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Nov 18, 2011

Data on Dual-Acting Antimalarials Made Public to Spur Development Efforts Worldwide

  • Researchers are making data on a new chemical class of dual-acting antimalarial compounds available to researchers worldwide to spur drug development. Discovered through an international collaboration, the imidazolopiperazine compounds are highly active against both liver and blood stages of Plasmodium parasites in mouse models of infection, providing potential for development as both therapeutic and prophylactic candidates.

    The discovery program was led by Scripps Research Institute researchers, working in collaboration with the Novartis Institutes for BioMedical Research through its Genomics Institute of the Novartis Research Foundation (GNF), and an international team of scientists. Their work suggested that the compounds act via a different mechanism of action to other antimalarial drug classes, and impact on a parasite gene of unknown function, which has been given the name P. falciparum Cyclic Amine Resistance Locus (pfcarl).

    "We have been making all of our data available to the community to spur drug development," comments Elizabeth Winzeler, Ph.D., GNF department head and lead investigator. "The data on all of the compounds that were tested will eventually be released, and this will allow people at universities and research institutes around the world to mine this data, and to use it to guide their own drug discovery efforts." The collaborators’ studies and initial results are, meanwhile, described in ScienceExpress, in a paper titled “Imaging of Plasmodium Liver Stages to Drive Next-Generation Antimalarial Drug Discovery.”

    Plasmodium sporozoites transferred to a human by a mosquito bite find their way to the host liver, where they multiply asexually as exoerythrocytic forms (EEFs) before emerging into the blood stream, the researchers explain. The sporozoites of many species of Plasmodium reside in the liver for a period of a week or so, although some species can persist as dormant hypnozoites within the liver for years. Once the parasites do emerge from the liver into the blood, they infect red blood cells, in which they replicate before destroy blood cells when they emerge to infect new erythrocytes. It is at this point that the characteristic symptoms of malaria start to develop.

    Most antimalarial drug development activity is focused on red blood cell-stage parasites, and the only drugs that have notable activity against proliferating EEFs and hypnozoites are the 8-aminoquinolines, some of these can cause dangerous side-effects and lead to resistance. However, as the researchers point out, if malaria is ever to be eradicated, a drug would ideally treat infection during both the liver and blood stages. 

    To try and identify drugs that might be active against liver stage parasites as well as blood stage parasites, the team modified an in vitro assay using Plasmodium yoelii sporozoites and HepG2-A16-CD81EGFP liver cells to screen a library of thousands of  commercially available compounds that had previously been found to demonstrate blood-stage activity. This compound library included 2,715 independent scaffolds.

    Results of treating the infected cells were evaluated in terms of parasite size, rather than infection ratio, because infection ratio may have been affected by drug toxicity to host cells and host cell division during incubation, the team points out. Parasite size measurements also enabled the identification of compounds that arrest parasite growth and development.

    Two rounds of screening identified 275 compounds that were active against liver stage parasites, including scaffolds that are chemically distinct from known antimalarial pharmacophores. The researchers homed in on the imidazolopiperazine (IP) scaffold, which comprises a class of saturated cyclic amines with a predicted high probability of activity against both liver and blood stage parasites.

    Indeed all three members of the original dataset (Pf-5069, Pf-5179, and Pf-5466) shared the same IP saturated cyclic amine chemical core, were active both in the yoelii liver assay, and also against P. falciparum blood stage cultures in vitro, including the multidrug-resistant strain, W2. “The IP scaffold is attractive as a starting point for medicinal chemistry because it is structurally unrelated to known antimalarial scaffolds and is chemically tractable,” the authors  point out. Notably, the IP scaffold compounds didn’t demonstrate cross-resistance with malarial strains that carried mutations rendering them resistant to drugs liver stage drugs, including atovaquone and pyrimethamine.

    Encouragingly, IP scaffold compounds that had been modified in terms of blood-stage potency also retained their effectiveness in the P. yoelii liver stage assay, causing the parasites to arrest at near minimal detectable size. Tested molecules in the series of optimized scaffolds in fact demonstrated liver stage activity equivalent their activity against P. falciparum blood stages.

    The team synthesized 1.200 analogs of the IP scaffolds and optimized their blood stage potency to generate molecules that could then be tested in animal models of malaria. One of these, an optimized 8,8-dimethyl IP analog (GNF179) was active against the W2 strain, demonstrated in vitro metabolic stability and in vivo oral bioavailability, and a suitable terminal half life.

    Initial in vivo studies suggested GNF179 reduced Plasmodium berghei parasitemia levels by 99.7% with a single 100 mg/kg oral dose, and prolonged mouse survival by an average of 19 days. In contrast, the average survival times for P. berghei–infected mice treated using a single, 100 mg/kg oral dose of chloroquine or artesunate, were 12.5 days and 7 days, respectively.

    The team then tested the prophylactic effects of GNF179 in a rodent model of P. berghei sporozoite infection. Prior to parasite challenge, the mice were treated using either a single dose of atovaquone, GNF179, or the Phase I-stage spiroindolone drug NITD609, which was also discovered by a Winzeler-led research team.

    While NITD609 was slightly more potent against blood stages than GNF179 and was fully curative at a 100 mg/kg oral dose in a blood-stage animal model, a single, 15 mg/kg oral dose of GNF179 protected against an infectious P. berghei sporozoite challenge, unlike NITD609. Examination of infected, GNF179-treated cells using microscopy showed the compound caused the parasites to arrest at even earlier stages than atovaquone and antifolate drugs. GNF179 was also effective at arresting parasite growth when administered later in EEF development, unlike antifolates, which must be administered very early after parasite invasion to be active.

    Interestingly, GNF179 appears to exhibit a different mechanism than other drugs that act early in hepatic stage development, the researchers continue. Unlike cyclohexamine, GNF179 didn’t rapidly inhibit parasite protein biosynthesis, nor is it likely to target cytochrome bc1, which is a validated target for atovaquone, the authors note. And unlike electron chain transport inhibitors, members of the IP scaffold didn’t cause a shift in IC50 when tested against transgneic parasites expressing S. cerevisiae dihydroorotate dehydrogenase.

    Sequencing the genomes of P. falciparum strains that had been exposed to increasing concentrations of IP series drugs until resistance emerged showed no evidence of cross-resistance for mefloquine or artemisinin. In fact, only one gene (PFC097w), which the team has called Pfcarl, was mutated in all the resulting resistant strains, and all the mutations seen in the resistant clones impacted on evolutionarily conserved amino acids. “Although more work is needed to characterize this gene, our data suggest that the IPs have a mechanism of action distinct from known antimalarials and that pfcarl could represent a conserved drug target or resistance gene,” they conclude. 


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