Leslie W. Tari

Structural basis for bisphosphonate-mediated inhibition of isoprenoid biosynthesis

Farnesyl pyrophosphate synthetase (FPPS) synthesizes farnesyl pyrophosphate through successive condensations of isopentyl pyrophosphate with dimethylallyl pyrophosphate and geranyl pyrophosphate. Nitrogen-containing bisphosphonate drugs used to treat osteoclast-mediated bone resorption and tumor-induced hypercalcemia are potent inhibitors of the enzyme. Here we present crystal structures of substrate and bisphosphonate complexes of FPPS. The structures reveal how enzyme conformational changes organize conserved active site residues to exploit metal-induced ionization and substrate positioning for catalysis. The structures further demonstrate how nitrogen-containing bisphosphonates mimic a carbocation intermediate to inhibit the enzyme. Together, these FPPS complexes provide a structural template for the design of novel inhibitors that may prove useful for the treatment of osteoporosis and other clinical indications including cancer.

Structure and Mechanism of Phosphoenolpyruvate Carboxykinase

SCHEME 1 This conversion is the first committed step of gluconeogenesis in Escherichia coli and is part of the gluconeogenic pathway in virtually all organisms. In bacteria, such as E. coli, PCK is utilized during gluconeogenic growth when sugar levels are low (1). PCK is also an important enzyme in the glycolytic pathways of some organisms, such as Ascaris suum (2) and Trypanosoma cruzi (3), where it forms OAA from PEP, which in turn enters the citric acid cycle. In humans and other mammals, PCK is a central enzyme in carbohydrate metabolism, helping to regulate the blood glucose level. Gluconeogenic tissues, such as kidney and liver, convert lactate and other non-carbohydrate molecules to glucose, which in turn is released into the blood. The importance of PCK to carbohydrate metabolism in humans is such that it has been suggested as a potential drug target in the treatment of non-insulindependent diabetes mellitus (4). PCKs have been traditionally classified according to nucleotide specificity, with the ATP-dependent enzymes found in bacteria, yeast, Trypanomastid parasites and plants, and GTP-dependent PCKs in a variety of other eukaryotes and mammals (5). There are both important differences and similarities between ATPand GTPdependent PCKs. With the exception of bacterial PCKs, which are monomeric (6), most enzymes of the ATP-dependent class are multimeric, with two (7), four (8, 9), or six (10) subunits per enzyme, while known members of the GTP-dependent class are exclusively monomeric. While enzymes of either the ATPor GTP-dependent classes show significant (40–80%) amino acid sequence identity within their respective groups (11, 12), there is no significant overall sequence homology between the two classes of enzyme. Despite this lack of overall homology, both groups of PCKs contain similar NTP and oxaloacetate binding “consensus motifs” in their active sites, which likely play similar roles in substrate binding (which will be described in this review). Also, both GTP-dependent and ATP-dependent PCKs have been shown to possess lysinyl (13–17), argininyl (18, 19), and histidinyl (20, 21) residues at or near their active sites. The differences in nucleotide specificity and kinetic properties between ATPand GTP-dependent PCKs have led to the suggestion that they may be potential therapeutic targets in parasitic nematodes (22) and in Trypanomastid parasites such as T. cruzi (11). This minireview will focus on new structural results derived from the recent crystal structure determinations of native E. coli PCK (23, 24), its complex with ATP-Mg-oxalate (25), and the implications for the active site residues and catalytic mechanism of E. coli and other ATPand GTP-dependent PCKs. We also suggest revised nucleotide-binding sites and possible active site residues for the GTP-dependent PCK family. Other aspects of GTP-dependent PCK enzymology and genetics have been recently reviewed (26).

Pyrrolopyrimidine inhibitors of DNA gyrase B (GyrB) and topoisomerase IV (ParE). Part I: Structure guided discovery and optimization of dual targeting agents with potent, broad-spectrum enzymatic activity.

The bacterial topoisomerases DNA gyrase (GyrB) and topoisomerase IV (ParE) are essential enzymes that control the topological state of DNA during replication. The high degree of conservation in the ATP-binding pockets of these enzymes make them appealing targets for broad-spectrum inhibitor development. A pyrrolopyrimidine scaffold was identified from a pharmacophore-based fragment screen with optimization potential. Structural characterization of inhibitor complexes conducted using selected GyrB/ParE orthologs aided in the identification of important steric, dynamic and compositional differences in the ATP-binding pockets of the targets, enabling the design of highly potent pyrrolopyrimidine inhibitors with broad enzymatic spectrum and dual targeting activity.

Tricyclic GyrB/ParE (TriBE) inhibitors: a new class of broad-spectrum dual-targeting antibacterial agents.

Increasing resistance to every major class of antibiotics and a dearth of novel classes of antibacterial agents in development pipelines has created a dwindling reservoir of treatment options for serious bacterial infections. The bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV, are validated antibacterial drug targets with multiple prospective drug binding sites, including the catalytic site targeted by the fluoroquinolone antibiotics. However, growing resistance to fluoroquinolones, frequently mediated by mutations in the drug-binding site, is increasingly limiting the utility of this antibiotic class, prompting the search for other inhibitor classes that target different sites on the topoisomerase complexes. The highly conserved ATP-binding subunits of DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as excellent candidates for the development of dual-targeting antibacterial agents with broad-spectrum potential. However, to date, no natural product or small molecule inhibitors targeting these sites have succeeded in the clinic, and no inhibitors of these enzymes have yet been reported with broad-spectrum antibacterial activity encompassing the majority of Gram-negative pathogens. Using structure-based drug design (SBDD), we have created a novel dual-targeting pyrimidoindole inhibitor series with exquisite potency against GyrB and ParE enzymes from a broad range of clinically important pathogens. Inhibitors from this series demonstrate potent, broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens of clinical importance, including fluoroquinolone resistant and multidrug resistant strains. Lead compounds have been discovered with clinical potential; they are well tolerated in animals, and efficacious in Gram-negative infection models.

Crystal Structures of Active Fully Assembled Substrate- and Product-Bound Complexes of UDP-N-Acetylmuramic Acid:l-Alanine Ligase (MurC) from Haemophilus influenzae

UDP-N-acetylmuramic acid:L-alanine ligase (MurC) catalyzes the addition of the first amino acid to the cytoplasmic precursor of the bacterial cell wall peptidoglycan. The crystal structures of Haemophilus influenzae MurC in complex with its substrate UDP-N-acetylmuramic acid (UNAM) and Mg(2+) and of a fully assembled MurC complex with its product UDP-N-acetylmuramoyl-L-alanine (UMA), the nonhydrolyzable ATP analogue AMPPNP, and Mn(2+) have been determined to 1.85- and 1.7-A resolution, respectively. These structures reveal a conserved, three-domain architecture with the binding sites for UNAM and ATP formed at the domain interfaces: the N-terminal domain binds the UDP portion of UNAM, and the central and C-terminal domains form the ATP-binding site, while the C-terminal domain also positions the alanine. An active enzyme structure is thus assembled at the common domain interfaces when all three substrates are bound. The MurC active site clearly shows that the gamma-phosphate of AMPPNP is positioned between two bound metal ions, one of which also binds the reactive UNAM carboxylate, and that the alanine is oriented by interactions with the positively charged side chains of two MurC arginine residues and the negatively charged alanine carboxyl group. These results indicate that significant diversity exists in binding of the UDP moiety of the substrate by MurC and the subsequent ligases in the bacterial cell wall biosynthesis pathway and that alterations in the domain packing and tertiary structure allow the Mur ligases to bind sequentially larger UNAM peptide substrates.

Pyrrolopyrimidine inhibitors of DNA gyrase B (GyrB) and topoisomerase IV (ParE), Part II: development of inhibitors with broad spectrum, Gram-negative antibacterial activity.

The structurally related bacterial topoisomerases DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as prime candidates for the development of broad spectrum antibacterial agents. However, GyrB/ParE targeting antibacterials with spectrum that encompasses robust Gram-negative pathogens have not yet been reported. Using structure-based inhibitor design, we optimized a novel pyrrolopyrimidine inhibitor series with potent, dual targeting activity against GyrB and ParE. Compounds were discovered with broad antibacterial spectrum, including activity against Pseudomonas aeruginosa, Acinetobacter baumannii and Escherichia coli. Herein we describe the SAR of the pyrrolopyrimidine series as it relates to key structural and electronic features necessary for Gram-negative antibacterial activity.

Structural Basis for Iron Binding and Release by a Novel Class of Periplasmic Iron-Binding Proteins Found in Gram-Negative Pathogens

We have determined the 1.35- and 1.45-A structures, respectively, of closed and open iron-loaded forms of Mannheimia haemolytica ferric ion-binding protein A. M. haemolytica is the causative agent in the economically important and fatal disease of cattle termed shipping fever. The periplasmic iron-binding protein of this gram-negative bacterium, which has homologous counterparts in many other pathogenic species, performs a key role in iron acquisition from mammalian host serum iron transport proteins and is essential for the survival of the pathogen within the host. The ferric (Fe(3+)) ion in the closed structure is bound by a novel asymmetric constellation of four ligands, including a synergistic carbonate anion. The open structure is ligated by three tyrosyl residues and a dynamically disordered solvent-exposed anion. Our results clearly implicate the synergistic anion as the primary mediator of global protein conformation and provide detailed insights into the molecular mechanisms of iron binding and release in the periplasm.

Benzimidazole and imidazole inhibitors of histone deacetylases: Synthesis and biological activity.

A series of N-hydroxy-3-[3-(1-substituted-1H-benzoimidazol-2-yl)-phenyl]-acrylamides (5a-5ab) and N-hydroxy-3-[3-(1,4,5-trisubstituted-1H-imidazol-2-yl)-phenyl]-acrylamides (12a-s) were designed, synthesized, and found to be nanomolar inhibitors of human histone deacetylases. Multiple compounds bearing an N1-piperidine demonstrate EC(50)s of 20-100 nM in human A549, HL60, and PC3 cells, in vitro and in vivo hyperacetylation of histones H3 and H4, and induction of p21(waf). Compound 5x displays efficacy in human tumor xenograft models.

Crystal structure of Pasteurella haemolytica ferric ion-binding protein A reveals a novel class of bacterial iron-binding proteins

Pasteurellosis caused by the Gram-negative pathogen Pasteurella haemolytica is a serious disease leading to death in cattle. To scavenge growth-limiting iron from the host, the pathogen utilizes the periplasmic ferric ion-binding protein A (PhFbpA) as a component of an ATP-binding cassette transport pathway. We report the 1.2-Å structure of the iron-free (apo) form of PhFbpA, which is a member of the transferrin structural superfamily. The protein structure adopts a closed conformation, allowing us to reliably assign putative iron-coordinating residues. Based on our analysis, PhFbpA utilizes a unique constellation of binding site residues and anions to octahedrally coordinate an iron atom. A surprising finding in the structure is the presence of two formate anions on opposite sides of the iron-binding pocket. The formate ions tether the N- and C-terminal domains of the protein and stabilize the closed structure, also providing clues as to probable candidates for synergistic anions in the iron-loaded state. PhFbpA represents a new class of bacterial iron-binding proteins.

High-affinity binding by the periplasmic iron-binding protein from Haemophilus influenzae is required for acquiring iron from transferrin.

The periplasmic iron-binding protein, FbpA (ferric-ion-binding protein A), performs an essential role in iron acquisition from transferrin in Haemophilus influenzae. A series of site-directed mutants in the metal-binding amino acids of FbpA were prepared to determine their relative contribution to iron binding and transport. Structural studies demonstrated that the mutant proteins crystallized in an open conformation with the iron atom associated with the C-terminal domain. The iron-binding properties of the mutant proteins were assessed by several assays, including a novel competitive iron-binding assay. The relative ability of the proteins to compete for iron was pH dependent, with a rank order at pH 6.5 of wild-type, Q58L, H9Q>H9A, E57A>Y195A, Y196A. The genes encoding the mutant FbpA were introduced into H. influenzae and the resulting strains varied in the level of ferric citrate required to support growth on iron-limited medium, suggesting a rank order for metal-binding affinities under physiological conditions comparable with the competitive binding assay at pH 6.5 (wild-type=Q58L>H9Q>H9A, E57A>Y195A, Y196A). Growth dependence on human transferrin was only obtained with cells expressing wild-type, Q58L or H9Q FbpAs, proteins with stability constants derived from the competition assay >2.0x10(18) M(-1). These results suggest that a relatively high affinity of iron binding by FbpA is required for removal of iron from transferrin and its transport across the outer membrane.