Potent dual inhibitors of Plasmodium falciparum M1 and M17 aminopeptidases through optimization of S1 pocket interactions

Malaria remains a global health problem, and though international efforts for treatment and eradication have made some headway, the emergence of drug-resistant parasites threatens this progress. Antimalarial therapeutics acting via novel mechanisms are urgently required. P. falciparum M1 and M17 are neutral aminopeptidases which are essential for parasite growth and development. Previous work in our group has identified inhibitors capable of dual inhibition of PfA-M1 and PfA-M17, and revealed further regions within the protease S1 pockets that could be exploited in the development of ligands with improved inhibitory activity. Herein, we report the structure-based design and synthesis of novel hydroxamic acid analogues that are capable of potent inhibition of both PfA-M1 and PfA-M17. Furthermore, the developed compounds potently inhibit Pf growth in culture, including the multi-drug resistant strain Dd2. The ongoing development of dual PfA-M1/PfA-M17 inhibitors continues to be an attractive strategy for the design of novel antimalarial therapeutics

Determination of selectivity and potential for drug resistance of novel antimalarial compounds from nature-inspired synthetic libraries

As malaria, caused by Plasmodium spp., continues to afflict millions of people worldwide, there is a dire need for the discovery of novel, inexpensive antimalarial drugs. Although there are effective drugs on the market, the consistent development of drug resistant species has decreased their efficacy, further emphasizing that novel therapeutic measures are urgently needed. Natural products provide the most diverse reservoir for the discovery of unique chemical scaffolds with the potential to effectively combat malarial infections, but, due to their complex structures, they often pose extreme challenges to medicinal chemists during pharmacokinetic optimization. In our laboratory we have performed unbiased, cell-based assays of numerous synthetic compounds from chemical libraries enriched with nature-like elements. This screening has led to the discovery of many original chemical scaffolds with promising antimalarial properties. In an attempt to further characterize these scaffolds, the most promising compounds were assayed in order to determine their cytotoxic effects on mammalian cells. In addition, the development of a drug resistant parasite line of Plasmodium falciparum to the most promising compound was done in order to determine the relative probability for parasite resistance development

Two-pronged attack: dual inhibition of Plasmodium falciparum M1 and M17 metalloaminopeptidases by a novel series of hydroxamic acid-based inhibitors

Plasmodium parasites, the causative agents of malaria, have developed resistance to most of our current antimalarial therapies, including artemisinin combination therapies which are widely described as our last line of defense. Antimalarial agents with a novel mode of action are urgently required. Two Plasmodium falciparum aminopeptidases, PfA-M1 and PfA-M17, play crucial roles in the erythrocytic stage of infection and have been validated as potential antimalarial targets. Using compound-bound crystal structures of both enzymes, we have used a structure-guided approach to develop a novel series of inhibitors capable of potent inhibition of both PfA-M1 and PfA-M17 activity and parasite growth in culture. Herein we describe the design, synthesis, and evaluation of a series of hydroxamic acid-based inhibitors and demonstrate the compounds to be exciting new leads for the development of novel antimalarial therapeutics

Non-bisphosphonate inhibitors of isoprenoid biosynthesis identified via computer-aided drug design.

The relaxed complex scheme, a virtual-screening methodology that accounts for protein receptor flexibility, was used to identify a low-micromolar, non-bisphosphonate inhibitor of farnesyl diphosphate synthase. Serendipitously, we also found that several predicted farnesyl diphosphate synthase inhibitors were low-micromolar inhibitors of undecaprenyl diphosphate synthase. These results are of interest because farnesyl diphosphate synthase inhibitors are being pursued as both anti-infective and anticancer agents, and undecaprenyl diphosphate synthase inhibitors are antibacterial drug leads

Global funding trends for malaria research in sub-Saharan Africa: a systematic analysis

Background Total domestic and international funding for malaria is inadequate to achieve WHO global targets in burden reduction by 2030. We describe the trends of investments in malaria-related research in sub-Saharan Africa and compare investment with national disease burden to identify areas of funding strength and potentially neglected populations. We also considered funding for malaria control. Methods Research funding data related to malaria for 1997–2013 were sourced from existing datasets, from 13 major public and philanthropic global health funders, and from funding databases. Investments (reported in US$) were considered by geographical area and compared with data on parasite prevalence and populations at risk in sub- Saharan Africa. 45 sub-Saharan African countries were ranked by amount of research funding received. Findings We found 333 research awards totalling US$814·4 million. Public health research covered $308·1 million (37·8$275·2 million (33·8%). Tanzania ($107·8 million [13·2$97·9 million [12·0%]), and Kenya ($92·9 million [11·4%]) received the highest sum of research investment and the most research awards. Malawi, Tanzania, and Uganda remained highly ranked after adjusting for national gross domestic product. Countries with a reasonably high malaria burden that received little research investment or funding for malaria control included Central African Republic (ranked 40th) and Sierra Leone (ranked 35th). Congo (Brazzaville) and Guinea had reasonably high malaria mortality, yet Congo (Brazzaville) ranked 38th and Guinea ranked 25th, thus receiving little investment. Interpretation Some countries receive reasonably large investments in malaria-related research (Tanzania, Kenya, Uganda), whereas others receive little or no investments (Sierra Leone, Central African Republic). Research investments are typically highest in countries where funding for malaria control is also high. Investment strategies should consider more equitable research and operational investments across countries to include currently neglected and susceptible populations

Investigating the antimalarial properties of small-molecule compounds and exploring Plasmodium falciparum hexokinase as a potential therapeutic target

Malaria, a deadly tropical disease transmitted by infected mosquitos, is caused by Plasmodium parasites. Plasmodium falciparum is the most pathogenic form, responsible for >95% of mortality. Continual development of therapeutic drug resistance necessitates the search for novel antimalarial therapeutics. In an aim to find novel anti-malarial therapeutics, the five small-molecule compounds were screened for their ability to kill P. falciparum parasites. The compounds were purified from Cinnamosma fragnans, a plant endemic to Madagascar commonly used as an antimalaria treatment. A 72-hour dose response assay was performed with the asexual stages of the parasite, and the percent inhibition of each drug was determined relative to the negative DMSO-control. CMOS was the most potent with an IC50 of 0.4148 micromolar, followed by CM18 with an IC50 of 0.9858 micromolar. In a target-based approach, this second part of the project focuses on characterizing the biochemical properties of P. falciparum hexokinase (PfHk) and studying how its properties change through the intra-erythrocytic life stages of P. falciparum. The parasite's single hexokinase enzyme is solely responsible for the conversion of glucose to glucose-6-phosphate, the necessary substrate for production of ATP via glycolysis or generation of reducing equivalents (NADPH) and ribose-5-phosphate via the pentose phosphate pathway. Glycolytic flux in the parasite has been shown to be regulated during the parasite's pathogenic, erythrocytic stages, with PfHK activity being suggested as the rate-limiting step. We are exploring the idea that regulated post-translational modification of PfHK and/or turnover is responsible for its catalytic activity. To test this hypothesis, polyclonal antisera was generated, which specifically recognizes PfHK. Western blotting of P. falciparum whole-cell lysate, under reducing conditions, recognizes a single band of ~55 kDa as is predicted. However, under native conditions, a single band of ~220 kDa is detected, suggestive of PfHK forming a homotetramer in vivo, which has not been described for other eukaryotic HKs. Recently published structural studies of recombinant PvHK in the Morris lab supports this observation (1). Furthermore, using our PfHK antisera we have successfully immunopurified the protein from the parasite and are in process of identifying post-translational modifications and possible binding partners. Immunofluorescence assays (IFA) were also performed to determine the location and expression of PfHk in the different life stages. Results from the IFA support previous hypotheses that the enzyme is cytosolic and is expressed in all stages. Additionally, SeaHorse XF and kinetic assays were performed to determine how the glycolytic flux and the activity of PfHk changes through the life cycle of the parasite, respectively. The results from the kinetic experiments show that trophozoites have the highest HK content with the lowest turnover rate; whereas the gametocytes had unmeasurable PfHk activity. The SeaHorse XF assays also showed that the asexual stages had measurable glycolytic flux relative to the sexual stages, which had a very low glycolytic flux. Thus, even though HK is expressed in the matured sexual stages, it is not active. Therefore, unraveling the mechanism by which PfHk activity is regulated in sexual stages is a promising next step to understanding how PfHk could be targeted in novel antimalarial therapeutics.Advanced Undergraduate Research awardHonors Arts and Science Competitive Research ScholarshipsA one-year embargo was granted for this item.Academic Major: Biochemistr