Discovery of MK-1084: An Orally Bioavailable and Low-Dose KRASG12C Inhibitor.

Oncogenic mutations in the RAS gene account for 30% of all human tumors; more than 60% of which present as KRAS mutations at the hotspot codon 12. After decades of intense pursuit, a covalent inhibition strategy has enabled selective targeting of this previously "undruggable" target. Herein, we disclose our journey toward the discovery of MK-1084, an orally bioavailable and low-dose KRASG12C covalent inhibitor currently in phase I clinical trials (NCT05067283). We leveraged structure-based drug design to identify a macrocyclic core structure, and hypothesis-driven optimization of biopharmaceutical properties to further improve metabolic stability and tolerability.

Design-rules for stapled peptides with in vivo activity and their application to Mdm2/X antagonists.

Although stapled α-helical peptides can address challenging targets, their advancement is impeded by poor understandings for making them cell permeable while avoiding off-target toxicities. By synthesizing >350 molecules, we present workflows for identifying stapled peptides against Mdm2(X) with in vivo activity and no off-target effects. Key insights include a clear correlation between lipophilicity and permeability, removal of positive charge to avoid off-target toxicities, judicious anionic residue placement to enhance solubility/behavior, optimization of C-terminal length/helicity to enhance potency, and optimization of staple type/number to avoid polypharmacology. Workflow application gives peptides with >292x improved cell proliferation potencies and no off-target cell proliferation effects ( > 3800x on-target index). Application of these 'design rules' to a distinct Mdm2(X) peptide series improves ( > 150x) cellular potencies and removes off-target toxicities. The outlined workflow should facilitate therapeutic impacts, especially for those targets such as Mdm2(X) that have hydrophobic interfaces and are targetable with a helical motif.

Metabolism and Excretion of Therapeutic Peptides: Current Industry Practices, Perspectives, and Recommendations.

Therapeutic peptides (TPeps) have expanded from the initial endogenous peptides to complex modified peptides through medicinal chemistry efforts for almost a century. Different from small molecules and large proteins, the diverse submodalities of TPeps have distinct structures and carry different absorption, distribution, metabolism, and excretion (ADME) properties. There is no distinct regulatory guidance for the industry on conducting ADME studies (what, how, and when) for TPeps. Therefore, the Peptide ADME Working Group sponsored by the Translational and ADME Sciences Leadership Group of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) was formed with the goal to develop a white paper focusing on metabolism and excretion studies to support discovery and development of TPeps. In this paper, the key learnings from an IQ industry survey and U.S. Food and Drug Administration/European Medicines Agency submission documents of TPeps approved between 2011 and 2022 are outlined in detail. In addition, a comprehensive assessment of in vitro and in vivo metabolism and excretion studies, mitigation strategies for TPep metabolism, analytical tools to conduct studies, regulatory status, and Metabolites in Safety Testing considerations are provided. Finally, an industry recommendation on conducting metabolism and excretion studies is proposed for regulatory filing of TPeps. SIGNIFICANCE STATEMENT: This white paper presents current industry practices for metabolism and excretion studies of therapeutic peptides based on an industry survey, regulatory submission documents, and expert opinions from the participants in the Peptide Absorption, Distribution, Metabolism, and Excretion Working Group of the International Consortium for Innovation and Quality in Pharmaceutical Development. The group also provides recommendations on the Metabolites in Safety Testing considerations and metabolism and excretion studies for regulatory filing of therapeutic peptides.

Rapid label-free cell-based Approach Membrane Permeability Assay using MALDI-hydrogen-deuterium exchange mass spectrometry for peptides.

Peptide therapeutics are a growing modality in the pharmaceutical industry and expanding these therapeutics to hit intracellular targets would require establishing cell permeability. Rapid measurement target-agnostic cell permeability of peptides is still analytically challenging. In this study, we demonstrate the development of a rapid high-throughput label-free methodology based on a MALDI-hydrogen-deuterium exchange mass spectrometry (MALDI-HDX-MS) approach to rank-order peptide cell membrane permeability using live THP-1 and AsPc-1 cells. Peptides were incubated in the presence of live cells and their permeability into the cells over time was measured by MALDI-HDX-MS. A differential hydrogen-deuterium exchange approach was used to distinguish the peptides outside of the cells from those inside. The peptides on the outside of the cells were labeled using sufficiently short exposure to deuterium oxide, while the peptides inside of the cells were protected from labeling as a result of permeation into the cells. The deuterium labeled and peak area ratios of unlabeled peptides were compared and plotted over time. The developed methodology, referred to as Cell-based Approach Membrane Permeability Assay (CAMPA), was applied to study an array of 24 diverse peptides including cell-penetrating peptides, stapled and macrocyclic peptides. The cell membrane permeability results observed by CAMPA were corroborated by previously reported in literature data. The CAMPA MALDI-MS analysis was fully automated including MS data processing using internally developed Python scripts. Moreover, CAMPA was demonstrated to be useful for differentiating passive and active cell transportation by using an endocytosis inhibitor in cell incubation media for selected peptides.

Discovery and Structure-Based Design of Macrocyclic Peptides Targeting STUB1.

Recent evidence suggests that deletion of STUB1─a pivotal negative regulator of interferon-γ sensing─may potentially clear malignant cells. However, current studies rely primarily on genetic approaches, as pharmacological inhibitors of STUB1 are lacking. Identifying a tool compound will be a step toward validating the target in a broader therapeutic sense. Herein, screening more than a billion macrocyclic peptides resulted in STUB1 binders, which were further optimized by a structure-enabled in silico design. The strategy to replace the macrocyclic peptides' hydrophilic and solvent-exposed region with a hydrophobic scaffold improved cellular permeability while maintaining the binding conformation. Further substitution of the permeability-limiting terminal aspartic acid with a tetrazole bioisostere retained the binding to a certain extent while improving permeability, suggesting a path forward. Although not optimal for cellular study, the current lead provides a valuable template for further development into selective tool compounds for STUB1 to enable target validation.

Accelerated Identification of Cell Active KRAS Inhibitory Macrocyclic Peptides using Mixture Libraries and Automated Ligand Identification System (ALIS) Technology.

Macrocyclic peptides can disrupt previously intractable protein-protein interactions (PPIs) relevant to oncology targets such as KRAS. Early hits often lack cellular activity and require meticulous improvement of affinity, permeability, and metabolic stability to become viable leads. We have validated the use of the Automated Ligand Identification System (ALIS) to screen oncogenic KRASG12D (GDP) against mass-encoded mini-libraries of macrocyclic peptides and accelerate our structure-activity relationship (SAR) exploration. These mixture libraries were generated by premixing various unnatural amino acids without the need for the laborious purification of individual peptides. The affinity ranking of the peptide sequences provided SAR-rich data sets that led to the selection of novel potency-enhancing substitutions in our subsequent designs. Additional stability and permeability optimization resulted in the identification of peptide 7 that inhibited pERK activity in a pancreatic cancer cell line. More broadly, this methodology offers an efficient alternative to accelerate the fastidious hit-to-lead optimization of PPI peptide inhibitors.

NanoClick: A High Throughput, Target-Agnostic Peptide Cell Permeability Assay.

Macrocyclic peptides open new opportunities to target intracellular protein-protein interactions (PPIs) that are often considered nondruggable by traditional small molecules. However, engineering sufficient membrane permeability into these molecules is a central challenge for identifying clinical candidates. Currently, there is a lack of high-throughput assays to assess peptide permeability, which limits our capacity to engineer this property into macrocyclic peptides for advancement through drug discovery pipelines. Accordingly, we developed a high throughput and target-agnostic cell permeability assay that measures the relative cumulative cytosolic exposure of a peptide in a concentration-dependent manner. The assay was named NanoClick as it combines in-cell Click chemistry with an intracellular NanoBRET signal. We validated the approach using known cell penetrating peptides and further demonstrated a correlation to cellular activity using a p53/MDM2 model system. With minimal change to the peptide sequence, NanoClick enables the ability to measure uptake of molecules that enter the cell via different mechanisms such as endocytosis, membrane translocation, or passive permeability. Overall, the NanoClick assay can serve as a screening tool to uncover predictive design rules to guide structure-activity-permeability relationships in the optimization of functionally active molecules.

Discovery of cell active macrocyclic peptides with on-target inhibition of KRAS signaling.

Macrocyclic peptides have the potential to address intracellular protein-protein interactions (PPIs) of high value therapeutic targets that have proven largely intractable to small molecules. Here, we report broadly applicable lessons for applying this modality to intracellular targets and specifically for advancing chemical matter to address KRAS, a protein that represents the most common oncogene in human lung, colorectal and pancreatic cancers yet is one of the most challenging targets in human disease. Specifically, we focused on KRpep-2d, an arginine-rich KRAS-binding peptide with a disulfide-mediated macrocyclic linkage and a protease-sensitive backbone. These latter redox and proteolytic labilities obviated cellular activity. Extensive structure-activity relationship studies involving macrocyclic linker replacement, stereochemical inversion, and backbone α-methylation, gave a peptide with on-target cellular activity. However, we uncovered an important generic insight - the arginine-dependent cell entry mechanism limited its therapeutic potential. In particular, we observed a strong correlation between net positive charge and histamine release in an ex vivo assay, thus making this series unsuitable for advancement due to the potentially fatal consequences of mast cell degranulation. This observation should signal to researchers that cationic-mediated cell entry - an approach that has yet to succeed in the clinic despite a long history of attempts - carries significant therapy-limiting safety liabilities. Nonetheless, the cell-active molecules identified here validate a unique inhibitory epitope on KRAS and thus provide valuable molecular templates for the development of therapeutics that are desperately needed to address KRAS-driven cancers - some of the most treatment-resistant human malignancies.

Cytochrome b5 enhances androgen synthesis by rapidly reducing the CYP17A1 oxy-complex in the lyase step.

Cytochrome P450 17A1 (CYP17A1) catalyzes the synthesis of androgens from the steroid precursors pregnenolone and progesterone in a two-step reaction process: allylic hydroxylation and carbo-carbon bond scission. Cytochrome b5 (Cyt-b5 ) is a stimulator of the second lyase reaction, but the chemical mechanism is unclear. We have shown previously that this stimulatory effect requires redox active Cyt-b5 . To investigate the origin of the lyase reaction enhancement by electron transfer from Cyt-b5 , we measured the reduction rates of oxy-ferrous substrate-bound CYP17A1 by Cyt-b5 and by cytochrome P450 reductase (CPR) coincorporated in Nanodiscs using stopped flow spectroscopy. We observed that Cyt-b5 reduces oxy-ferrous CYP17A1 10-fold faster than CPR, with the rate similar to that observed in a ternary complex of all three proteins.

Human Cytochrome CYP17A1: The Structural Basis for Compromised Lyase Activity with 17-Hydroxyprogesterone.

The multifunctional enzyme, cytochrome P450 (CYP17A1), plays a crucial role in the production of androgens, catalyzing two key reactions on pregnenolone (PREG) and progesterone (PROG), the first being a 17-hydroxylation to generate 17-OH PREG and 17-OH PROG, with roughly equal efficiencies. The second is a C-C bond scission or "lyase" reaction in which the C17-C20 bond is cleaved, leading to the eventual production of powerful androgens, whose involvement in the proliferation of prostate cancer has generated intense interest in developing inhibitors of CYP17A1. For humans, the significance of the C-C bond cleavage of 17-OH PROG is lessened, because it is about 50 times less efficient than for 17-OH PREG in terms of kcat/Km. Recognizing the need to clarify relevant reaction mechanisms involved with such transformations, we first report studies of solvent isotope effects, results of which are consistent with a Compound I mediated PROG hydroxylase activity, yet exclude this intermediate as a participant in the formation of androstenedione (AD) via the lyase reaction. This finding is also supported by a combination of cryoreduction and resonance Raman spectroscopy that traps and structurally characterizes the key hemiketal reaction intermediates. Adding to a previous study of PREG and 17-OH PREG metabolism, the current work provides definitive evidence for a more facile protonation of the initially formed ferric peroxo-intermediate for 17-OH PROG-bound CYP17A1, compared to the complex with 17-OH PREG. Importantly, Raman characterization also reveals an H-bonding interaction with the terminal oxygen of the peroxo fragment, rather than with the proximal oxygen, as is present for 17-OH PREG. These factors would favor a diminished lyase activity of the sample with 17-OH PROG relative to the complex with 17-OH PREG, thereby providing a convincing structural explanation for the dramatic differences in activity for these lyase substrates in humans.

Evidence that cytochrome b5 acts as a redox donor in CYP17A1 mediated androgen synthesis.

Cytochrome P450 17A1 (CYP17A1) is an important drug target for castration resistant prostate cancer. It is a bi-functional enzyme, catalyzing production of glucocorticoid precursors by hydroxylation of pregnene-nucleus, and androgen biosynthesis by a second CC lyase step, at the expense of glucocorticoid production. Cytochrome b5 (cyt b5) is known to be a key regulator of the androgen synthesis reaction in vivo, by a mechanism that is not well understood. Two hypotheses have been proposed for the mechanism by which cyt b5 increases androgen biosynthesis. Cyt b5 could act as an allosteric effector, binding to CYP17A1 and either changing its selective substrate affinity or altering the conformation of the P450 to increase the catalytic rate or decrease unproductive uncoupling channels. Alternatively, cyt b5 could act as a redox donor for supply of the second electron in the P450 cycle, reducing the oxyferrous complex to form the reactive peroxo-intermediate. To understand the mechanism of lyase enhancement by cyt b5, we generated a redox-inactive form of cyt b5, in which the heme is replaced with a Manganese-protoporphyrin IX (Mn-b5), and investigated enhancement of androgen producing lyase reaction by CYP17A1. Given the critical significance of a stable membrane anchor for all of the proteins involved and the need for controlled stoichiometric ratios, we employed the Nanodisc system for this study. The redox inactive form was observed to have no effect on the lyase reaction, while reactions with the normal heme-iron containing cyt b5 were enhanced ∼5 fold as compared to reactions in the absence of cyt b5. We also performed resonance Raman measurements on ferric CYP17A1 bound to Mn-b5. Upon addition of Mn-b5 to Nanodisc reconstituted CYP17A1, we observed clear evidence for the formation of a b5-CYP17A1 complex, as noted by changes in the porphyrin modes and alteration in the proximal FeS vibrational frequency. Thus, although Mn-b5 binds to CYP17A1, it is unable to enhance the lyase reaction, strongly suggesting that cyt b5 has a redox effector role in enhancement of the CYP17A1 mediated lyase reaction necessary for androgen synthesis.

Kinetic solvent isotope effect in steady-state turnover by CYP19A1 suggests involvement of Compound 1 for both hydroxylation and aromatization steps.

CYP19A1, or human aromatase catalyzes the conversion of androgens to estrogens in a three-step reaction through the formation of 19-hydroxy and 19-aldehyde intermediates. While the first two steps of hydroxylation are thought to proceed through a high-valent iron-oxo species, controversy exists surrounding the identity of the reaction intermediate that catalyzes the lyase and aromatization reaction. We investigated the kinetic isotope effect on the steady-state turnover of Nanodisc-incorporated human CYP19A1 to explore the mechanisms of this reaction. Our experiments reveal a significant (∼ 2.5) kinetic solvent isotope effect for the C10-C19 lyase reaction, similar to that of the first two hydroxylation steps (2.7 and 1.2). These data implicate the involvement of Compound 1 as a reactive intermediate in the final aromatization step of CYP19A1.