My laboratory uses a wide variety of highly interdisciplinary approaches to study infectious diseases. These approaches include the use of genomic technologies and bioinformatics for the study of Plasmodium falciparum, RNA viruses, and diseases of unknown etiology in organisms ranging from insects to people. These research targets are served by a core interest in developing new technologies and therapeutics for biomedical applications.
The majority of the world's infectious disease burden, especially in children under 5, can be attributed to just a handful of pathogens. Among these, Plasmodium falciparum ranks as the most vicious and one of the most neglected afflictions known to humankind. Despite the failures of the past, there are renewed efforts working toward global eradication of this parasite. A major component of this effort will focus on treating diseased individuals. To assist in these efforts, there continues to be a need for new, cheap antimalarial therapeutics.
Drug discovery. In collaboration with Kip Guy (University of California, San Francisco and St. Jude Children's Research Hospital), we have engaged in an intense hit-to-lead development program, whereby compounds with antimalarial activity are further developed to evaluate their realistic potential for becoming a therapeutic. The majority of antimalarial compounds discovered in high-throughput screening assays against whole parasites have no known target. Therefore, identification and characterization of molecular targets are important components of our program.
A primary method for identifying a target is to implement large-scale culture systems in which parasites are kept under continuous drug pressure. Given a large starting population, rare genetic mutants that are resistant or less susceptible to the drug pressure will be selected and may be recovered. Individual clones can then be subjected to ultradeep sequencing to identify all genetic changes that occurred with drug pressure. By comparing individual clones, we can separate passenger mutations from driver mutations and identify the genetic determinants. Knowledge of the drug target or mechanism of resistance then allows rational optimization of the lead series of compounds.
We used this experimental logic to identify the target of SJ733, a dihydroisoquinoline with potent antimalarial activity. We identified the target as the protein product of the gene PfATP4, a sodium pump responsible for extruding sodium from the parasite. Collectively, our data and those of our collaborators strongly support a model whereby SJ733 blocks sodium extrusion. Animal testing has revealed rapid clearance of parasites by SJ733, faster than would be predicted by in vitro work alone, and this effect appears to be a result of structural changes in the infected red blood cell when treated with SJ733. We believe that a rapid-clearing drug has tremendous potential for radical cure of blood-stage malaria. SJ733 was awarded preclinical status by the MMV (Medicines for Malaria Venture) organization (funded by the Bill & Melinda Gates Foundation), and we expect human safety testing to commence at St. Jude in 2015–2016.
Plasmodium cell biology. For nearly 20 years since the discovery of the apicoplast, the function of this unique and essential plastid organelle in blood-stage Plasmodium parasites has eluded researchers. Recently, we showed that parasites that have lost their apicoplast can be "rescued" by the addition of isoprenoid precursor IPP (isopentenyl pyrophosphate), demonstrating that the sole function of the apicoplast in blood-stage parasites is the biosynthesis of isoprenoid precursors. This discovery resolves a long-standing mystery in Plasmodium biology and presents new opportunities for investigation into the apicoplast and its role in malaria pathogenesis. Given the importance of the apicoplast for drug development, this insight also provides the ability to identify essential and specific apicoplast targets for therapeutic intervention and opens new avenues for the study of this intriguing "weak spot" of the parasite.
Leveraging the ability of IPP to rescue parasites treated with drugs that target the apicoplast, my laboratory implemented a screen of available antimalarial compounds with no known target and discovered a single potent compound rescued in this manner. By selection and genome sequencing, we identified the target as PfIspD – an enzyme of the non-mevalonate pathway, which is the essential biosynthetic pathway for isoprenoids in this parasite. By exogenous expression, we demonstrated that the compound directly targets the enzyme and blocks its activity. To date this enzyme had never been successfully targeted in malaria prior to this work. We are now attempting to optimize the compound and understand its mode of inhibition to generate additional leads for downstream development.
Plasmodium genomics. Our laboratory seeks to discover and dissect fundamental regulatory mechanisms that govern Plasmodium development. Several years ago, we first described the asexual blood-cycle transcriptome at the 1-hour resolution. To augment this, we have recently used ribosome profiling to complete a similar study examining the translational dynamics of the parasite. We have discovered that most genes are translated in a manner tightly coupled to transcription, although we find special examples whereby translational regulatory mechanisms are clearly at work. To better understand translational dynamics, we have developed a P. falciparum in vitro translation system that will allow fine-grained dissection.
Virology and Viral Discovery
A second focus of our lab is the pursuit of viral agents associated with diseases of unknown etiology. We have recently pioneered the use of next-generation sequencing (NGS) in the context of critical care. NGS allows us to discover viruses that have little if any obvious similarity to known viruses and helps determine the underlying causes of a variety of unknown ailments in both humans and animals. By combining detailed sequencing reads with the cutting-edge software and computing power available to the lab, researchers can readily translate this information to a fast, actionable diagnosis. In one recent case, we diagnosed, within 48 hours, a critically ill 14-year-old boy, which led to appropriate treatment and his rapid recovery.
Beyond diagnosis of human disease, we have also investigated numerous nonhuman diseases to identify and characterize their etiological agents, including, among others, avian bornavirus in psittacines, novel arenaviruses in boas and pythons, and a novel nidovirus in reptiles. In addition to discovery efforts, my laboratory is also engaged in further characterization of specific RNA viruses, especially picornaviruses. For example, we have developed deep-sequencing and proteomic approaches to uncover molecular determinants of picornavirus replication and recombination.
The Bill & Melinda Gates Foundation, the National Institutes of Health, and the David and Lucile Packard Foundation have provided partial support for these projects.
As of April 29, 2016