UC Santa Cruz

Drug discovery efforts have favored small molecules that can be described by Lipinski's "Rule of 5" (Ro5). Compounds that fit into this Ro5 are under 500 in their molecular weight (MW), have an octanol-water partition coefficient of less than five, have fewer than five hydrogen bond donors (HBDs), and fewer than ten hydrogen bond acceptors (HBAs). While some have published variations on the Ro5 since its description by Lipinski in 1997 and though the Ro5 was simply a summary of orally active drugs at the time, the Ro5 has often been used as design parameters in drug discovery. Drugs that conform to the Ro5 are typically likely to be orally bioavailable and have favorable ADME properties (absorption, distribution, metabolism, and excretion). However, confining drug design to fit into this set of criteria restricts drug targets to proteins with well-defined active sites and leaves a large portion of the proteome "undruggable." To expand the druggable proteome, there has been a recent surge in antibody-based drugs. These drugs are larger and able to target proteins without a well-defined active site. However, these expensive molecules are not orally bioavailable, and they are so large that they are only capable of targeting extracellular receptors. In this drug paradigm, intracellular protein disease targets either lacking small molecule binding sites or participating in protein-protein interaction are left without treatment.Further and critical expansion of the druggable proteome can be achieved with both natural product-inspired cyclic peptides and degradation-triggering proteolysis targeting chimeras (PROTACs). The intermediate size of these types of compounds lends the ability to affect intracellular disease targets while maintaining cellular permeability and oral bioavailability. Both molecule types can also bind to their protein targets at sites other than a deep pocket, overcoming this limitation common to typical small molecule drugs. The overall goal of my dissertation is to investigate the biological activity and physicochemical properties of these two "beyond Rule of 5" (bRo5) therapeutics capable of targeting previously "undruggable" protein disease targets.

Chapter one investigates the bioactivity and permeability of the cyclic peptide natural product cordyheptapeptide A and several synthetic derivatives. These natural product-inspired derivatives reveal that it is crucial to consider both bioactivity and permeability when optimizing a natural product. Additionally, using a combination of high-content screening and biochemical assay, I identify the intracellular target of cordyheptapeptide A as the eukaryotic translation elongation factor eEF1α. This is a critical disease target that is upregulated in many cancers and has yet to be successfully inhibited by a small molecule that adheres to the Ro5. Importantly, this work highlights that cyclic peptides dominated by aromatic and lipophilic sidechains, like cordyheptapeptide A, have the capacity to inhibit intracellular drug targets with sub-micromolar potencies.

In chapters two and three, I explore the physicochemical properties of PROTACs. These heterobifunctional molecules catalytically trigger the degradation of a protein target. PROTACs are typically more specific, more potent, and produce fewer off-target effects than typical Ro5 small molecule drugs. Our knowledge of PROTAC bioactivity is rapidly growing, but there is an urgent need to better understand these compounds' physicochemical properties. Chapter two uses two label-free mass spectrometry related assays to determine the passive permeability of several previously published PROTACs and examine how different structural features contribute to this permeability. I also demonstrate that amide-to-ester substitutions increase PROTAC permeability, PROTACs can form intramolecular hydrogen bonds that could be important for their permeability, and that PROTAC bioactivity is affected by permeability. However, strong target binding and ternary complex formation can overcome permeability deficits.

Chapter three expands on the findings from chapter two and investigates permeability improvements gained from an amide to ester substitution in compounds with a wide range of calculated lipophilicities and several different linkers. I describe how esters increase the permeability of PROTACs over a broad range of linkers and lipophilicities, but not for compounds that already have high lipophilicities (>4.5) that are moving into the range of lacking aqueous solubility. We also discovered that while amide-containing PROTACs are the most stable in plasma, ester-containing compounds in which the ester is near a larger drug-like side chain see only minimal hydrolysis in plasma. Combined, chapters two and three offer design guidelines for developing permeable PROTACs, insights into how their structural features affect their permeability, and strategies to improve these compounds' permeabilities.

Overall, this dissertation emphasizes the need to 1) consider the relationship between bioactivity and permeability when optimizing new drug compounds and 2) expand our current drug discovery efforts to include bRo5 compounds to treat previously "undruggable" diseases.