J. Richard Miller

A class of selective antibacterials derived from a protein kinase inhibitor pharmacophore

As the need for novel antibiotic classes to combat bacterial drug resistance increases, the paucity of leads resulting from target-based antibacterial screening of pharmaceutical compound libraries is of major concern. One explanation for this lack of success is that antibacterial screening efforts have not leveraged the eukaryotic bias resulting from more extensive chemistry efforts targeting eukaryotic gene families such as G protein-coupled receptors and protein kinases. Consistent with a focus on antibacterial target space resembling these eukaryotic targets, we used whole-cell screening to identify a series of antibacterial pyridopyrimidines derived from a protein kinase inhibitor pharmacophore. In bacteria, the pyridopyrimidines target the ATP-binding site of biotin carboxylase (BC), which catalyzes the first enzymatic step of fatty acid biosynthesis. These inhibitors are effective in vitro and in vivo against fastidious Gram-negative pathogens including Haemophilus influenzae. Although the BC active site has architectural similarity to those of eukaryotic protein kinases, inhibitor binding to the BC ATP-binding site is distinct from the protein kinase-binding mode, such that the inhibitors are selective for bacterial BC. In summary, we have discovered a promising class of potent antibacterials with a previously undescribed mechanism of action. In consideration of the eukaryotic bias of pharmaceutical libraries, our findings also suggest that pursuit of a novel inhibitor leads for antibacterial targets with active-site structural similarity to known human targets will likely be more fruitful than the traditional focus on unique bacterial target space, particularly when structure-based and computational methodologies are applied to ensure bacterial selectivity.

Structural basis for effectiveness of siderophore-conjugated monocarbams against clinically relevant strains of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that causes nosocomial infections for which there are limited treatment options. Penicillin-binding protein PBP3, a key therapeutic target, is an essential enzyme responsible for the final steps of peptidoglycan synthesis and is covalently inactivated by β-lactam antibiotics. Here we disclose the first high resolution cocrystal structures of the P. aeruginosa PBP3 with both novel and marketed β-lactams. These structures reveal a conformational rearrangement of Tyr532 and Phe533 and a ligand-induced conformational change of Tyr409 and Arg489. The well-known affinity of the monobactam aztreonam for P. aeruginosa PBP3 is due to a distinct hydrophobic aromatic wall composed of Tyr503, Tyr532, and Phe533 interacting with the gem-dimethyl group. The structure of MC-1, a new siderophore-conjugated monocarbam complexed with PBP3 provides molecular insights for lead optimization. Importantly, we have identified a novel conformation that is distinct to the high-molecular-weight class B PBP subfamily, which is identifiable by common features such as a hydrophobic aromatic wall formed by Tyr503, Tyr532, and Phe533 and the structural flexibility of Tyr409 flanked by two glycine residues. This is also the first example of a siderophore-conjugated triazolone-linked monocarbam complexed with any PBP. Energetic analysis of tightly and loosely held computed hydration sites indicates protein desolvation effects contribute significantly to PBP3 binding, and analysis of hydration site energies allows rank ordering of the second-order acylation rate constants. Taken together, these structural, biochemical, and computational studies provide a molecular basis for recognition of P. aeruginosa PBP3 and open avenues for future design of inhibitors of this class of PBPs.

Discovery of Antibacterial Biotin Carboxylase Inhibitors by Virtual Screening and Fragment-Based Approaches.

As part of our effort to inhibit bacterial fatty acid biosynthesis through the recently validated target biotin carboxylase, we employed a unique combination of two emergent lead discovery strategies. We used both de novo fragment-based drug discovery and virtual screening, which employs 3D shape and electrostatic property similarity searching. We screened a collection of unbiased low-molecular-weight molecules and identified a structurally diverse collection of weak-binding but ligand-efficient fragments as potential building blocks for biotin carboxylase ATP-competitive inhibitors. Through iterative cycles of structure-based drug design relying on successive fragment costructures, we improved the potency of the initial hits by up to 3000-fold while maintaining their ligand-efficiency and desirable physicochemical properties. In one example, hit-expansion efforts resulted in a series of amino-oxazoles with antibacterial activity. These results successfully demonstrate that virtual screening approaches can substantially augment fragment-based screening approaches to identify novel antibacterial agents.

Structural evidence for substrate-induced synergism and half-sites reactivity in biotin carboxylase

Bacterial acetyl‐CoA carboxylase is a multifunctional biotin‐dependent enzyme that consists of three separate proteins: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT). Acetyl‐CoA carboxylase is a potentially attractive target for novel antibiotics because it catalyzes the first committed step in fatty acid biosynthesis. In the first half‐reaction, BC catalyzes the ATP‐dependent carboxylation of BCCP. In the second half‐reaction, the carboxyl group is transferred from carboxybiotinylated BCCP to acetyl‐CoA to produce malonyl‐CoA. A series of structures of BC from several bacteria crystallized in the presence of various ATP analogs is described that addresses three major questions concerning the catalytic mechanism. The structure of BC bound to AMPPNP and the two catalytically essential magnesium ions resolves inconsistencies between the kinetics of active‐site BC mutants and previously reported BC structures. Another structure of AMPPNP bound to BC shows the polyphosphate chain folded back on itself, and not in the correct (i.e., extended) conformation for catalysis. This provides the first structural evidence for the hypothesis of substrate‐induced synergism, which posits that ATP binds nonproductively to BC in the absence of biotin. The BC homodimer has been proposed to exhibit half‐sites reactivity where the active sites alternate or “flip‐flop” their catalytic cycles. A crystal structure of BC showed the ATP analog AMPPCF2P bound to one subunit while the other subunit was unliganded. The liganded subunit was in the closed or catalytic conformation while the unliganded subunit was in the open conformation. This provides the first structural evidence for half‐sites reactivity in BC.

Structural comparison of chromosomal and exogenous dihydrofolate reductase from Staphylococcus aureus in complex with the potent inhibitor trimethoprim

Dihydrofolate reductase (DHFR) is the enzyme responsible for the NADPH‐dependent reduction of 5,6‐dihydrofolate to 5,6,7,8‐tetrahydrofolate, an essential cofactor in the synthesis of purines, thymidylate, methionine, and other key metabolites. Because of its importance in multiple cellular functions, DHFR has been the subject of much research targeting the enzyme with anticancer, antibacterial, and antimicrobial agents. Clinically used compounds targeting DHFR include methotrexate for the treatment of cancer and diaminopyrimidines (DAPs) such as trimethoprim (TMP) for the treatment of bacterial infections. DAP inhibitors of DHFR have been used clinically for >30 years and resistance to these agents has become widespread. Methicillin‐resistant Staphylococcus aureus (MRSA), the causative agent of many serious nosocomial and community acquired infections, and other gram‐positive organisms can show resistance to DAPs through mutation of the chromosomal gene or acquisition of an alternative DHFR termed “S1 DHFR.” To develop new therapies for health threats such as MRSA, it is important to understand the molecular basis of DAP resistance. Here, we report the crystal structure of the wild‐type chromosomal DHFR from S. aureus in complex with NADPH and TMP. We have also solved the structure of the exogenous, TMP resistant S1 DHFR, apo and in complex with TMP. The structural and thermodynamic data point to important molecular differences between the two enzymes that lead to dramatically reduced affinity of DAPs to S1 DHFR. These differences in enzyme binding affinity translate into reduced antibacterial activity against strains of S. aureus that express S1 DHFR. Proteins 2009.

Discovery of novel antibacterials

Importance of the field: Antibiotics have existed in the environment for millennia, but it has only been in the past 80 years that humans have used them systematically to treat infections. This battle between humans and bacteria has led to an alarming increase in resistance to all clinically useful antibacterial agents. Thus, there is an imperative need for new agents to combat these resistant strains of bacteria. Areas covered in this review: The topics covered include natural product screening, identification and validation of new antibacterial targets and approaches for the discovery and optimization of antibacterial compounds. Last, an assessment of the major challenges facing antibacterial discovery is presented. What the reader will gain: The current strategies and methodologies for discovering and designing new antibacterial agents are evaluated as to their potential for generating the next round of therapeutics. Each topic is presented in a general, basic manner and will hopefully be a useful resource for students and newcomers to the field. Take home message: New antibacterial agents are desperately needed to fight the increasing number of antibiotic resistant pathogenic bacteria. New methodologies as well as traditional approaches should both be used for discovering antibiotics to meet this serious medical need.

The use of biochemical and biophysical tools for triage of high-throughput screening hits - A case study with Escherichia coli phosphopantetheine adenylyltransferase.

High‐throughput screening is utilized by pharmaceutical researchers and, increasingly, academic investigators to identify agents that act upon enzymes, receptors, and cellular processes. Screening hits include molecules that specifically bind the target and a greater number of non‐specific compounds. It is necessary to ‘triage’ these hits to identify the subset worthy of further exploration. As part of our antibacterial drug discovery effort, we applied a suite of biochemical and biophysical tools to accelerate the triage process. We describe application of these tools to a series of 9‐oxo‐4,9‐dihydropyrazolo[5,1‐b]quinazoline‐2‐carboxylic acids (PQ) hits from a screen of Escherichia coli phosphopantetheine adenylyltransferase (PPAT). Initial confirmation of specific binding to phosphopantetheine adenylyltransferase was obtained using biochemical and biophysical tools, including a novel orthogonal assay, isothermal titration calorimetry, and saturation transfer difference NMR. To identify the phosphopantetheine adenylyltransferase sub‐site bound by these inhibitors, two techniques were utilized: steady‐state enzyme kinetics and a novel 19F NMR method in which fluorine‐containing fragments that bind the ATP and/or phosphopantetheine sites serve as competitive reporter probes. These data are consistent with PQs binding the ATP sub‐site. In addition to identification of a series of PPAT inhibitors, the described hit triage process is broadly applicable to other enzyme targets in which milligram quantities of purified target protein are available.

Evaluation of pharmacokinetic/pharmacodynamic relationships of PD-0162819, a biotin carboxylase inhibitor representing a new class of antibacterial compounds, using in vitro infection models.

ABSTRACT The present study investigated the pharmacokinetic/pharmacodynamic (PK/PD) relationships of a prototype biotin carboxylase (BC) inhibitor, PD-0162819, against Haemophilus influenzae 3113 in static concentration time-kill (SCTK) and one-compartment chemostat in vitro infection models. H. influenzae 3113 was exposed to PD-0162819 concentrations of 0.5 to 16× the MIC (MIC = 0.125 μg/ml) and area-under-the-curve (AUC)/MIC ratios of 1 to 1,100 in SCTK and chemostat experiments, respectively. Serial samples were collected over 24 h. For efficacy driver analysis, a sigmoid maximum-effect (Emax) model was fitted to the relationship between bacterial density changes over 24 h and corresponding PK/PD indices. A semimechanistic PK/PD model describing the time course of bacterial growth and death was developed. The AUC/MIC ratio best explained efficacy (r2 = 0.95) compared to the peak drug concentration (Cmax)/MIC ratio (r2 = 0.76) and time above the MIC (T>MIC) (r2 = 0.88). Static effects and 99.9% killing were achieved at AUC/MIC values of 500 and 600, respectively. For time course analysis, the net bacterial growth rate constant, maximum bacterial density, and maximum kill rate constant were similar in SCTK and chemostat studies, but PD-0162819 was more potent in SCTK than in the chemostat (50% effective concentration [EC50] = 0.046 versus 0.34 μg/ml). In conclusion, basic PK/PD relationships for PD-0162819 were established using in vitro dynamic systems. Although the bacterial growth parameters and maximum drug effects were similar in SCTK and the chemostat system, PD-0162819 appeared to be more potent in SCTK, illustrating the importance of understanding the differences in preclinical models. Additional studies are needed to determine the in vivo relevance of these results.