Fernando de Assis Batista

The appearance of multi‐drug resistant strains of malaria poses a major challenge to human health and validated drug targets are urgently required. To define a protein's function in vivo and thereby validate it as a drug target, highly specific tools are required that modify protein function with minimal cross‐reactivity. While modern genetic approaches often offer the desired level of target specificity, applying these techniques is frequently challenging—particularly in the most dangerous malaria parasite, Plasmodium falciparum. Our hypothesis is that such challenges can be addressed by incorporating mutant proteins within oligomeric protein complexes of the target organism in vivo. In this manuscript, we provide data to support our hypothesis by demonstrating that recombinant expression of mutant proteins within P. falciparum leverages the native protein oligomeric state to influence protein function in vivo, thereby providing a rapid validation of potential drug targets. Our data show that interference with aspartate metabolism in vivo leads to a significant hindrance in parasite survival and strongly suggest that enzymes integral to aspartate metabolism are promising targets for the discovery of novel antimalarials.

Malaria remains a major threat to human health, as strains resistant to current therapeutics are discovered. Efforts in finding new drug targets are hampered by the lack of sufficiently specific tools to provide target validation prior to initiating expensive drug discovery projects. Thus, new approaches that can rapidly enable drug target validation are of significant interest. In this manuscript we present the crystal structure of malate dehydrogenase from Plasmodium falciparum (PfMDH) at 2.4 Å resolution and structure-based mutagenic experiments interfering with the inter-oligomeric interactions of the enzyme. We report decreased thermal stability, significantly decreased specific activity and kinetic parameters of PfMDH mutants upon mutagenic disruption of either oligomeric interface. In contrast, stabilization of one of the interfaces resulted in increased thermal stability, increased substrate/cofactor affinity and hyperactivity of the enzyme towards malate production at sub-millimolar substrate concentrations. Furthermore, the presented data show that our designed PfMDH mutant could be used as specific inhibitor of the wild type PfMDH activity, as mutated PfMDH copies were shown to be able to self-incorporate into the native assembly upon introduction in vitro, yielding deactivated mutant:wild-type species. These data provide an insight into the role of oligomeric assembly in regulation of PfMDH activity and reveal that recombinant mutants could be used as probe tool for specific modification of the wild type PfMDH activity, thus offering the potential to validate its druggability in vivo without recourse to complex genetics or initial tool compounds. Such tool compounds often lack specificity between host or pathogen proteins (or are toxic in in vivo trials) and result in difficulties in assessing cause and effect—particularly in cases when the enzymes of interest possess close homologs within the human host. Furthermore, our oligomeric interference approach could be used in the future in order to assess druggability of other challenging human pathogen drug targets.

Keywords Calsenilin; diagnostic; follicular thyroid lesion; KChIP-3; thyroid nodules

Gastrointestinal stromal tumors (GISTs) are rare mesenchymal malignancies of the gastrointestinal tract. Most GISTs harbor a c-KIT (80%) or a PDGFRα (10%) mutation that leads to constitutive activation of the tyrosine kinase receptor. Response to treatment with tyrosine kinase inhibitors (TKIs) is dependent on mutational status of the tumor. The most common mutation in PDGFRα, D842V, is known to be imatinib resistant. Almost all other PDGFRα mutations are imatinib sensitive. We describe two patients with a PDGFRα exon 18 mutated GIST responding to treatment with TKIs. One of these patients has a p.M844_S847 deletion, not previously described in relation with TKI treatment response. Mutations in circulating tumor DNA were detectable with digital droplet PCR in serial plasma samples taken during treatment and correlated with treatment response of both patients. Computer 3D-modeling of the PDGFRα kinase domain of these two variants revealed no direct interference in imatinib or sunitinib binding and no effect in its activity in contrast to the reported structure of the imatinib resistant D842V mutation.

The validation of drug targets in malaria and other human diseases remains a highly challenging process. In the vast majority of cases, highly specific small molecule tools to inhibit a proteins function in vivo are simply not available. Additionally, the use of genetic tools in the analysis of malarial pathways is challenging. These issues result in difficulties in specifically modulating a hypothetical drug target’s function in vivo. Thus, new approaches to identify a protein’s function in vivo are required to provide target validation in the drug discovery process. Oligomerisation is the assembly of two or more copies of a single protein into one object and this self-assembly is present in approximately one third of all protein structures. Thus, oligomerisation plays a central role in the generation of functional biomolecules. A key feature of oligomerisation is that the oligomeric interfaces between the individual parts of the final assembly are highly specific. However, these interface regions of essential plasmodial proteins have not yet been systematically explored or exploited as a method to dissect biochemical pathways in vivo. This mini review will describe initial efforts to exploit oligomerisation surfaces in drug target validation.

Identification and Validation of Novel Drug Targets for the Treatment of Plasmodium falciparum Malaria: New Insights | InTechOpen, Published on: 2016-11-30. Authors: Sergey Lunev, Fernando A. Batista, Soraya S. Bosch, et