Structures of glyceraldehyde 3-phosphate dehydrogenase in Neisseria gonorrhoeae and Chlamydia trachomatis.

Neisseria gonorrhoeae (Ng) and Chlamydia trachomatis (Ct) are the most commonly reported sexually transmitted bacteria worldwide and usually present as co-infections. Increasing resistance of Ng to currently recommended dual therapy of azithromycin and ceftriaxone presents therapeutic challenges for syndromic management of Ng-Ct co-infections. Development of a safe, effective, and inexpensive dual therapy for Ng-Ct co-infections is an effective strategy for the global control and prevention of these two most prevalent bacterial sexually transmitted infections. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a validated drug target with two approved drugs for indications other than antibacterials. Nonetheless, any new drugs targeting GAPDH in Ng and Ct must be specific inhibitors of bacterial GAPDH that do not inhibit human GAPDH, and structural information of Ng and Ct GAPDH will aid in finding such selective inhibitors. Here, we report the X-ray crystal structures of Ng and Ct GAPDH. Analysis of the structures demonstrates significant differences in amino acid residues in the active sites of human GAPDH from those of the two bacterial enzymes suggesting design of compounds to selectively inhibit Ng and Ct is possible. We also describe an efficient in vitro assay of recombinant GAPDH enzyme activity amenable to high-throughput drug screening to aid in identifying inhibitory compounds and begin to address selectivity.

Fragment-Based Screening of a Natural Product Library against 62 Potential Malaria Drug Targets Employing Native Mass Spectrometry.

Natural products are well known for their biological relevance, high degree of three-dimensionality, and access to areas of largely unexplored chemical space. To shape our understanding of the interaction between natural products and protein targets in the postgenomic era, we have used native mass spectrometry to investigate 62 potential protein targets for malaria using a natural-product-based fragment library. We reveal here 96 low-molecular-weight natural products identified as binding partners of 32 of the putative malarial targets. Seventy-nine (79) fragments have direct growth inhibition on Plasmodium falciparum at concentrations that are promising for the development of fragment hits against these protein targets. This adds a fragment library to the published HTS active libraries in the public domain.

In vitro efficacy of bumped kinase inhibitors against Besnoitia besnoiti tachyzoites.

Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a chronic and debilitating disease that causes systemic and skin manifestations and sterility in bulls. Neither treatments nor vaccines are currently available. In the search for therapeutic candidates, calcium-dependent protein kinases have arisen as promising drug targets in other apicomplexans (e.g. Neospora caninum, Toxoplasma gondii, Plasmodium spp. and Eimeria spp.) and are effectively targeted by bumped kinase inhibitors. In this study, we identified and cloned the gene coding for BbCDPK1. The impact of a library of nine bumped kinase inhibitor analogues on the activity of recombinant BbCDPK1 was assessed by luciferase assay. Afterwards, those were further screened for efficacy against Besnoitiabesnoiti tachyzoites grown in Marc-145 cells. Primary tests at 5µM revealed that eight compounds exhibited more than 90% inhibition of invasion and proliferation. The compounds BKI 1294, 1517, 1553 and 1571 were further characterised, and EC99 (1294: 2.38µM; 1517: 2.20µM; 1553: 3.34µM; 1571: 2.78µM) were determined by quantitative real-time polymerase chain reaction in 3-day proliferation assays. Exposure of infected cultures with EC99 concentrations of these drugs for up to 48h was not parasiticidal. The lack of parasiticidal action was confirmed by transmission electron microscopy, which showed that bumped kinase inhibitor treatment interfered with cell cycle regulation and non-disjunction of tachyzoites, resulting in the formation of large multi-nucleated complexes which co-existed with viable parasites within the parasitophorous vacuole. However, it is possible that, in the face of an active immune response, parasite clearance may occur. In summary, bumped kinase inhibitors may be effective drug candidates to control Besnoitiabesnoiti infection. Further in vivo experiments should be planned, as attainment and maintenance of therapeutic blood plasma levels in calves, without toxicity, has been demonstrated for BKIs 1294, 1517 and 1553.

Novel Bumped Kinase Inhibitors Are Safe and Effective Therapeutics in the Calf Clinical Model for Cryptosporidiosis.

Cryptosporidiosis, caused by the apicomplexan parasite Cryptosporidium parvum, is a diarrheal disease that has produced a large global burden in mortality and morbidity in humans and livestock. There are currently no consistently effective parasite-specific pharmaceuticals available for this disease. Bumped kinase inhibitors (BKIs) specific for parasite calcium-dependent protein kinases (CDPKs) have been shown to reduce infection in several parasites having medical and veterinary importance, including Toxoplasma gondii, Plasmodium falciparum, and C. parvum In the present study, BKIs were screened for efficacy against C. parvum infection in the neonatal mouse model. Three BKIs were then selected for safety and clinical efficacy evaluation in the calf model for cryptosporidiosis. Significant BKI treatment effects were observed for virtually all clinical and parasitological scoring parameters, including diarrhea severity, oocyst shedding, and overall health. These results provide proof of concept for BKIs as therapeutic drug leads in an animal model for human cryptosporidiosis.

Open Source Drug Discovery with the Malaria Box Compound Collection for Neglected Diseases and Beyond.

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts.

Bioaffinity Mass Spectrometry Screening.

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS or ESI-FTMS) was used to screen 192 natural product extracts and a 659-member natural product-based fragment library for bindings to a potential malaria drug target, Plasmodium falciparum Rab11a (PfRab11a, PF13_0119). One natural product extract and 11 fragments showed binding activity. A new natural product, arborside E, was identified from the active extract of Psydrax montigena as a weak binder. Its binding activity and inhibitory activity against PfRab11a were confirmed by ESI-FTMS titration experiments and an orthogonal enzyme assay.

High-throughput screening against thioredoxin glutathione reductase identifies novel inhibitors with potential therapeutic value for schistosomiasis.

BACKGROUND: Schistosomiasis, a parasitic disease also known as bilharzia and snail fever, is caused by different species of flatworms, such as Schistosoma mansoni (S. mansoni). Thioredoxin glutathione reductase (TGR) from S. mansoni (SmTGR) is a well-characterized drug target for schistosomiasis, yet no anti-SmTGR compounds have reached clinical trials, suggesting that therapeutic development against schistosomiasis might benefit from additional scaffolds targeting this enzyme. METHODS: A high-throughput screening (HTS) assay in vitro against SmTGR was developed and applied to a diverse compound library. SmTGR activity was quantified with ThioGlo®, a reagent that fluoresces upon binding to the free sulfhydryl groups of the reaction product GSH (reduced glutathione). RESULTS: We implemented an HTS effort against 59,360 synthetic compounds. In the primary screening, initial hits (928 or 1.56 %) showing greater than 90 % inhibition on SmTGR activity at a final concentration of 10 µM for each compound were identified. Further tests were carried out to confirm the effects of these hits and to explore the concentration-dependent response characteristics. As a result, 74 of them (0.12 %) representing 17 chemical scaffolds were confirmed and showed a great concentration-dependent inhibitory trend against SmTGR, including structures previously shown to be lethal to schistosomal growth. Of these, two scaffolds displayed a limited structure-activity relationship. When tested in cultured larvae, 39 compounds had cidal activity in 48 h, and five of them killed larvae completely at 3.125 µM. Of these, three compounds also killed adult worms ex vivo at concentrations between 5 µM and 10 µM. CONCLUSION: These confirmed hits may serve as starting points for the development of novel therapeutics to combat schistosomiasis.

Trypanosoma brucei glycogen synthase kinase-3, a target for anti-trypanosomal drug development: a public-private partnership to identify novel leads.

BACKGROUND: Trypanosoma brucei, the causative agent of Human African Trypanosomiasis (HAT), expresses two proteins with homology to human glycogen synthase kinase 3ß (HsGSK-3) designated TbruGSK-3 short and TbruGSK-3 long. TbruGSK-3 short has previously been validated as a potential drug target and since this enzyme has also been pursued as a human drug target, a large number of inhibitors are available for screening against the parasite enzyme. A collaborative industrial/academic partnership facilitated by the World Health Organisation Tropical Diseases Research division (WHO TDR) was initiated to stimulate research aimed at identifying new drugs for treating HAT. METHODOLOGY/PRINCIPAL FINDINGS: A subset of over 16,000 inhibitors of HsGSK-3 ß from the Pfizer compound collection was screened against the shorter of two orthologues of TbruGSK-3. The resulting active compounds were tested for selectivity versus HsGSK-3ß and a panel of human kinases, as well as in vitro anti-trypanosomal activity. Structural analysis of the human and trypanosomal enzymes was also performed. CONCLUSIONS/SIGNIFICANCE: We identified potent and selective compounds representing potential attractive starting points for a drug discovery program. Structural analysis of the human and trypanosomal enzymes also revealed hypotheses for further improving selectivity of the compounds.

Integrated dataset of screening hits against multiple neglected disease pathogens.

New chemical entities are desperately needed that overcome the limitations of existing drugs for neglected diseases. Screening a diverse library of 10,000 drug-like compounds against 7 neglected disease pathogens resulted in an integrated dataset of 744 hits. We discuss the prioritization of these hits for each pathogen and the strong correlation observed between compounds active against more than two pathogens and mammalian cell toxicity. Our work suggests that the efficiency of early drug discovery for neglected diseases can be enhanced through a collaborative, multi-pathogen approach.

Identification of inhibitors for putative malaria drug targets among novel antimalarial compounds.

The efficacy of most marketed antimalarial drugs has been compromised by evolution of parasite resistance, underscoring an urgent need to find new drugs with new mechanisms of action. We have taken a high-throughput approach toward identifying novel antimalarial chemical inhibitors of prioritized drug targets for Plasmodium falciparum, excluding targets which are inhibited by currently used drugs. A screen of commercially available libraries identified 5655 low molecular weight compounds that inhibit growth of P. falciparum cultures with EC(50) values below 1.25µM. These compounds were then tested in 384- or 1536-well biochemical assays for activity against nine Plasmodium enzymes: adenylosuccinate synthetase (AdSS), choline kinase (CK), deoxyuridine triphosphate nucleotidohydrolase (dUTPase), glutamate dehydrogenase (GDH), guanylate kinase (GK), N-myristoyltransferase (NMT), orotidine 5'-monophosphate decarboxylase (OMPDC), farnesyl pyrophosphate synthase (FPPS) and S-adenosylhomocysteine hydrolase (SAHH). These enzymes were selected using TDRtargets.org, and are believed to have excellent potential as drug targets based on criteria such as their likely essentiality, druggability, and amenability to high-throughput biochemical screening. Six of these targets were inhibited by one or more of the antimalarial scaffolds and may have potential use in drug development, further target validation studies and exploration of P. falciparum biochemistry and biology.

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.