Exquisite selectivity of griselimycin extends to beta subunit of DNA polymerases from Gram-negative bacterial pathogens.

Griselimycin, a cyclic depsidecapeptide produced by Streptomyces griseus, is a promising lead inhibitor of the sliding clamp component of bacterial DNA polymerases (β-subunit of Escherichia coli DNA pol III). It was previously shown to inhibit the Mycobacterium tuberculosis β-clamp with remarkably high affinity and selectivity - the peptide lacks any interaction with the human sliding clamp. Here, we used a structural genomics approach to address the prospect of broader-spectrum inhibition, in particular of β-clamps from Gram-negative bacterial targets.

Characterization of an Monofunctional Phosphomethylpyrimidine Kinase That Is Inhibited by Pyridoxal Phosphate.

Thiamin and its phosphate derivatives are ubiquitous molecules involved as essential cofactors in many cellular processes. The biosynthesis of thiamin employs the parallel synthesis of 4-methyl-5-(2-hydroxyethyl)thiazole (THZ-P) and 4-amino-2-methyl-5(diphosphooxymethyl) pyrimidine (HMP) pyrophosphate (HMP-PP), which are coupled to generate thiamin phosphate. Most organisms that can biosynthesize thiamin employ a kinase (HMPK or ThiD) to generate HMP-PP.

Crystal structure and biophysical characterization of IspD from Burkholderia thailandensis and Mycobacterium paratuberculosis.

The methylerythritol phosphate (MEP) pathway is a metabolic pathway that produces the isoprenoids isopentyl pyrophosphate and dimethylallyl pyrophosphate. Notably, the MEP pathway is present in bacteria and not in mammals, which makes the enzymes of the MEP pathway attractive targets for discovering new anti-infective agents due to the reduced chances of off-target interactions leading to side effects. There are seven enzymes in the MEP pathway, the third of which is IspD.

Crystal structures of glutamyl-tRNA synthetase from Elizabethkingia anopheles and E. meningosepticum.

Elizabethkingia bacteria are globally emerging pathogens that cause opportunistic and nosocomial infections, with up to 40% mortality among the immunocompromised. Elizabethkingia species are in the pipeline of organisms for high-throughput structural analysis at the Seattle Structural Genomics Center for Infectious Disease (SSGCID). These efforts include the structure-function analysis of potential therapeutic targets.

Molecular mechanism for strengthening E-cadherin adhesion using a monoclonal antibody.

E-cadherin (Ecad) is an essential cell-cell adhesion protein with tumor suppression properties. The adhesive state of Ecad can be modified by the monoclonal antibody 19A11, which has potential applications in reducing cancer metastasis. Using X-ray crystallography, we determine the structure of 19A11 Fab bound to Ecad and show that the antibody binds to the first extracellular domain of Ecad near its primary adhesive motif: the strand-swap dimer interface.

Broad-spectrum in vitro activity of macrophage infectivity potentiator inhibitors against Gram-negative bacteria and Leishmania major.

Background: The macrophage infectivity potentiator (Mip) protein, which belongs to the immunophilin superfamily, is a peptidyl-prolyl cis/trans isomerase (PPIase) enzyme. Mip has been shown to be important for virulence in a wide range of pathogenic microorganisms. It has previously been demonstrated that small-molecule compounds designed to target Mip from the Gram-negative bacterium Burkholderia pseudomallei bind at the site of enzymatic activity of the protein, inhibiting the in vitro activity of Mip.

Crystal structures of FolM alternative dihydrofolate reductase 1 from Brucella suis and Brucella canis.

Members of the bacterial genus Brucella cause brucellosis, a zoonotic disease that affects both livestock and wildlife. Brucella are category B infectious agents that can be aerosolized for biological warfare. As part of the structural genomics studies at the Seattle Structural Genomics Center for Infectious Disease (SSGCID), FolM alternative dihydrofolate reductases 1 from Brucella suis and Brucella canis were produced and their structures are reported.

Evolutionary Diversification of Host-Targeted Effectors Proteins Derived from a Conserved FicTA Toxin-Antitoxin Module.

Proteins containing a FIC domain catalyze AMPylation and other post-translational modifications (PTMs). In bacteria, they are typically part of FicTA toxin-antitoxin modules that control conserved biochemical processes such as topoisomerase activity, but they have also repeatedly diversified into host-targeted virulence factors. Among these, effector proteins (Beps) comprise a particularly diverse ensemble of FIC domains that subvert various host cellular functions.

Naegleria fowleri: Protein structures to facilitate drug discovery for the deadly, pathogenic free-living amoeba.

Naegleria fowleri is a pathogenic, thermophilic, free-living amoeba which causes primary amebic meningoencephalitis (PAM). Penetrating the olfactory mucosa, the brain-eating amoeba travels along the olfactory nerves, burrowing through the cribriform plate to its destination: the brain's frontal lobes. The amoeba thrives in warm, freshwater environments, with peak infection rates in the summer months and has a mortality rate of approximately 97%.

Pyrazole-Based Lactate Dehydrogenase Inhibitors with Optimized Cell Activity and Pharmacokinetic Properties.

Lactate dehydrogenase (LDH) catalyzes the conversion of pyruvate to lactate, with concomitant oxidation of reduced nicotinamide adenine dinucleotide as the final step in the glycolytic pathway. Glycolysis plays an important role in the metabolic plasticity of cancer cells and has long been recognized as a potential therapeutic target. Thus, potent, selective inhibitors of LDH represent an attractive therapeutic approach.

Discovery of Adamantane Carboxamides as Ebola Virus Cell Entry and Glycoprotein Inhibitors.

We identified and explored the structure-activity-relationship (SAR) of an adamantane carboxamide chemical series of Ebola virus (EBOV) inhibitors. Selected analogs exhibited half-maximal inhibitory concentrations (EC values) of ∼10-15 nM in (VSV) pseudotyped EBOV (pEBOV) infectivity assays, low hundred nanomolar EC activity against wild type EBOV, aqueous solubility >20 mg/mL, and attractive metabolic stability in human and nonhuman liver microsomes. X-ray cocrystallographic characterizations of a lead compound with the EBOV glycoprotein (GP) established the EBOV GP as a target for direct compound inhibitory activity and further provided relevant structural models that may assist in identifying optimized therapeutic candidates.

Toward a structome of Acinetobacter baumannii drug targets.

Acinetobacter baumannii is well known for causing hospital-associated infections due in part to its intrinsic antibiotic resistance as well as its ability to remain viable on surfaces and resist cleaning agents. In a previous publication, A. baumannii strain AB5075 was studied by transposon mutagenesis and 438 essential gene candidates for growth on rich-medium were identified.

Crystal structures of thiamine monophosphate kinase from Acinetobacter baumannii in complex with substrates and products.

Thiamine monophosphate kinase (ThiL) catalyzes the last step of thiamine pyrophosphate (TPP) synthesis, the ATP-dependent phosphorylation of thiamine monophosphate (TMP) to thiamine pyrophosphate. We solved the structure of ThiL from the human pathogen A. baumanii in complex with a pair of substrates TMP and a non-hydrolyzable adenosine triphosphate analog, and in complex with a pair of products TPP and adenosine diphosphate.

Enzymatic and Structural Characterization of the Glucokinase.

Infection with the free-living amoeba leads to life-threatening primary amoebic meningoencephalitis. Efficacious treatment options for these infections are limited, and the mortality rate is very high (∼98%). Parasite metabolism may provide suitable targets for therapeutic design.

Characterization and structure determination of prolyl-tRNA synthetase from Pseudomonas aeruginosa and development as a screening platform.

Pseudomonas aeruginosa is an opportunistic multi-drug resistant pathogen implicated as a causative agent in nosocomial and community acquired bacterial infections. The gene encoding prolyl-tRNA synthetase (ProRS) from P. aeruginosa was overexpressed in Escherichia coli and the resulting protein was characterized.

Crystal structure of chorismate mutase from Burkholderia thailandensis.

Burkholderia thailandensis is often used as a model for more virulent members of this genus of proteobacteria that are highly antibiotic-resistant and are potential agents of biological warfare that are infective by inhalation. As part of ongoing efforts to identify potential targets for the development of rational therapeutics, the structures of enzymes that are absent in humans, including that of chorismate mutase from B. thailandensis, have been determined by the Seattle Structural Genomics Center for Infectious Disease.

Discovery and Optimization of Potent, Cell-Active Pyrazole-Based Inhibitors of Lactate Dehydrogenase (LDH).

We report the discovery and medicinal chemistry optimization of a novel series of pyrazole-based inhibitors of human lactate dehydrogenase (LDH). Utilization of a quantitative high-throughput screening paradigm facilitated hit identification, while structure-based design and multiparameter optimization enabled the development of compounds with potent enzymatic and cell-based inhibition of LDH enzymatic activity. Lead compounds such as 63 exhibit low nM inhibition of both LDHA and LDHB, submicromolar inhibition of lactate production, and inhibition of glycolysis in MiaPaCa2 pancreatic cancer and A673 sarcoma cells.