The impact of whole genome sequence data on drug discovery--a malaria case study.

BACKGROUND: Identification and validation of a drug discovery target is a prominent step in drug development. In the post-genomic era it is possible to reevaluate the association of a gene with a specific biological function to see if a homologous gene can subsume this role. This concept has special relevance to drug discovery in human infectious diseases, like malaria. A trophozoite cysteine protease (falcipain-1) from the papain family, thought to be responsible for the degradation of erythrocyte hemoglobin, has been considered a promising target for drug discovery efforts owing to the antimalarial activity of peptide based covalent cysteine protease inhibitors. This led to the development of non-peptidic non-covalent inhibitors of falcipain-1 and their characterization as antimalarials. It is now clear from sequencing efforts that the malaria genome contains more than one cysteine protease and that falcipain-1 is not the most important contributor to hemoglobin degradation. Rather, falcipain-2 and falcipain-3 appear to account for the majority of cysteine hemoglobinase activity in the plasmodium trophozoite. MATERIALS AND METHODS: We have modeled the falcipain-2 cysteine protease from one of the major human malaria species, Plasmodium falciparum and compared it to our original work on falcipain-1. As with falcipain-1, computa-tional screening of the falcipain-2 active site was conducted using DOCK. Using structural superpositions within the protease family and evolutionary analysis of substrate specificity sites, we focused on the commonalities and the protein specific features to direct our drug discovery effort. RESULTS: Since 1993, the size of the Available Chemicals Directory had increased from 55313 to 195419 unique chemical structures. For falcipain-2, eight inhibitors were identified with IC50's against the enzyme between 1 and 7 microM. Application of three of these inhibitors to infected erythrocytes cured malaria in culture, but parasite death did not correlate with food vacuole abnormalities associated with the activity of mechanistic inhibitors of cysteine proteases like the epoxide E64. CONCLUSIONS: Using plasmodial falcipain proteases, we show how a protein family perspective can influence target discovery and inhibitor design. We suspect that parallel drug discovery programs where a family of targets is considered, rather than serial programs built on a single therapeutic focus, will become the dominant industrial paradigm. Economies of scale in assay development and in compound synthesis are expected owing to the functional and structural features of individual family members. One of the remaining challenges in post-genomic drug discovery is that inhibitors of one target are likely to show some activity against other family members. This lack of specificity may lead to difficulties in functional assignments and target validation as well as a complex side effect profile.     

HDAC inhibitors in parasitic diseases

Parasitic diseases cause significant global morbidity and mortality, particularly in underdeveloped regions of the world. Malaria alone causes ~800000 deaths each year, with children and pregnant women being at highest risk. There is no licensed vaccine available for any human parasitic disease and drug resistance is compromising the efficacy of many available anti-parasitic drugs. This is driving drug discovery research on new agents with novel modes of action. Histone deacetylase (HDAC) inhibitors are being investigated as drugs for a range of diseases, including cancers and infectious diseases such as HIV/AIDS, and several parasitic diseases. This review focuses on the current state of knowledge of HDAC inhibitors targeted to the major human parasitic diseases malaria, schistosomiasis, trypanosomiasis, toxoplasmosis and leishmaniasis. Insights are provided into the unique challenges that will need to be considered if HDAC inhibitors are to be progressed towards clinical development as potential new anti-parasitic drugs.     

An Image-Based Drug Susceptibility Assay Targeting the Placental Sequestration of Plasmodium falciparum-Infected Erythrocytes

Placental malaria is a significant cause of all malaria-related deaths globally for which no drugs have been developed to specifically disrupt its pathogenesis. To facilitate the discovery of antimalarial drugs targeting the cytoadherence process of Plasmodium-infected erythrocytes in the placenta microvasculature, we have developed an automated image-based assay for high-throughput screening for potent cytoadherence inhibitors in vitro. Parasitized erythrocytes were drug-treated for 24 h and then allowed to adhere on a monolayer of placental BeWo cells prior to red blood cell staining with glycophorin A antibodies. Upon image-acquisition, drug effects were quantified as the proportion of treated parasitized erythrocytes to BeWo cells compared to the binding of untreated iRBCs. We confirmed the reliability of this new assay by comparing the binding ratios of CSA- and CD36-panned parasites on the placental BeWo cells, and by quantifying the effects of chondroitin sulfate A, brefeldin A, and artemisinin on the binding. By simultaneously examining the drug effects on parasite viability, we could discriminate between cytoadherence-specific inhibitors and other schizonticidal compounds. Taken together, our data establish that the developed assay is highly suitable for drug studies targeting placental malaria, and will facilitate the discovery and rapid development of new therapies against malaria.     

Comparative folate metabolism in humans and malaria parasites (part I): pointers for malaria treatment from cancer chemotherapy

New inhibitors are urgently needed to overcome the burgeoning problem of drug resistance in the treatment of Plasmodium falciparum infection. Targeting the folate pathway has proved to be a powerful strategy for drug development against rapidly multiplying systems such as cancer cells and microorganisms. Antifolates have long been used for malaria treatment but, despite their success, much less is known about parasite folate metabolism than about that of the human host. In this article, we focus on folate enzymes used clinically as anticancer drug targets, in addition to those that have potential to be used as drug targets, for which there are inhibitors at various stages of development. We discuss how this information could lead to the identification of new targets in malaria parasites.     

Metallo-aminopeptidase inhibitors

Aminopeptidases are enzymes that selectively hydrolyze an amino acid residue from the N-terminus of proteins and peptides. They are important for the proper functioning of prokaryotic and eukaryotic cells, but very often are central players in the devastating human diseases like cancer, malaria and diabetes. The largest aminopeptidase group include enzymes containing metal ion(s) in their active centers, which often determines the type of inhibitors that are the most suitable for them. Effective ligands mostly bind in a non-covalent mode by forming complexes with the metal ion(s). Here, we present several approaches for the design of inhibitors for metallo-aminopeptidases. The optimized structures should be considered as potential leads in the drug discovery process against endogenous and infectious diseases.     

Expression of Functional Plasmodium falciparum Enzymes Using a Wheat Germ Cell-Free System

One decade after the sequencing of the Plasmodium falciparum genome, 95% of malaria proteins in the genome cannot be expressed in traditional cell-based expression systems, and the targets of the best new leads for antimalarial drug discovery are either not known or not available in functional form. For a disease that kills up to 1 million people per year, routine expression of recombinant malaria proteins in functional form is needed both for the discovery of new therapeutics and for identification of targets of new drugs. We tested the general utility of cell-free systems for expressing malaria enzymes. Thirteen test enzyme sequences were reverse amplified from total RNA, cloned into a plant-like expression vector, and subjected to cell-free expression in a wheat germ system. Protein electrophoresis and autoradiography confirmed the synthesis of products of expected molecular masses. In rare problematic cases, truncated products were avoided by using synthetic genes carrying wheat codons. Scaled-up production generated 39 to 354 μg of soluble protein per 10 mg of translation lysate. Compared to rare proteins where cell-based systems do produce functional proteins, the cell-free yields are comparable or better. All 13 test products were enzymatically active, without failure. This general path to produce functional malaria proteins should now allow the community to access new tools, such as biologically active protein arrays, and lead to the discovery of new chemical functions, structures, and inhibitors of previously inaccessible malaria gene products.     

Drug discovery for malaria: a very challenging and timely endeavor

The prevalence of resistance to known antimalarial drugs has resulted in the expansion of antimalarial drug discovery efforts. Academic and nonprofit institutions are partnering with the pharmaceutical industry to develop new antimalarial drugs. Several new antimalarial agents are undergoing clinical trials, mainly those resurrected from previous antimalarial drug discovery programs. Novel antimalarials are being advanced through the drug development process, of course, with the anticipated high failure rate typical of drug discovery. Many of these are summarized in this review. Mechanisms for funding antimalarial drug discovery and genomic information to aid drug target selection have never been better. It remains to be seen whether ongoing efforts will be sufficient for reducing malaria burden in the developing world.     

The potent antiplasmodial calmodulin-antagonist trifluoperazine inhibits plasmodium falciparum calcium-dependent protein kinase 4

Due to their critical involvement in the execution of the malaria parasite developmental pattern both in the mosquito vector and in the human host, Plasmodium calcium-dependent protein kinases (CDPKs) are considered promising candidates for the development of new tools to block malaria transmission. We report here that the phenothiazine trifluoperazine non-competitively inhibits Plasmodium falciparum CDPK4 in the micromolar range while other calmodulin antagonists only marginally affect the enzyme activity, and we propose the inhibition mechanism. Our results demonstrate that selective enzyme inhibition is achievable by targeting its calmodulin-like domain. This observation could be exploited for the discovery of innovative phenothiazine-based CDPK inhibitors of potential medical interest.     

Inhibitors of Plasmepsin II-potential antimalarial agents

In order to overcome the problem of drug resistance in malaria, it appears wise to concentrate drug discovery efforts toward new structural classes and new mechanisms of action. We report our results, targeting Plasmepsin II, a Plasmodium falciparum aspartic protease active in hemoglobin degradation, a parasite specific catabolic pathway. The results show that the new structural class is not only inhibiting PMII in vitro but is also active in a P. falciparum infected human red blood cell assay.     

Isoprenoid Precursor Biosynthesis Is the Essential Metabolic Role of the Apicoplast during Gametocytogenesis in Plasmodium falciparum

The malaria parasite harbors a relict plastid called the apicoplast and its discovery opened a new avenue for drug discovery and development due to its unusual, nonmammalian metabolism. The apicoplast is essential during the asexual intraerythrocytic and hepatic stages of the parasite, and there is strong evidence supporting its essential metabolic role during the mosquito stages of the parasite. Supply of the isoprenoid building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) is the essential metabolic function of the apicoplast during the asexual intraerythrocytic stages. However, the metabolic role of the apicoplast during gametocyte development, the malaria stages transmitted to the mosquito, remains unknown. In this study, we showed that production of IPP for isoprenoid biosynthesis is the essential metabolic function of the apicoplast during gametocytogenesis, by obtaining normal gametocytes lacking the apicoplast when supplemented with IPP. When IPP supplementation was removed early in gametocytogenesis, developmental defects were observed, supporting the essential role of isoprenoids for normal gametocytogenesis. Furthermore, mosquitoes infected with gametocytes lacking the apicoplast developed fewer and smaller oocysts that failed to produce sporozoites. This finding further supports the essential role of the apicoplast in establishing a successful infection in the mosquito vector. Our study supports isoprenoid biosynthesis as a valid drug target for development of malaria transmission-blocking inhibitors.     

Malaria: Targeting parasite and host cell kinomes

Malaria still remains one of the deadliest infectious diseases, and has a tremendous morbidity and mortality impact in the developing world. The propensity of the parasites to develop drug resistance, and the relative reluctance of the pharmaceutical industry to invest massively in the developments of drugs that would offer only limited marketing prospects, are major issues in antimalarial drug discovery. Protein kinases (PKs) have become a major family of targets for drug discovery research in a number of disease contexts, which has generated considerable resources such as kinase-directed libraries and high throughput kinase inhibition assays. The phylogenetic distance between malaria parasites and their human host translates into important divergences in their respective kinomes, and most Plasmodium kinases display atypical properties (as compared to mammalian PKs) that can be exploited towards selective inhibition. Here, we discuss the taxon-specific kinases possessed by malaria parasites, and give an overview of target PKs that have been validated by reverse genetics, either in the human malaria parasite Plasmodium falciparum or in the rodent model Plasmodium berghei. We also briefly allude to the possibility of attacking Plasmodium through the inhibition of human PKs that are required for survival of this obligatory intracellular parasite, and which are targets for other human diseases.     

The development of a whole-cell based medium throughput screening system for the discovery of human aldosterone synthase (CYP11B2) inhibitors: old drugs disclose new applications for the therapy of congestive heart failure, myocardial fibrosis and hypertension

Cytochrome P450 enzymes play an important role in steroid hormone biosynthesis of the human adrenal gland, e.g., the production of cortisol and aldosterone. Aldosterone, the most important human mineralocorticoid, is involved in the regulation of the salt and water homeostasis of the body and thus in the regulation of blood pressure, whereas cortisol is the most important glucocorticoid of the human body. CYP11B-dependent steroid hydroxylases are drug development targets, and since they are very closely related enzymes, the discovery of selective inhibitors has been subject to intense investigations for several years. Here we report the development of a whole-cell medium throughput screening technology for the discovery of CYP11B2 inhibitors. The new screening system displayed high reproducibility and was applied to investigate a library of pharmacologically active compounds. 1268 compounds were investigated during this study which revealed 5 selective inhibitors of CYP11B2 (after validation against CYP11B1). The new inhibitors of CYP11B2 are already existing drugs that could be used either in the treatment of hyperaldosteronism-related diseases or as lead compounds that could further be optimised to achieve safer and selective inhibitors of aldosterone synthase. Article from the Special issue on 'Targeted Inhibitors'.     

Recent advances in malaria drug discovery

This digest covers some of the most relevant progress in malaria drug discovery published between 2010 and 2012. There is an urgent need to develop new antimalarial drugs. Such drugs can target the blood stage of the disease to alleviate the symptoms, the liver stage to prevent relapses, and the transmission stage to protect other humans. The pipeline for the blood stage is becoming robust, but this should not be a source of complacency, as the current therapies set a high standard. Drug discovery efforts directed towards the liver and transmission stages are in their infancy but are receiving increasing attention as targeting these stages could be instrumental in eradicating malaria.     

Strategies for senolytic drug discovery

Senolytics are a category of drugs that reduce the impact of cellular senescence, an effect associated with a range of chronic and age-related diseases. Since the discovery of the first senolytics in 2015, the number of known senolytic agents has grown dramatically. This review discusses the broad categories of known senolytics—kinase inhibitors, Bcl-2 family protein inhibitors, naturally occurring polyphenols, heat shock protein inhibitors, BET family protein inhibitors, P53 stabilizers, repurposed anti-cancer drugs, cardiac steroids, PPAR-alpha agonists, and antibiotics. The approaches used to screen for new senolytics are articulated including a range of methods to induce senescence, different target cell types, various senolytic assays, and markers. The choice of methods can greatly influence the outcomes of a screen, with high-quality screens featuring robust systems, adequate controls, and extensive validation in alternate assays. Recent advances in single-cell analysis and computational methods for senolytic identification are also discussed. There is significant potential for further drug discovery, but this will require additional research into drug targets and mechanisms of actions and their subsequent rigorous evaluation in pre-clinical models and human trials.     

Paradigm shifts in malaria parasite biochemistry and anti-malarial chemotherapy

A fatty acid synthesis (FAS) pathway was recently discovered and established in the obligate human parasite Plasmodium falciparum. Its inhibition by triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether) leads to its classification as a type II FAS. Humans, the vertebrate host for the malarial parasite utilize type I FAS, which is not inhibited by triclosan. This discovery thus paves the way for novel approaches to the treatment of malaria. In direct contrast to the delayed-death phenotype associated with poisoning of the apicoplast using certain other drugs, the rapid and striking action of triclosan suggests the possibility of developing new drug(s) for the treatment of malaria.     

In Vitro Resistance Selections for Plasmodium falciparum Dihydroorotate Dehydrogenase Inhibitors Give Mutants with Multiple Point Mutations in the Drug-binding Site and Altered Growth

Malaria is a preventable and treatable disease; yet half of the world''s population lives at risk of infection, and an estimated 660,000 people die of malaria-related causes every year. Rising drug resistance threatens to make malaria untreatable, necessitating both the discovery of new antimalarial agents and the development of strategies to identify and suppress the emergence and spread of drug resistance. We focused on in-development dihydroorotate dehydrogenase (DHODH) inhibitors. Characterizing resistance pathways for antimalarial agents not yet in clinical use will increase our understanding of the potential for resistance. We identified resistance mechanisms of Plasmodium falciparum (Pf) DHODH inhibitors via in vitro resistance selections. We found 11 point mutations in the PfDHODH target. Target gene amplification and unknown mechanisms also contributed to resistance, albeit to a lesser extent. These mutant parasites were often hypersensitive to other PfDHODH inhibitors, which immediately suggested a novel combination therapy approach to preventing resistance. Indeed, a combination of wild-type and mutant-type selective inhibitors led to resistance far less often than either drug alone. The effects of point mutations in PfDHODH were corroborated with purified recombinant wild-type and mutant-type PfDHODH proteins, which showed the same trends in drug response as the cognate cell lines. Comparative growth assays demonstrated that two mutant parasites grew less robustly than their wild-type parent, and the purified protein of those mutants showed a decrease in catalytic efficiency, thereby suggesting a reason for the diminished growth rate. Co-crystallography of PfDHODH with three inhibitors suggested that hydrophobic interactions are important for drug binding and selectivity.     

Identification and Characterization of Hundreds of Potent and Selective Inhibitors of Trypanosoma brucei Growth from a Kinase-Targeted Library Screening Campaign

In the interest of identification of new kinase-targeting chemotypes for target and pathway analysis and drug discovery in Trypanosomal brucei, a high-throughput screen of 42,444 focused inhibitors from the GlaxoSmithKline screening collection was performed against parasite cell cultures and counter-screened against human hepatocarcinoma (HepG2) cells. In this way, we have identified 797 sub-micromolar inhibitors of T. brucei growth that are at least 100-fold selective over HepG2 cells. Importantly, 242 of these hit compounds acted rapidly in inhibiting cellular growth, 137 showed rapid cidality. A variety of in silico and in vitro physicochemical and drug metabolism properties were assessed, and human kinase selectivity data were obtained, and, based on these data, we prioritized three compounds for pharmacokinetic assessment and demonstrated parasitological cure of a murine bloodstream infection of T. brucei rhodesiense with one of these compounds (NEU-1053). This work represents a successful implementation of a unique industrial-academic collaboration model aimed at identification of high quality inhibitors that will provide the parasitology community with chemical matter that can be utilized to develop kinase-targeting tool compounds. Furthermore these results are expected to provide rich starting points for discovery of kinase-targeting tool compounds for T. brucei, and new HAT therapeutics discovery programs.     

Developing kinase inhibitors for malaria: an opportunity or liability?

Highly druggable and essential to almost all aspects of cellular life, the protein and phosphoinositide kinase gene families offer a wealth of potential targets for pharmacological modulation for both noncommunicable and infectious diseases. Despite the success of kinase inhibitors in oncology and other disease indications, targeting kinases comes with significant challenges. Key hurdles for kinase drug discovery include selectivity and acquired resistance. The phosphatidylinositol 4-kinase beta inhibitor MMV390048 showed good efficacy in Phase 2a clinical trials, demonstrating the potential of kinase inhibitors for malaria treatment. Here we argue that the potential benefits of Plasmodium kinase inhibitors outweigh the risks, and we highlight the opportunity for designed polypharmacology to reduce the risk of resistance.