibacterial activity of these inhibitors and is therefore a viable target for antibacterial drug design.

Direct Identification of Enzyme Active Site Residues by Solid-State REDOR NMR: Application to KDO8P Synthase

In this communication we report the first direct identification of active site residues of the enzyme 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDO8PS). This is accomplished by the application of rotational-echo double-resonance1 (REDOR) solid-state NMR experiments to lyophilized binary complexes of uniformly 15N-labeled KDO8PS with each of its natural substrates, phosphoenolpyruvate (PEP) and D-arabinose-5-phosphate (A5P). Its complex with the mechanism-based inhibitor, 2 (Scheme 1), the most potent inhibitor known to date2 (Ki ) 0.4 μM), is studied as well and its binding is compared to that of the natural substrates. The REDOR technique1 is based on selective reintroduction of (magnetic) dipolar interactions between pairs of specific types of nuclei, and in favorable cases enables their accurate measurement from which distance constraints can be obtained.3 Its application for the study of protein-ligand interactions is achieved by incorporation of stable isotopes at known positions in the protein that are proximate to (labeled) ligand moieties. In previous studies, REDOR measured distances were shown to be in excellent agreement with X-ray data.3a,b Moreover, 31P{13C} REDOR was employed to investigate the conformation of the bound inhibitor glyphosate in the enzyme-substrate-inhibitor ternary complex of 5-enolpyruvoylshikimate-3-phosphate synthase (EPSPS),3c for which no X-ray structure is available. REDOR, e.g., 31P{13C} and 15N {31P}, was also used for the determination of structural constraints in the above ternary complex,3d and to directly monitor transient enzyme-intermediate complexes of EPSPS3e and of UDP-GlcNac enolpyruvoyl transferase (Mur Z).3f The enzyme KDO8PS catalyzes the condensation reaction between A5P and PEP to form KDO8P and inorganic phosphate (Pi, Scheme 1).4 This important enzymatic reaction plays a crucial role in the assembly process of lipopolysaccharides of most Gramnegative bacteria,5 and is therefore an attractive target for the design of novel antibacterial drugs. Interestingly, unlike most PEPutilizing enzymes that cleave the high-energy P-O bond of PEP, KDO8PS and its family members (Mur Z, EPSPS, and 3-deoxyD-arabino-heptulosonate-7-phosphate synthase6 (DAHPS)) cleave the C-O bond of PEP.7 While the mechanisms of EPSPS8a (targeted by the herbicide glyphosate) and Mur Z8b (targeted by the antibiotic fosfomycin) were unambiguously characterized, the mechanisms of KDO8PS and DAHPS continue to be uncertain. Recent studies9a,2 on KDO8PS, including the synthesis and evaluation of the first acyclic bisubstrate inhibitor (2),2 supported a mechanism involving the formation of an acyclic bisphosphate intermediate 1 (Scheme 1).9b Mutagenesis studies10 identified in E. coli KDO8PS two vital cysteines, Cys38 and Cys166, yet their specific role still remains unclear. In addition, since to date neither the crystal structure nor the identity of the active site residues of KDO8PS are known, rational design of potent inhibitors for this enzyme is impaired. As part of our study on the structurefunction relationship of KDO8PS, [u-15N]KDO8PS was prepared and interactions between 15N enzyme labels to 31P nuclei of the ligands were examined by REDOR NMR.11 The 15N NMR CPMAS echo spectrum of [u-15N]KDO8PS binary complex with PEP (Figure 1a) shows resolved peaks for the nitrogen atoms of 10 side-chain arginine (guanidino: internal , 60 ppm; terminal η, 49 ppm), 22 lysine ( 9.0 ppm) residues, and an intense amide peak (95 ppm). Peaks of six side-chain histidines (δ1 224; 2 143 ppm) are not shown. In addition, detailed inspection of this spectrum reveals fine structure for the arginine (56.0 ppm), and a high field shoulder for lysine (3.0 ppm), attributed to a single arginine and to a single lysine residue residing in a distinct chemical environment. Interestingly, these two peaks are also present in the 15N CPMAS reference spectra of the other two complexes, KDO8PS-A5P and KDO8PS-2 (spectra not shown), implying that they arise from chemically distinct environments inherent to the enzyme itself, and are therefore not a result of interaction with a specific ligand. As will be shown below, these residues will be associated with specific recognition and binding of PEP by the enzyme.

Structure of Haemophilus influenzae Fe+3-binding protein reveals convergent evolution within a superfamily

The first crystal structure of the iron-transporter ferric ion-binding protein from Haemophilus influenzae (hFBP), at 1.6 A resolution, reveals the structural basis for iron uptake and transport required by several important bacterial pathogens. Paradoxically, although hFBP belongs to a protein superfamily which includes human transferrin, iron binding in hFBP and transferrin appears to have developed independently by convergent evolution. Structural comparison of hFBP with other prokaryotic periplasmic transport proteins and the eukaryotic transferrins suggests that these proteins are related by divergent evolution from an anion-binding common ancestor, not from an iron-binding ancestor. The iron binding site of hFBP incorporates a water and an exogenous phosphate ion as iron ligands and exhibits nearly ideal octahedral metal coordination. FBP is highly conserved, required for virulence, and is a nodal point for free iron uptake in several Gram-negative pathogenic bacteria, thus providing a potential target for broad-spectrum antibacterial drug design against human pathogens such as H. influenzae, Neisseria gonorrhoeae, and Neisseria meningitidis.

New strategies for antibacterial drug design

New strategies for antibacterial drug design

A disulfide bridge near the active site of carbapenem-hydrolyzing class A beta-lactamases might explain their unusual substrate profile.

Bacterial resistance to beta-lactam antibiotics, a clinically worrying and recurrent problem, is often due to the production of beta-lactamases, enzymes that efficiently hydrolyze the amide bond of the beta-lactam nucleus. Imipenem and other carbapenems escape the activity of most active site serine beta-lactamases and have therefore become very popular drugs for antibacterial chemotherapy in the hospital environment. Their usefulness is, however, threatened by the appearance of new beta-lactamases that efficiently hydrolyze them. This study is focused on the structure and properties of two recently described class A carbapenemases, produced by Serratia marcescens and Enterobacter cloacae strains and leads to a better understanding of the specificity of beta-lactamases. In turn, this will contribute to the design of better antibacterial drugs. Three-dimensional models of the two class A carbapenemases were constructed by homology modeling. They suggested the presence, near the active site of the enzymes, of a disulfide bridge (C69-C238) whose existence was experimentally confirmed. Kinetic parameters were measured with the purified Sme-1 carbapenemase, and an attempt was made to explain its specific substrate profile by analyzing the structures of minimized Henri-Michaelis complexes and comparing them to those obtained for the "classical" TEM-1 beta-lactamase. The peculiar substrate profile of the carbapenemases appears to be strongly correlated with the presence of the disulfide bridge between C69 and C238.

Drugs form new type of complex with DNA

Researchers at the University of Texas at Austin have discovered that a widely used class of drugs forms a previously unknown type of complex with DNA. The findings may have implications for the rational design of new antibacterial and anticancer drugs with higher potency and fewer side effects than existing drugs of the same class. The new type of complex forms between DNA and a class of quinolone and quinobenzoxazine drugs of great therapeutic importance. The quinolones include commercial antibacterial agents like Ciprofloxacin (marketed by Miles Inc.) and Norfloxacin (marketed by Merck & Co.). The quinobenzoxazines are quinolone derivatives that have shown promising anticancer activity. According to Miles, Ciprofloxacin is one of the most widely prescribed antibiotics, with 1994 worldwide sales of about $1.3 billion. Merck declines to provide sales figures on Norfloxacin. The precise mechanism of action of the quinolones and quinobenzoxazines has been uncertain. But Jun-Yao Fan, Daekyu Sun, Hongtao Yu,...