Insights into the Development of Chemotherapeutics Targeting PFKFB Enzymes

The PFKFB enzymes control the primary checkpoint in the glycolytic pathway and are implicated in a multitude of diseases: from cancer, to schizophrenia, to diabetes, and heart disease. The inducible isoform, PFKFB3, is known to be associated with the upregulation of glycolysis in many cancers. The first study within this work investigates the potential for using tier-based approaches of virtual screening to target small molecule kinases, with PFKFB3 serving as a case study. For this investigation, bioactive compounds for PFKFB3 were identified from a compound library of 1364 compounds via high-throughput screening, with bioactive compounds being further characterized as either competitive or non-competitive for F6P. Using the F6P-competitive compounds, several structure based docking programs were assessed individually and in conjunction with a pharmacophore screening. The results showed that the tiered virtual screening approach, using pharmacophore screening in addition to structure-based docking, improved enrichments rates in 80% of cases, reduced CPU costs up to 7-fold, and lessened variability among different structure-based docking methods. The second study investigates the structural and kinetic characteristics of citrate inhibition on the heart PFKFB isoenzyme, PFKFB2. High levels of citrate, an intermediate of the TCA cycle, signify an abundance of biosynthetic precursors and that additional glucose need not be degraded for this purpose. Previous studies have noted that citrate acts as an important negative feed-back mechanism to limit glycolytic activity by inhibiting PFKFB enzymes, yet the structural and mechanistic details of citrate’s inhibition had not been determined. To study the molecular basis for citrate inhibition, the three-dimensional structures of the human and bovine PFKFB2 orthologues were solved, each in complex with citrate. For both cases, citrate primarily occupied the binding site of Fructose-6-phosphate (F6P), competitively blocking F6P from binding. Additionally, a carboxy arm of citrate extended into the γ-phosphate binding site of ATP, sterically and electrostatically blocking the catalytic binding mode for ATP. In the human orthologue, which utilized AMPPNP as an ATP analogue, conformational changes were observed in the 2-kinase domain as well as the binding mode for AMPPNP. This study gives new insights as to how the citrate-mediate negative feedback loop influences glycolytic flux through PFKFB enzymes

Binding Affinity and Specificity of SH2 Domain Interactions in Receptor Tyrosine Kinase Signaling Networks

Receptor tyrosine kinase (RTK) signaling mechanisms play a central role in intracellular signaling and control development of multicellular organisms, cell growth, cell migration, and programmed cell death. Dysregulation of these signaling mechanisms results in defects of development and diseases such as cancer. Control of this network relies on the specificity and selectivity of Src Homology 2 (SH2) domain interactions with phosphorylated target peptides. In this work, we review and identify the limitations of current quantitative understanding of SH2 domain interactions, and identify severe limitations in accuracy and availability of SH2 domain interaction data. We propose a framework to address some of these limitations and present new results which improve the quality and accuracy of currently available data. Furthermore, we supplement published results with a large body of negative interactions of high-confidence extracted from rejected data, allowing for improved modeling and prediction of SH2 interactions. We present and analyze new experimental results for the dynamic response of downstream signaling proteins in response to RTK signaling. Our data identify differences in downstream response depending on the character and dose of the receptor stimulus, which has implications for previous studies using high-dose stimulation. We review some of the methods used in this work, focusing on pitfalls of clustering biological data, and address the high-dimensional nature of biological data from high-throughput experiments, the failure to consider more than one clustering method for a given problem, and the difficulty in determining whether clustering has produced meaningful results