A review of >70 recent case studies of hit-to-clinical candidate programs will be summarized.  Among the topics discussed, we will present the physical chemical properties as well as the methods most widely used to find hits that result in clinical compounds.  Finally, we will focus attention on those case studies which are considered bRo5, and discuss the underlying features that led to the success of those compounds.

Beyond Rule-of-5 (bRo5) compounds are increasingly used in drug discovery. I present our analysis of 37 target proteins that have bRo5 drugs or clinical candidates. We found three general types of targets that can benefit from bRo5 drugs and I will describe each category.

PROTACs residing far beyond oral druggable space (MW 1000–1500 Da) are able-enter cells and induce target degradation. NMR spectroscopy and MD simulations suggest that cell permeability correlates to the ability of PROTACs to adopt folded conformations in which their polar surface area and size are reduced in an apolar environment. In addition, MD simulations and other computational techniques show promise for the prediction of PROTAC cell permeability.

NCATS through the ASPIRE (A Specialized Platform for Innovative Research Exploration) program seeks to transform the design-synthesize-test cycle through the development of new algorithms and workflows to capture data from automated chemical synthesis and biological testing systems to predict and inform the next iteration of new chemical entities. ASPIRE will enhance the ability to discover and develop new chemistries towards previously undrugged biological targets (i.e. those with no known drugs to modulate their function) across many human diseases and conditions.

Cystine-knot peptides (CKPs). a new modality for drug discovery, have a knot fold and a characteristic three-disulfide bond pattern. CKPs have great potential to target intracellular protein-protein interactions due to their relatively larger 3D framework compared to traditional 4-15 amino acid macrocyclic peptides. However, cell entry of CKPs remains elusive. Most attempts rely on cell penetrating peptides. I describe lipidation of cystine-knot peptides, an alternative to improving cell permeability.

We developed a new approach for synthesizing and screening ten-thousands of macrocyclic compounds. In brief, chemical building blocks are combinatoriallly reacted in 1536-well plates using acoustic liquid transfer. The macrocycles are produced at picomole scale and without purification, allowing a large throughput. Over the course of the last year, we were able to identify nanomolar binders to several targets, that we can now show as examples.

We evaluated how well models designed for judging the intestinal absorption of compounds, perfomed on predicting the absorption of bRo5 compounds. We evaluated a large, structurally diverse dataset of ∼1000 compounds with In silico (quantitative structure–activity relationship), in vitro (Caco-2), and in vivo (rat) models to statistically evaluate the models’ predictive performance against this human intestinal absorption dataset.

High resolution LCMS with CID MS/MS fragmentation is commonly used to elucidate the structure of small molecules and their metabolites. Here we present the limitations of CID fragmentation and the complementarity of Electron Activated Dissociation for structural elucidation. Additionally, increases in sensitivity via hybrid ion trapping coupled to high resolution TOF MS/MS greatly increases the sensitivity of these techniques for qualitative and quantitative workflows commonly seen in DMPK. 

We are using a range of experimental and theoretical methods to establish factors that limit the permeation of bRo5 peptides through membranes. Using long-timescale replica-exchange molecular dynamics simulations, we have investigated the importance of peptide conformation for permeation and, in a second study, we have developed an empirical machine-learning model for macrocycle permeation.

While Amgen’s Lumakras has beaten other RAS inhibitors to the market, it faces challenges. This class will likely be compromised by escape mutations and the undruggable nature of RAS.  Targeted K-RAS degradation can overcome this limitation, but PROTACs employing cereblon, VHL, or similar ligases will be less effective. We identified ligands for a novel membrane ubiquitin ligase, which are highly effective in degrading the membrane-associated K-RAS target. Progress in this novel K-RAS PROTAC will be presented.

PROteolysis-TArgeting Chimeras (PROTACs) have emerged as an innovative drug development platform. However, their use has been limited by the availability of few E3 ligase ligands to recruit different E3 ligases to mediate the degradation of targeted proteins. Here we report the discovery of a nature compound that functions as a new covalent Keap1 E3 ligase ligand. Using this natural compound, we have generated a number of PROTACs that target CDK9 and ALK to Keap1 for degradation. We have found that these PROTACs can potently degrade CDK9 and ALK in tumor cells, resulting in induction of apoptosis of these cells.

Three features separate targeted protein degradation from previous drug discovery modalities; its catalytic mechanism to achieve higher efficacy, its ability to target previously undruggable proteins such as non-enzyme targets, and its potential to deliver drug activity to selective tissues. All three features depend on the E3 ligands that bind to a specific E3 ubiquitin ligase and bring target protein into the close proximity through either the mode of molecular glue or bifunctional degrader. Human cells express estimated more than 600 E3 ubiquitin ligases, yet nearly all current advanced degraders use the same E3 ligand binding to cereblon. I will discuss our rationale and efforts in discovering novel E3 ligands for targeted protein degradation.

Two types of molecular glue will be discussed, one is inducing the formation of a complex with a higher binding affinity (stabilizer), another one is inducing protein-protein interaction for degradation and inhibiting the function of targeted protein (degrading agent).