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Reaching for the High Hanging Fruit: Working Towards Better Drug Discovery

  • Author(s): Pye, Cameron
  • Advisor(s): Rubin, Seth
  • et al.
Abstract

The following work encompasses three distinct avenues of modern drug discovery: high throughput screening against an attractive cancer target, pharmacokinetic investigations towards better design rules for therapeutics, and a survey of natural products as a continued source for new chemical entities.

The retinoblastoma (Rb) tumor suppressor protein negatively regulates cell proliferation by binding and inhibiting E2F transcription factors. Rb inactivation occurs in cancer cells upon Cyclin-dependent kinase (Cdk) phosphorylation, which induces E2F release and activation of cell cycle genes. We present a strategy for activating phosphorylated Rb with molecules that bind Rb directly and enhance affinity for E2F. We developed a fluorescence polarization assay that can detect the effect of exogenous compounds on modulating affinity of Rb for the E2F transactivation domain. We found that a peptide capable of disrupting the compact inactive Rb conformation increases affinity of the repressive Rb-E2F complex. Our results demonstrate the feasibility of discovering novel molecules that target the cell cycle and proliferation through directly targeting Rb rather than upstream kinase activity.

Macrocyclic peptides are considered large enough to inhibit “undruggable” targets, but the design of passively cell-permeable molecules in this space remains a challenge due to the poorly understood role of molecular size on passive membrane permeability. Using split-pool combinatorial synthesis, we constructed a library of cyclic, per-N-methlyated peptides spanning a wide range of calculated lipohilicities (0 < AlogP < 8) and molecular weights (~800Da < MW < ~1200Da). Analysis by the Parallel Artificial Membrane Permeability Assay (PAMPA) revealed a steep drop-off in apparent passive permeability with increasing size, in stark disagreement with current permeation models. This observation, corroborated by a set of natural products, helps define criteria for achieving permeability in larger molecular size regimes and suggests an operational cut-off beyond which passive permeability is constrained by a sharply increasing penalty on membrane permeation.

Understanding of the capacity of the natural world to produce secondary metabolites is important to a broad range of fields, including drug discovery, ecology, biosynthesis and chemical biology among others. Both the absolute number and the rate of discovery of natural products have increased significantly in recent years. However, there is a perception and concern that the fundamental novelty of these discoveries is decreasing relative to previously known natural products. This study presents a quantitative examination of the field from the perspective of both number of compounds and compound novelty using a dataset of all published microbial and marine-derived natural products. This analysis aimed to explore a number of key questions, such as how the rate of discovery of new natural products has changed over the past decades, how the average natural product structural novelty has changed as a function of time, whether exploring novel taxonomic space affords an advantage in terms of novel compound discovery and if it is possible to estimate how close we are to having described all of the chemical space covered by natural products. Our analyses demonstrate that most natural products being published today bear structural similarity to previously published compounds, and that the range of scaffolds readily accessible from nature is limited. However, the analysis also shows that the field continues to discover appreciable numbers of natural products with no structural precedent. Together these results suggest that the development of innovative discovery methods will continue to yield compounds with unique structural and biological properties.

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