W. C. Van Voorhis

The Seattle Structural Genomics Center for Infectious Disease (SSGCID)

The NIAID-funded Seattle Structural Genomics Center for Infectious Disease (SSGCID) is a consortium established to apply structural genomics approaches to potential drug targets from NIAID priority organisms for biodefense and emerging and re-emerging diseases. The mission of the SSGCID is to determine approximately 400 protein structures over the next five years. In order to maximize biomedical impact, ligand-based drug-lead discovery campaigns will be pursued for a small number of high-impact targets. Here we review the centers target selection processes, which include pro-active engagement of the infectious disease research and drug therapy communities to identify drug targets, essential enzymes, virulence factors and vaccine candidates of biomedical relevance to combat infectious diseases. This is followed by a brief overview of the SSGCID structure determination pipeline and ligand screening methodology. Finally, specifics of our resources available to the scientific community are presented. Physical materials and data produced by SSGCID will be made available to the scientific community, with the aim that they will provide essential groundwork benefiting future research and drug discovery.

Therapy and Prophylaxis of Systemic Protozoan Infections

SummaryThis article summarises current therapy and prophylaxis for Pneumocystis carinii, Toxoplasma gondii, Leishmania species, African trypanosomes (Trypanosoma brucei gambiense and T. b. rhodesiense), and American trypanosome (Trypanosoma cruzi) infections. Each agent and the disease it causes is briefly reviewed, and current data on the structure, mode of action, indications for treatment, dosage, administration, duration of therapy, efficacy, toxicity, and necessary monitoring during therapy are discussed for each drug. Drugs considered include cotrimoxazole (trimethoprim + sulfamethoxazole), pentamidine, dapsone (diaphenylsulfone), trimetrexate, eflornithine (DFMO), and primaquine/clindamycin and pyrimethamine/sulphonamide combinations for Pneumocystis pneumonia; pyrimethamine/sulfadiazine, spiramycin, and clindamycin for toxoplasmosis; pentavalent antimonials (‘Pentostam’ and ‘Glucantime’), pentamidine, amphotericin B, allopurinol, ketoconazole, and itraconazole for leishmaniasis; suramin, pentamidine, melarsoprol, tryparsamide, Mel W, berenil, and eflornithine (DFMO) for African trypanosomiasis; and nifurtimox, benznidazole and gentian violet for American trypanosomiasis.

Expression of proteins in Escherichia coli as fusions with maltose-binding protein to rescue non-expressed targets in a high-throughput protein-expression and purification pipeline

The rescue of protein-expression levels by cloning genes into MBP-fusion vector is described.

Structure of a ribulose 5‐phosphate 3‐epimerase from Plasmodium falciparum

The crystal structure of Pfal009167AAA, a putative ribulose 5‐phosphate 3‐epimerase (PfalRPE) from Plasmodium falciparum, has been determined to 2 Å resolution. RPE represents an exciting potential drug target for developing antimalarials because it is involved in the shikimate and the pentose phosphate pathways. The structure is a classic TIM‐barrel fold. A coordinated Zn ion and a bound sulfate ion in the active site of the enzyme allow for a greater understanding of the mechanism of action of this enzyme. This structure is solved in the framework of the Structural Genomics of Pathogenic Protozoa (SGPP) consortium. Proteins 2006.

Structure of nitrilotriacetate monooxygenase component B from Mycobacterium thermoresistibile

The 1.6 Å resolution crystal structure of nitrilotriacetate monooxygenase component B (NTA-MoB) from M. thermoresistibile is presented, revealing a highly conserved C-terminal tail that may modulate the activity of NTA-MoB in mycobacteria.

Invasion of hepatocytes by Plasmodium sporozoites requires cGMP-dependent protein kinase and calcium dependent protein kinase 4

Invasion of hepatocytes by sporozoites is essential for Plasmodium to initiate infection of the mammalian host. The parasites subsequent intracellular differentiation in the liver is the first developmental step of its mammalian cycle. Despite their biological significance, surprisingly little is known of the signalling pathways required for sporozoite invasion. We report that sporozoite invasion of hepatocytes requires signalling through two second‐messengers – cGMP mediated by the parasites cGMP‐dependent protein kinase (PKG), and Ca2+, mediated by the parasites calcium‐dependent protein kinase 4 (CDPK4). Sporozoites expressing a mutated form of Plasmodium berghei PKG or carrying a deletion of the CDPK4 gene are defective in invasion of hepatocytes. Using specific and potent inhibitors of Plasmodium PKG and CDPK4, we demonstrate that PKG and CDPK4 are required for sporozoite motility, and that PKG regulates the secretion of TRAP, an adhesin that is essential for motility. Chemical inhibition of PKG decreases parasite egress from hepatocytes by inhibiting either the formation or release of merosomes. In contrast, genetic inhibition of CDPK4 does not significantly decrease the number of merosomes. By revealing the requirement for PKG and CDPK4 in Plasmodium sporozoite invasion, our work enables a better understanding of kinase pathways that act in different Plasmodium stages.

Structure of the prolyl-tRNA synthetase from the eukaryotic pathogen Giardia lamblia.

The genome of the human intestinal parasite Giardia lamblia contains only a single aminoacyl-tRNA synthetase gene for each amino acid. The Giardia prolyl-tRNA synthetase gene product was originally misidentified as a dual-specificity Pro/Cys enzyme, in part owing to its unexpectedly high off-target activation of cysteine, but is now believed to be a normal representative of the class of archaeal/eukaryotic prolyl-tRNA synthetases. The 2.2 Å resolution crystal structure of the G. lamblia enzyme presented here is thus the first structure determination of a prolyl-tRNA synthetase from a eukaryote. The relative occupancies of substrate (proline) and product (prolyl-AMP) in the active site are consistent with half-of-the-sites reactivity, as is the observed biphasic thermal denaturation curve for the protein in the presence of proline and MgATP. However, no corresponding induced asymmetry is evident in the structure of the protein. No thermal stabilization is observed in the presence of cysteine and ATP. The implied low affinity for the off-target activation product cysteinyl-AMP suggests that translational fidelity in Giardia is aided by the rapid release of misactivated cysteine.

Solution-state NMR structure and biophysical characterization of zinc-substituted rubredoxin B (Rv3250c) from Mycobacterium tuberculosis

One third of the world’s human population is infected with M. tuberculosis, the etiological agent responsible for tuberculosis (TB). Here, the solution structure of the small iron-binding protein from this organism, rubredoxin B (Rv3250c), is reported in the zinc-substituted form.

Structures of a putative ζ-class glutathione S-transferase from the pathogenic fungus Coccidioides immitis

The pathogenic fungus C. immitis causes coccidioidomycosis, a potentially fatal disease. Here, apo and glutathione-bound crystal structures of a previously uncharacterized protein from C. immitis that appears to be a ζ-class glutathione S-transferase are presented.

Structure of aldose reductase from Giardia lamblia

The 1.75 Å resolution crystal structure of aldose reductase from G. lamblia, the etiological agent of giardiasis, is reported.