Novel multifunctional degraders of EP300/CBP and IKZF1/3 with potent anti-myeloma activity Free

Immunomodulatory imide drugs (IMiDs) are well-established therapies for patients with multiple myeloma (MM) (Hartley-Brown 2024). The primary mechanism of IMiD action occurs through binding to the E3 ligase cereblon and allosterically enabling the degradation of the key myeloma transcription factors IKZF1 (Ikaros) and IKZF3 (Aiolos) (Hartley-Brown 2024). As a scaffold for binding to cereblon, IMiDs serve as a common moiety in the design of targeted protein degraders.

E1A-associated protein p300 (EP300) and CREB-binding protein (CBP) are paralogs possessing histone acetyltransferase (HAT) activity and harbor bromodomains (BRDs) that serve to regulate the HAT activity. EP300 and CBP also function in large protein complexes that regulate the transcription of thousands of genes, including those critical for promoting growth and transformation. Inhibitors of the BRDs of EP300 and CBP are known to reduce the activity of key drivers of MM progression and maintenance, including IRF4 and MYC (Nicosia 2023). OPN-6602 is a potent oral small molecule inhibitor of the BRDs of EP300 and CBP that is currently in a Phase 1 clinical study to evaluate efficacy and safety in patients with relapsed/refractory MM (NCT06433947).

Preclinical MM studies that combine IMiDs with OPN-6602 demonstrate synergy with high xenograft tumor response, including complete regressions and improved response durability (Matusow 2024). Based on these foundational studies, next generation chimeras that combine an IMiD moiety with derivatives of OPN-6602 have been designed and interrogated in MM models. The hypothesis is that these multifunctional chimeras will cause degradation of all four key proteins, namely EP300, CBP, IKZF1 and IKZF3, and thus have the potential for being active as a single agent in hematological malignancies such as myeloma and lymphoma.

Over 200 chimeric compounds were synthesized and characterized through a funnel of functional assays. Individual cell lines were developed that expressed one of the four target proteins with an appended HiBiT tag, enabling high throughput profiling of target protein degradation. HiBiT-determined DC₅₀ values were validated with Western blotting to confirm reduced protein levels and viability assays in human MM cell lines were used to stratify anti-proliferative activities. qPCR was employed to quantify effects on gene expression, and binding of chimeras to protein targets was demonstrated using cell-free AlphaScreen.

From this screening funnel, a first-generation chimera, OPN-5667, was chosen for proof-of-concept study in the OPM-2 MM xenograft model. OPN-5667 exhibited a pan-degrader profile with preference for EP300 over CBP in vitro, showing HiBiT DC₅₀ for EP300, CBP, IKZF1 and IKZF3 of 15nM, 110nM, 20nM and 36nM, respectively. At nanomolar concentrations, OPN-5667 reduced target protein expression in Jurkat T cells and downregulated the expression of IRF4, MYC, and BCL2 genes in OPM-2 cells. These effects were consistent with an IC₅₀ of 50nM in suppressing OPM-2 cell proliferation. Following 2 weeks of daily intra-peritoneal dosing in the murine OPM-2 xenograft model, 30mg/kg OPN-5667 resulted in 117% tumor growth inhibition on average, with tumor regression in all animals and 3 out of 6 animals exhibiting complete regression.

Based on the efficacy of OPN-5667, a second-generation chimera, OPN-5877, was designed and synthesized. Overall, OPN-5877 demonstrated improved and balanced potency with DC₅₀ for EP300, CBP, IKZF1 and IKZF3 of 7nM, 6nM, 0.12nM and <0.5nM, respectively. OPN-5877 dosed at 1nM in vitro resulted in barely detectable levels of all four target proteins by Western blotting, and greater than 50% downregulation of the expression of IRF4, MYC, and BCL2 genes. Proliferation assays in 2 MM cell lines, OPM-2 and NCI-H929, showed comparable IC₅₀ of 140pM and 150pM, respectively. Importantly, IC₅₀ of binding of OPN-5877 to both EP300 and CBP BRDs was determined to be a modest 150nM. This set of data suggested that each molecule of OPN-5877 resulted in the degradation of many molecules of the target proteins.

Further chemical optimization towards a development candidate is ongoing. This polypharmacology approach has potential to yield a novel therapeutic with multifunctional activity that targets several oncogenic pathways concurrently in the same cancer cell, thus enabling effective single agent anti-cancer activity.