In the Medicines for Malaria Venture MMV

In 2011 the Medicines for Malaria Venture (MMV) distributed the Open-Access Malaria Box to accelerate antimalarial drug discovery (Spangenberg et al., 2013). The Malaria Box consists of 400 structurally diverse compounds, curated from >20,000 hits generated from large-scale screens, that inhibit the growth of blood-stage Plasmodium falciparum parasites (Gamo et al., 2010, Guiguemde et al., 2010, Rottmann et al., 2010). A major goal of sharing these compounds was to facilitate elucidation of their antimalarial mechanism of action and open new classes of validated chemical scaffolds and drug targets. Compounds that disrupt isoprenoid metabolism can be detected by “rescue” of their growth inhibition upon supplementation of isoprenoids in the growth media (Yeh and Derisi, 2011). Previously, we and two other groups screened the Malaria Box for compounds whose growth inhibition was rescued by addition of IPP, and identified MMV008138 (Bowman et al., 2014, Wu et al., 2015). We and our collaborators demonstrated that MMV008138 inhibits IspD, an enzyme in the MEP pathway that produces IPP (Wu et al., 2015). Using a quantitative high-throughput screen (qHTS), we report a second compound in the Malaria Box, MMV019313, that shows an IPP rescue phenotype not identified in screens performed by other groups (Bowman et al., 2014, DeRisi, 2014, Van Voorhis et al., 2016). We demonstrate that the target of MMV019313 is the P. falciparum FPPS/GGPPS, a cytosolic isoprenoid synthase that utilizes IPP and the key branchpoint enzyme in isoprenoid biosynthesis in parasites. MMV019313 represents the first new class of specific non-bisphosphonate inhibitors of PfFPPS/GGPPS.
Results
Discussion FPPS and GGPPS are key branchpoint enzymes in isoprenoid biosynthesis. Human Pamidronate Disodium mg contain separate FPPS and GGPPS enzymes. An important class of clinical drugs, nitrogen-containing bisphosphonates, inhibits human FPPS in osteoclasts and blocks their function and proliferation (Kavanagh et al., 2006). Because osteoclasts are responsible for bone resorption, bisphosphonates are highly effective for treatment of osteoporosis and other bone-remodeling diseases. Bisphosphonates are chemically stable analogs of inorganic pyrophosphate containing a P-C-P bond in place of the phosphodiester, which accounts for both its inhibition of FPPS (acting as an analog of the allylic diphosphate substrate) and its high selectivity for osteoclasts (depositing in bone mineral, which is composed of calcium and phosphate). Unfortunately the charge state of bisphosphonates is a major liability in other therapeutic applications, as they are poorly bioavailable, rapidly cleared by the kidney, and do not achieve therapeutic levels in serum for the treatment of non-bone diseases (Cremers et al., 2005, Sinigaglia et al., 2007). Due to these limitations, there have been efforts to develop modified bisphosphonates or non-bisphosphonate FPPS inhibitors with improved pharmacokinetic properties for soft-tissue cancer and infectious disease applications (Chen et al., 2013, Jahnke et al., 2010, Liu et al., 2014, Marzinzik et al., 2015, Zhang et al., 2009). As an antimicrobial, there is the additional concern that broad inhibition of human FPPS could result in toxicity (Kotz, 2010). PfFPPS/GGPPS, the molecular target of MMV019313 as demonstrated in this study, closely resembles mammalian FPPS enzymes in sequence, structure, and inhibition by bisphosphonates (Artz et al., 2011, Jordão et al., 2013, No et al., 2012). Like human FPPS, it is a central node in cellular isoprenoid biosynthesis vulnerable to drug inhibition (Artz et al., 2011, Kavanagh et al., 2006, Luckman et al., 1998). In Plasmodium, FPP and GGPP are required for the biosynthesis of prenylated proteins, the prenyl modification of ubiquinone, and other isoprenoid products, such that inhibition of PfFPPS/GGPPS disrupts multiple cellular pathways (Chakrabarti et al., 2002, de Macedo et al., 2002, Gabriel et al., 2015, Gisselberg et al., 2017, Suazo et al., 2016, Tonhosolo et al., 2005, Tonhosolo et al., 2009). Previously lipophilic bisphosphonates modified with an alkyl chain to increase their cell permeability were shown to inhibit PvFPPS/GGPPS and clear both blood- and liver-stage Plasmodium parasites in mouse infection models (No et al., 2012, Singh et al., 2010). Inhibition of isoprenoid biosynthesis likely does not block gametocyte development (Lell et al., 2003, Van Voorhis et al., 2016). Importantly, these results validated Plasmodium FPPS/GGPPS as an antiparasitic drug target for both acute malaria treatment and malaria chemoprophylaxis.