Harnessing evolutionary fitness in Plasmodium falciparum for drug discovery and suppressing resistance

Drug resistance emerges in an ecological context where fitness costs restrict the diversity of escape pathways. These pathways are targets for drug discovery, and here we demonstrate that we can identify small-molecule inhibitors that differentially target resistant parasites. Combining wild-type and mutant-type inhibitors may prevent the emergence of competitively viable resistance. We tested this hypothesis with a clinically derived chloroquine-resistant (CQr) malaria parasite and with parasites derived by in vitro selection with Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitors. We screened a chemical library against CQs and CQr lines and discovered a drug-like compound (IDI-3783) that was potent only in the CQr line. Surprisingly, in vitro selection of Plasmodium falciparum resistant to IDI-3783 restored CQ sensitivity, thereby indicating that CQ might once again be useful as a malaria therapy. In parallel experiments, we selected P. falciparum lines resistant to structurally unrelated PfDHODH inhibitors (Genz-666136 and DSM74). Both selections yielded resistant lines with the same point mutation in PfDHODH:E182D. We discovered a compound (IDI-6273) more potent against E182D than wild-type parasites. Selection of the E182D mutant with IDI-6273 yielded a reversion to the wild-type protein sequence and phenotype although the nucleotide sequence was different. Importantly, selection with a combination of Genz-669178, a wild-type PfDHODH inhibitor, and IDI-6273, a mutant-selective PfDHODH inhibitor, did not yield resistant parasites. These two examples demonstrate that the compromise between resistance and evolutionary fitness can be exploited to design therapies that prevent the emergence and spread of resistant organisms.Organismic and Evolutionary Biolog

Diversity-Oriented Synthesis Yields a Novel Lead for the Treatment of Malaria

Here, we describe the discovery of a novel antimalarial agent using phenotypic screening of Plasmodium falciparum asexual blood-stage parasites. Screening a novel compound collection created using diversity-oriented synthesis (DOS) led to the initial hit. Structure–activity relationships guided the synthesis of compounds having improved potency and water solubility, yielding a subnanomolar inhibitor of parasite asexual blood-stage growth. Optimized compound 27 has an excellent off-target activity profile in erythrocyte lysis and HepG2 assays and is stable in human plasma. This compound is available via the molecular libraries probe production centers network (MLPCN) and is designated ML238.Chemistry and Chemical Biolog

Diversity-Oriented Synthesis Probe Targets Plasmodium falciparum Cytochrome b Ubiquinone Reduction Site and Synergizes With Oxidation Site Inhibitors

Background. The emergence and spread of drug resistance to current antimalarial therapies remains a pressing concern, escalating the need for compounds that demonstrate novel modes of action. Diversity-Oriented Synthesis (DOS) libraries bridge the gap between conventional small molecule and natural product libraries, allowing the interrogation of more diverse chemical space in efforts to identify probes of novel parasite pathways. Methods. We screened and optimized a probe from a DOS library using whole-cell phenotypic assays. Resistance selection and whole-genome sequencing approaches were employed to identify the cellular target of the compounds. Results. We identified a novel macrocyclic inhibitor of Plasmodium falciparum with nanomolar potency and identified the reduction site of cytochrome b as its cellular target. Combination experiments with reduction and oxidation site inhibitors showed synergistic inhibition of the parasite. Conclusions. The cytochrome b oxidation center is a validated antimalarial target. We show that the reduction site of cytochrome b is also a druggable target. Our results demonstrating a synergistic relationship between oxidation and reduction site inhibitors suggests a future strategy for new combination therapies in the treatment of malaria

Divergent Kinetics Differentiate the Mechanism of Action of Two HDAC Inhibitors

Histone deacetylases (HDACs) play diverse roles in many diseases including cancer, sarcopenia, and Alzheimer’s. Different isoforms of HDACs appear to play disparate roles in the cell and are associated with specific diseases; as such, a substantial effort has been made to develop isoform-selective HDAC inhibitors. Our group focused on developing HDAC1/HDAC2-specific inhibitors as a cancer therapeutic. In the course of characterizing the mechanism of inhibition of a novel HDAC1/2-selective inhibitor, it was determined that it did not exhibit classical Michaelis–Menten kinetic behavior; this result is in contrast to the seminal HDAC inhibitor SAHA. Enzymatic assays, along with a newly developed binding assay, were used to determine the rates of binding and the affinities of both the HDAC1/2-selective inhibitor and SAHA. The mechanism of action studies identified a potential conformational change required for optimal binding by the selective inhibitor. A model of this putative conformational change is proposed

Diversity-Oriented Synthesis-Facilitated Medicinal Chemistry: Toward the Development of Novel Antimalarial Agents

Here, we describe medicinal chemistry that was accelerated by a diversity-oriented synthesis (DOS) pathway, and in vivo studies of our previously reported macrocyclic antimalarial agent that derived from the synthetic pathway. Structure–activity relationships that focused on both appendage and skeletal features yielded a nanomolar inhibitor of P. falciparum asexual blood-stage growth with improved solubility and microsomal stability and reduced hERG binding. The build/couple/pair (B/C/P) synthetic strategy, used in the preparation of the original screening library, facilitated medicinal chemistry optimization of the antimalarial lead