It is difficult to find a more devastating illness packaged into a single word than “cancer”. Cancer is in fact a large collection of illnesses that share similar behaviors and outcomes. Cancers arise from lesions in cell-cycle regulation that ultimately allow aberrant cell proliferation which may result in tumor growth, angiogenesis, metastasis, and a destructive takeover of surrounding tissues which may inevitably lead to the death of the organism. The challenge to overcoming cancer, and why a “cure” for cancer is a misnomer, is that at the root of this family of diseases is an evolutionarily advantageous mechanism: cell survival and proliferation. Without these traits we ourselves would not survive. With these traits comes the risk of cancer. There is no cure for cancer because we cannot dissolve the very thing that allows us to live as the complex multi-cellular organisms that we are. We can however harness a better understanding of the pathways that regulate cell proliferation and cancer, and pursue therapeutic strategies that are intelligently created with this knowledge. While I touched on both of these areas throughout my years at UCSC, this piece focuses on my contributions toward understand the mechanism of a critical cell-cycle repressor. Chapter 2 describes my research toward understanding the importance of chromatin-modifying complexes in the transcriptional regulation of the cell-cycle.

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.

The ability of a single cell to grow, replicate its genetic material, and divide into two identical daughter cells is a vital process to ensure the propagation of all life. This process is known as the cell division cycle (cell cycle) and is one of the most highly spatially and temporally regulated cellular processes. Misregulation of the cell cycle, particularly in ways that confer both a proliferative advantage and escape from ultimate growth control mechanisms like cellular senescence or apoptosis, can result in unrestrained cell division and tumor formation. In this study, I utilize structural biology, biochemistry, and biophysical data to demonstrate a novel mechanism regulating Rb phosphorylation state and to map pairwise interactions in a conserved cell-cycle regulatory complex called the MuvB Core. First, competition between CDK and PP1 for Rb access provides an efficient regulatory mechanism to dephosphorylate Rb during mitotic exit and times of genotoxic stress. Second, data is presented mapping domain contacts in the MuvB Core, and structure/function studies with MuvB Core suggest an atypical function in regulation of chromatin structure.

The study of the cell cycle, specifically, understanding mechanistic regulation is fundamental for the development of therapeutic treatment in the event of disease. Limitless cell proliferation is a hallmark of cancer and often tied to cell cycle control. My research studied Cyclin- dependent kinases (Cdks) and their role in cell proliferation at the G1-S phase restriction point. I was able to shed insight on cyclin binding kinetics using Biolayer Interferometry (BLI) and the influence different types of inhibitors have on association of Cdks and cyclins. I reveal that Type I inhibitors increase binding affinity of CycA for Cdk2, whereas Type II inhibitors decrease affinity but differ in how they alter the CycA kinetic profile. Moreover, I explore an allosteric binding site of Cdk4-CycD that may be responsible for structural rearrangement to an active kinase configuration. My research as a whole gives insight into the development of a Cdk2 specific inhibitor and highlight the use of allosteric sites to understand mechanistic control of the cell cycle for Cdk inhibitor treatment therapy.

The cell cycle is essential for the growth and development of all multi-cellular organisms and is temporally regulated to coordinate processes such as cell size growth, organelle expansion, DNA replication, expression of mitotic proteins, chromatin condensation, and mitosis. Cell cycle proliferation is tightly regulated by a number of mechanisms including the transcription of G1/S and G2/M genes. In mammals, DREAM and Rb-E2F complexes are considered master regulators of the cell cycle and differentiation yet the molecular mechanisms underlying how these complexes activate and repress genes are still emerging. DREAM and Rb-E2F complexes are promoter dominant transcription factors that bind to target genes near their transcription start sites and modulate their expression. Evidence suggests that these transcription factors interact with chromatin and chromatin-modifying enzymes to alter the chromatin state in promoters. This work explores how these transcription factors engage with chromatin to modulate gene expression.

The regulation of cell division is critical for ensuring the outcome of two viable daughter cells. Improper regulation of the cell cycle can lead to aberrant proliferation and tumor formation. The human pocket proteins retinoblastoma (Rb), p107, and p130 are critical regulators of the cell cycle and contribute to tumor suppression. Their growth suppressive function is achieved through the regulation of E2F transcription factors, but within the pocket protein family, there is specificity for the particular E2F transcription factors each negatively regulates. The molecular mechanisms governing this specificity remain unknown, as does how E2F specificity contributes to the different functions observed among pocket proteins. In this study, I explore the molecular determinants governing the interactions between pocket proteins and E2Fs, how this E2F specificity may influence the function of pocket proteins, as well as the evolution of tumor suppression characteristics in the protein Rb. Using structural biology, biochemistry and biophysical techniques, we identified key regions of Rb that evolved to regulate a specific subset of E2Fs, giving rise to the unique functional activities associated with Rb.

Intrinsically disordered transcription factor transactivation domains (TADs) function through structural plasticity, adopting ordered conformations when bound to transcriptional co-regulators. Many transcription factors contain a negative regulatory domain (NRD) that suppresses recruitment of transcriptional machinery through autoregulation of the TAD. We report the solution structure of an autoinhibited NRD-TAD complex within FoxM1, a critical activator of mitotic gene expression. We observe that while both the FoxM1 NRD and TAD are primarily intrinsically disordered domains, they associate and adopt a structured conformation. We identify how Plk1 and Cdk kinases cooperate to phosphorylate FoxM1, which releases the TAD into a disordered conformation that then associates with the TAZ2 or KIX domains of the transcriptional co-activator CBP. Our results support a mechanism of FoxM1 regulation in which the TAD undergoes switching between disordered and different ordered structures.