Discovery of GNE-502 as an orally bioavailable and potent degrader for estrogen receptor positive breast cancer.

Fulvestrant is an FDA-approved drug with a dual mechanism of action (MOA), acting as a full antagonist and degrader of the estrogen receptor protein. A significant limitation of fulvestrant is the dosing regimen required for efficacy. Due to its high lipophilicity and poor pharmacokinetic profile, fulvestrant needs to be administered through intramuscular injections which leads to injection site soreness. This route of administration also limits the dose and target occupancy in patients. We envisioned a best-in-class molecule that would function with the same dual MOA as fulvestrant, but with improved physicochemical properties and would be orally bioavailable. Herein we report our progress toward that goal, resulting in a new lead GNE-502 which addressed some of the liabilities of our previously reported lead molecule GNE-149.

GDC-9545 (Giredestrant): A Potent and Orally Bioavailable Selective Estrogen Receptor Antagonist and Degrader with an Exceptional Preclinical Profile for ER+ Breast Cancer.

Breast cancer remains a leading cause of cancer death in women, representing a significant unmet medical need. Here, we disclose our discovery efforts culminating in a clinical candidate, 35 (GDC-9545 or giredestrant). 35 is an efficient and potent selective estrogen receptor degrader (SERD) and a full antagonist, which translates into better antiproliferation activity than known SERDs (1, 6, 7, and 9) across multiple cell lines. Fine-tuning the physiochemical properties enabled once daily oral dosing of 35 in preclinical species and humans. 35 exhibits low drug-drug interaction liability and demonstrates excellent in vitro and in vivo safety profiles. At low doses, 35 induces tumor regressions either as a single agent or in combination with a CDK4/6 inhibitor in an ESR1Y537S mutant PDX or a wild-type ERα tumor model. Currently, 35 is being evaluated in Phase III clinical trials.

Antibody-mediated delivery of chimeric protein degraders which target estrogen receptor alpha (ERα).

Chimeric molecules which effect intracellular degradation of target proteins via E3 ligase-mediated ubiquitination (e.g., PROTACs) are currently of high interest in medicinal chemistry. However, these entities are relatively large compounds that often possess molecular characteristics which may compromise oral bioavailability, solubility, and/or in vivo pharmacokinetic properties. Accordingly, we explored whether conjugation of chimeric degraders to monoclonal antibodies using technologies originally developed for cytotoxic payloads might provide alternate delivery options for these novel agents. In this report we describe the construction of several degrader-antibody conjugates comprised of two distinct ERα-targeting degrader entities and three independent ADC linker modalities. We subsequently demonstrate the antigen-dependent delivery to MCF7-neo/HER2 cells of the degrader payloads that are incorporated into these conjugates. We also provide evidence for efficient intracellular degrader release from one of the employed linkers. In addition, preliminary data are described which suggest that reasonably favorable in vivo stability properties are associated with the linkers utilized to construct the degrader conjugates.

Discovery of a C-8 hydroxychromene as a potent degrader of estrogen receptor alpha with improved rat oral exposure over GDC-0927.

Phenolic groups are responsible for the high clearance and low oral bioavailability of the estrogen receptor alpha (ERα) clinical candidate GDC-0927. An exhaustive search for a backup molecule with improved pharmacokinetic (PK) properties identified several metabolically stable analogs, although in general at the expense of the desired potency and degradation efficiency. C-8 hydroxychromene 30 is the first example of a phenol-containing chromene that not only maintained excellent potency but also exhibited 10-fold higher oral exposure in rats. The improved in vivo clearance in rat was hypothesized to be the result of C-8 hydroxy group being sterically protected from glucuronide conjugation. The excellent potency underscores the possibility of replacing the presumed indispensable phenolic group at C-6 or C-7 of the chromene core. Co-crystal structures were obtained to highlight the change in key interactions and rationalize the retained potency.

Unexpected equivalent potency of a constrained chromene enantiomeric pair rationalized by co-crystal structures in complex with estrogen receptor alpha.

Despite tremendous progress made in the understanding of the ERα signaling pathway and the approval of many therapeutic agents, ER+ breast cancer continues to be a leading cause of cancer death in women. We set out to discover compounds with a dual mechanism of action in which they not only compete with estradiol for binding with ERα, but also can induce the degradation of the ERα protein itself. We were attracted to the constrained chromenes containing a tetracyclic benzopyranobenzoxepine scaffold, which were reported as potent selective estrogen receptor modulators (SERMs). Incorporation of a fluoromethyl azetidine side chain yielded highly potent and efficacious selective estrogen receptor degraders (SERDs), such as 16aa and surprisingly, also its enantiomeric pair 16ab. Co-crystal structures of the enantiomeric pair 16aa and 16ab in complex with ERα revealed default (mimics the A-D rings of endogenous ligand estradiol) and core-flipped binding modes, rationalizing the equivalent potency observed for these enantiomers in the ERα degradation and MCF-7 anti-proliferation assays.

From a novel HTS hit to potent, selective, and orally bioavailable KDM5 inhibitors.

A high-throughput screening (HTS) of the Genentech/Roche library identified a novel, uncharged scaffold as a KDM5A inhibitor. Lacking insight into the binding mode, initial attempts to improve inhibitor potency failed to improve potency, and synthesis of analogs was further hampered by the presence of a C-C bond between the pyrrolidine and pyridine. Replacing this with a C-N bond significantly simplified synthesis, yielding pyrazole analog 35, of which we obtained a co-crystal structure with KDM5A. Using structure-based design approach, we identified 50 with improved biochemical, cell potency and reduced MW and lower lipophilicity (LogD) compared with the original hit. Furthermore, 50 showed lower clearance than 9 in mice. In combination with its remarkably low plasma protein binding (PPB) in mice (40%), oral dosing of 50 at 5mg/kg resulted in unbound Cmax ∼2-fold of its cell potency (PC9 H3K4Me3 0.96µM), meeting our criteria for an in vivo tool compound from a new scaffold.

Metabolic plasticity underpins innate and acquired resistance to LDHA inhibition.

Metabolic reprogramming in tumors represents a potential therapeutic target. Herein we used shRNA depletion and a novel lactate dehydrogenase (LDHA) inhibitor, GNE-140, to probe the role of LDHA in tumor growth in vitro and in vivo. In MIA PaCa-2 human pancreatic cells, LDHA inhibition rapidly affected global metabolism, although cell death only occurred after 2 d of continuous LDHA inhibition. Pancreatic cell lines that utilize oxidative phosphorylation (OXPHOS) rather than glycolysis were inherently resistant to GNE-140, but could be resensitized to GNE-140 with the OXPHOS inhibitor phenformin. Acquired resistance to GNE-140 was driven by activation of the AMPK-mTOR-S6K signaling pathway, which led to increased OXPHOS, and inhibitors targeting this pathway could prevent resistance. Thus, combining an LDHA inhibitor with compounds targeting the mitochondrial or AMPK-S6K signaling axis may not only broaden the clinical utility of LDHA inhibitors beyond glycolytically dependent tumors but also reduce the emergence of resistance to LDHA inhibition.

Lead optimization of a pyrazolo[1,5-a]pyrimidin-7(4H)-one scaffold to identify potent, selective and orally bioavailable KDM5 inhibitors suitable for in vivo biological studies.

Starting with a lead [1,5-a]pyrimidin-7(4H)-one-containing molecule (1), we generated potent, selective and orally bioavailable KDM5 inhibitors. Using structure- and property-based approaches, we designed 48 with improved cell potency (PC9 H3K4Me3 EC50=0.34µM). Furthermore, 48 maintained suitable physiochemical properties and displayed an excellent pharmacokinetic (PK) profile in mice. When dosed orally in mice at 50mg/kg twice a day (BID), 48 showed an unbound maximal plasma concentration (Cmax) >15-fold over its cell EC50, thereby providing a robust chemical probe for studying KDM5 biological functions in vivo.

Fragment-Based Discovery of a Selective and Cell-Active Benzodiazepinone CBP/EP300 Bromodomain Inhibitor (CPI-637).

CBP and EP300 are highly homologous, bromodomain-containing transcription coactivators involved in numerous cellular pathways relevant to oncology. As part of our effort to explore the potential therapeutic implications of selectively targeting bromodomains, we set out to identify a CBP/EP300 bromodomain inhibitor that was potent both in vitro and in cellular target engagement assays and was selective over the other members of the bromodomain family. Reported here is a series of cell-potent and selective probes of the CBP/EP300 bromodomains, derived from the fragment screening hit 4-methyl-1,3,4,5-tetrahydro-2H-benzo[b][1,4]diazepin-2-one.

Infrared authentication of ginseng species: the use of the 2-6PC rule.

The quality of herbal products is important for ensuring efficacy and consumer safety. Traditional methods of authenticating herbs like ginseng via their morphology are hardly reliable. Different chemical constituents in herbs like ginseng tend to exhibit characteristic IR fingerprints that enable their identification. We previously introduced an IR-based protocol known as the "2-6PC rule" to categorize and identify ginseng and its products, as well as distinguishing it from morphological fakes. Here, we describe the use of this rule as a rapid and effective means of analyzing the IR spectral fingerprints of the biologically active components of ginseng, as well as distinguishing among its species. Our results show that Panax ginseng, P. quinquefolius, and P. notoginseng can be differentiated from each other. Our results also indicate the presence of starch, carbohydrates, calcium oxalate, and ginsenosides Re and Rg1 in commercial ginseng roots sold in Singapore. This work effectively demonstrates the usefulness of the 2-6PC rule as a rapid screening tool in the authentication of ginseng species.