Wesley C. Van Voorhis

Chemical genetics of Plasmodium falciparum

Malaria caused by Plasmodium falciparum is a disease that is responsible for 880,000 deaths per year worldwide. Vaccine development has proved difficult and resistance has emerged for most antimalarial drugs. To discover new antimalarial chemotypes, we have used a phenotypic forward chemical genetic approach to assay 309,474 chemicals. Here we disclose structures and biological activity of the entire library—many of which showed potent in vitro activity against drug-resistant P. falciparum strains—and detailed profiling of 172 representative candidates. A reverse chemical genetic study identified 19 new inhibitors of 4 validated drug targets and 15 novel binders among 61 malarial proteins. Phylochemogenetic profiling in several organisms revealed similarities between Toxoplasma gondii and mammalian cell lines and dissimilarities between P. falciparum and related protozoans. One exemplar compound displayed efficacy in a murine model. Our findings provide the scientific community with new starting points for malaria drug discovery.

Genomic-scale prioritization of drug targets: the TDR Targets database

The increasing availability of genomic data for pathogens that cause tropical diseases has created new opportunities for drug discovery and development. However, if the potential of such data is to be fully exploited, the data must be effectively integrated and be easy to interrogate. Here, we discuss the development of the TDR Targets database (http://tdrtargets.org), which encompasses extensive genetic, biochemical and pharmacological data related to tropical disease pathogens, as well as computationally predicted druggability for potential targets and compound desirability information. By allowing the integration and weighting of this information, this database aims to facilitate the identification and prioritization of candidate drug targets for pathogens.

Malaria medicines: a glass half full?

Despite substantial scientific progress over the past two decades, malaria remains a worldwide burden that causes hundreds of thousands of deaths every year. New, affordable and safe drugs are required to overcome increasing resistance against artemisinin-based treatments, treat vulnerable populations, interrupt the parasite life cycle by blocking transmission to the vectors, prevent infection and target malaria species that transiently remain dormant in the liver. In this Review, we discuss how the antimalarial drug discovery pipeline has changed over the past 10 years, grouped by the various target compound or product profiles, to assess progress and gaps, and to recommend priorities.

Protein farnesyl and N-myristoyl transferases: piggy-back medicinal chemistry targets for the development of antitrypanosomatid and antimalarial therapeutics

To accelerate progress in the development of therapeutics for protozoan parasitic diseases, we are studying enzymes active in co- and post-translational protein modification that are already the focus of drug development in other eukaryotic systems. Inhibitors of the protein farnesyltransferases (PFT) are well-established antitumour agents of low cytotoxicity and known pharmokinetic properties, while inhibitors of N-myristoyl transferase show both selectivity and specificity in the treatment of fungal infections. Here, we summarise the current evidence that supports the targeting of these ubiquitous eukaryotic enzymes for drug development against trypanosomatid infections and malaria.

Toxoplasma gondii calcium-dependent protein kinase 1 is a target for selective kinase inhibitors.

New drugs are needed to treat toxoplasmosis. Toxoplasma gondii calcium-dependent protein kinases (TgCDPKs) are attractive targets because they are absent in mammals. We show that TgCDPK1 is inhibited by low nanomolar levels of bumped kinase inhibitors (BKIs), compounds inactive against mammalian kinases. Cocrystal structures of TgCDPK1 with BKIs confirm that the structural basis for selectivity is due to the unique glycine gatekeeper residue in the ATP-binding site. We show that BKIs interfere with an early step in T. gondii infection of human cells in culture. Furthermore, we show that TgCDPK1 is the in vivo target of BKIs because T. gondii expressing a glycine to methionine gatekeeper mutant enzyme show significantly decreased sensitivity to BKIs. Thus, design of selective TgCDPK1 inhibitors with low host toxicity may be achievable.

Identification of Attractive Drug Targets in Neglected-Disease Pathogens Using an In Silico Approach

Background The increased sequencing of pathogen genomes and the subsequent availability of genome-scale functional datasets are expected to guide the experimental work necessary for target-based drug discovery. However, a major bottleneck in this has been the difficulty of capturing and integrating relevant information in an easily accessible format for identifying and prioritizing potential targets. The open-access resource TDRtargets.org facilitates drug target prioritization for major tropical disease pathogens such as the mycobacteria Mycobacterium leprae and Mycobacterium tuberculosis; the kinetoplastid protozoans Leishmania major, Trypanosoma brucei, and Trypanosoma cruzi; the apicomplexan protozoans Plasmodium falciparum, Plasmodium vivax, and Toxoplasma gondii; and the helminths Brugia malayi and Schistosoma mansoni. Methodology/Principal Findings Here we present strategies to prioritize pathogen proteins based on whether their properties meet criteria considered desirable in a drug target. These criteria are based upon both sequence-derived information (e.g., molecular mass) and functional data on expression, essentiality, phenotypes, metabolic pathways, assayability, and druggability. This approach also highlights the fact that data for many relevant criteria are lacking in less-studied pathogens (e.g., helminths), and we demonstrate how this can be partially overcome by mapping data from homologous genes in well-studied organisms. We also show how individual users can easily upload external datasets and integrate them with existing data in TDRtargets.org to generate highly customized ranked lists of potential targets. Conclusions/Significance Using the datasets and the tools available in TDRtargets.org, we have generated illustrative lists of potential drug targets in seven tropical disease pathogens. While these lists are broadly consistent with the research communitys current interest in certain specific proteins, and suggest novel target candidates that may merit further study, the lists can easily be modified in a user-specific manner, either by adjusting the weights for chosen criteria or by changing the criteria that are included.

Opsonic Potential, Protective Capacity, and Sequence Conservation of the Treponema pallidum subspecies pallidum Tp92

By means of a differential screening technique, a 92-kDa antigen, designated Tp92, was identified from Treponema pallidum subspecies pallidum. This protein is similar in sequence to the protective surface antigens D15 from Haemophilus influenzae and Oma87 from Pasteurella multocida. Amino acid sequence analyses revealed a cleavable N-terminal signal sequence and predicted the outer membrane location for Tp92. In support of this, antiserum raised against recombinant Tp92 promotes opsonization and phagocytosis of T. pallidum by rabbit macrophages, and anti-Tp92 reactivity is absent from washed treponemal preparations presumed to be lacking outer membranes. The Tp92 amino acid sequence is 95.5%-100% conserved among 11 strains representing 4 pathogenic treponemes, and immunization with recombinant Tp92 partially protected rabbits from subsequent T. pallidum challenge. These results demonstrate that Tp92 is an invariant, immunoprotective antigen that may be present on the surface of T. pallidum and may represent a potential vaccine candidate for syphilis.

Thematic review series: Lipid Posttranslational Modifications. Fighting parasitic disease by blocking protein farnesylation

Protein farnesylation is a form of posttranslational modification that occurs in most, if not all, eukaryotic cells. Inhibitors of protein farnesyltransferase (PFTIs) have been developed as anticancer chemotherapeutic agents. Using the knowledge gained from the development of PFTIs for the treatment of cancer, researchers are currently investigating the use of PFTIs for the treatment of eukaryotic pathogens. This “piggy-back” approach not only accelerates the development of a chemotherapeutic agent for protozoan pathogens but is also a means of mitigating the costs associated with de novo drug design. PFTIs have already been shown to be efficacious in the treatment of eukaryotic pathogens in animal models, including both Trypanosoma brucei, the causative agent of African sleeping sickness, and Plasmodium falciparum, one of the causative agents of malaria. Here, current evidence and progress are summarized that support the targeting of protein farnesyltransferase for the treatment of parasitic diseases.

The effects of protein farnesyltransferase inhibitors on trypanosomatids: inhibition of protein farnesylation and cell growth

Attachment of the prenyl groups farnesyl and geranylgeranyl to specific eukaryotic cell proteins by protein prenyltransferases is required for the functioning of a number of cellular processes including signal transduction. In this study it was found that previously reported inhibitors of mammalian protein farnesyltransferase (PFT) [those that mimic the substrate farnesyl pyrophosphate and those that mimic the protein acceptor of the farnesyl group (CaaX mimetic)] inhibit in vitro farnesylation catalyzed by partially purified Trypanosoma brucei (T. brucei) PFT. The most potent PFT inhibitors at concentrations of 3-10 microM inhibit the growth of insect (procyclic) and bloodstream forms of T. brucei. One of the PFT inhibitors was found to block the incorporation of radiolabeled mevalonic acid (the precursor of prenyl groups) into specific T. brucei proteins. This study also shows that protein prenylation occurs in the protozoan parasites Trypanosoma cruzi (T. cruzi) and Leishmania mexicana (L. mexicana). The growth of T. cruzi intracellular form (amastigote) is also sensitive to PFT inhibitors, whereas the insect form (epimastigote) is considerably more resistant to inhibition of protein farnesylation. On the other hand, growth of 3T3 fibroblast cells (host cells for amastigote growth) was not affected by up to 100 microM PFT inhibitors. The growth of L. mexicana insect form (promastigote) is modestly inhibited by protein farnesyltransferase inhibitors. These results suggest the potential for the development of PFT inhibitors for treating trypanosomiasis and leishmaniasis.

The tprK gene is heterogeneous among Treponema pallidum strains and has multiple alleles.

ABSTRACT We have previously shown that the TprK antigen of T. pallidum, Nichols strain, is predominantly expressed in treponemes obtained 10 days after infection and that the hydrophilic domain of TprK is a target of opsonic antibodies and confers significant protection against homologous challenge. The T. pallidum genome sequence reported the presence of a single copy of the tprK gene in the Nichols strain. In the present study we demonstrate size heterogeneity in the central portions of the TprK hydrophilic domains of 14 treponemal isolates. Sequence analysis of the central domains and the complete open reading frames (ORFs) of the tprK genes confirms this heterogeneity. Further, multiple tprK sequences were found in the Nichols-definedtprK locus in three isolates (Sea 81-4, Bal 7, and Bal 73-1). In contrast, only a single tprK sequence could be identified in this locus in the Nichols strain. Alignment of the DNA and deduced amino acid sequences of the whole tprK ORFs shows the presence of seven discrete variable domains flanked by highly conserved regions. We hypothesize that these heterogeneous regions may be involved in antigenic heterogeneity and, in particular, evasion of the immune response. The presence of different tprK alleles in the tprK locus strongly suggests the existence of genetically different subpopulations within treponemal isolates.