Small Molecule-induced Protein Degradation Research Articles

Abstract LBA002: Targeting GSPT1 by a novel cereblon E3 ligase modulator for the treatment of Acute Lymphoblastic Leukemia

Abstract Acute Lymphoblastic Leukemia (ALL), the most common childhood cancer and the second most common acute leukemia in adults, arises from clonal expansion of undifferentiated lymphoid precursor cells in bone marrow. Although the majority of childhood ALL cases harbor clonal genetic alterations of transcription factor (TF) gene mutations or rearrangements, TF alterations remain difficult therapeutic targets. Small molecule induced protein degradation is a novel strategy that can be applied to currently undruggable targets such as TF and fusion oncoproteins. In this paradigm, small molecule degraders (either PROTAC or Molecular Glue (MG)) redirect the cell’s endogenous ubiquitin proteasome system and induce ubiquitination of the target protein or non-native substrate of an E3 ligase (neosubstrate) and its subsequent proteasomal degradation. Recently, the CRBN E3 ligase modulator, CC90009 was reported to show potent antitumor activity in acute myeloid leukemia, leading to the identification of GSPT1 (G1 to S phase transition factor) as a CRBN neosubstrate. These findings suggest the potential of MGs to target unanticipated vulnerabilities in different malignancies. Here, using a structurally diverse and unique set of MGs (Molecular Glue Library (MGL)) with confirmed CRBN binding affinity, we sought to identify novel CRBN modulators through phenotypic and proteomic methods. Our screening of the MGL in a panel of representative acute leukemia cell lines, including the CRLF2-rearranged ALL cell line MHH-CALL-4 identified several active MGs with EC50 <5 μM. A lenalidomide competition assay and MHH-CALL-4 CRBN knock down cells confirmed the CRBN dependency of these MGs. Among these compounds, SJ6986, a thalidomide-driven sulfonamide showed potent cytotoxicity in over 10 ALL cell lines tested in vitro. TMT-MS proteomic analyses identified GSPT1/2 as the primary targets for this compound with high selectivity. We next tested SJ6986 activity in a panel of patient derived ALL xenografts (PDX) harboring rearrangement of IGH-CRLF2, EPOR, ATF7IP-JAK2 ex vivo. All the tested tumors were highly sensitive to SJ6986 with IC50s at nanomolar range. PK analyses in NSG mice indicated rapid absorption and over 80% oral bioavailability for SJ6986. PD studies in an IGH-CRLF2 PDX showed dose-dependent degradation of GSPT1 within 48 hours of treatment. Finally, we examined the antitumor activity of SJ6986 in 6 different PDX representative of high-risk subtypes of ALL including near haploid, low-hypodiploid, CRLF2-rearranged and EPOR-rearranged, in vivo for 28 days. SJ6986 was able to dramatically decrease the tumor burden at 1 mg/kg dose in most of the tumor models. Collectively, these results affirm that SJ6986 is a novel CRBN modulator and a potential therapeutic agent by targeting GSPT1 protein with high selectivity and potency for the treatment of ALL. Citation Format: Fatemeh Keramatnia, Yunchao Chang, Gisele Nishiguchi, Jaeki Min, Charles Mullighan, Marcus Fischer, Zoran Rankovic, Fatemeh Keramatnia. Targeting GSPT1 by a novel cereblon E3 ligase modulator for the treatment of Acute Lymphoblastic Leukemia [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA002.

Opportunities and Challenges of Small Molecule Induced Targeted Protein Degradation.

Proteolysis targeting chimeras (PROTAC) represents a new type of small molecule induced protein degradation technology that has emerged in recent years. PROTAC uses bifunctional small molecules to induce ubiquitination of target proteins and utilizes intracellular proteasomes for chemical knockdown. It complements the gene editing and RNA interference for protein knockdown. Compared with small molecule inhibitors, PROTAC has shown great advantages in overcoming tumor resistance, affecting the non-enzymatic function of target proteins, degrading undruggable targets, and providing new rapid and reversible chemical knockout tools. At the same time, its challenges and problems also need to be resolved as a fast-developing newchemical biology technology.

POS0006 NX-5948, A SELECTIVE DEGRADER OF BTK, SIGNIFICANTLY REDUCES INFLAMMATION IN A MODEL OF AUTOIMMUNE DISEASE

Background:Aberrant activation of B cells and autoantibody mediated tissue damage are hallmarks of autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Therefore, novel treatments that prevent autoantibody generation or antibody-mediated end organ tissue damage are of high interest. Bruton’s tyrosine kinase (BTK) transduces signals downstream of the B cell receptor (BCR), toll-like receptors, and Fc receptors in B cells and myeloid cells [1]. Overexpression of BTK in B cells leads to hyperactive BCR signaling, plasma cell generation, autoantibody secretion, and an SLE-like disease in mice [2]. Conversely, reducing BTK expression in B cells can ameliorate disease in Lyn-deficient mice.[3] BTK inhibitors, such as evobrutinib, have entered clinical studies for the treatment of autoimmune diseases.[4]Objectives:Small molecule-induced protein degradation offers a unique approach to target BTK; this approach simultaneously eliminates both BTK kinase activity and BTK-mediated scaffolding interactions in the signalosome. Chimeric Targeting Molecules (CTMs) are small molecules that catalyze ubiquitylation and proteasomal degradation of target proteins and are comprised of a ubiquitin ligase binding element (“harness”), a linker, and a target binding element (“hook”). NX-5948 is a CTM that contains a BTK hook linked to a cereblon (CRBN) harness. We examined the activity of NX-5948 in a collagen-induced arthritis model as part of an assessment of its potential as a drug candidate for autoimmune disease.Methods:Cellular degradation of BTK, Aiolos and Ikaros as well as induction of CD69 and CD86 was determined using flow cytometry. Degradation of BTK in CD-1 mice or cynomolgus monkey was determined using flow cytometry analysis. In a collagen-induced arthritis (CIA) model, mice were vaccinated with type II collagen and treated before the onset of symptoms. Serum cytokine and anti-type II collagen antibody levels were determined using Luminex and ELISA, respectively.Results:In human PBMCs, NX-5948 degrades BTK at sub-nanomolar concentrations and inhibits BCR signaling as measured by CD69 and CD86 induction in anti-IgM-stimulated B cells with similar potency. Oral administration of NX-5948 in mice leads to BTK degradation to <10% of baseline levels in circulating and splenic B cells. NX-5948 also promotes potent BTK degradation in cynomolgus monkeys, and it can suppress BTK levels to <10% of baseline levels after a single oral dose as low as 10 mg/kg.Unlike IMiD drugs such as lenalidomide, the CRBN harness of NX-5948 was designed to avoid the degradation of known CRBN neo-substrates Aiolos (IKZF3) and Ikaros (IKZF1). In primary human T cells, NX-5948 induces minimal degradation of Aiolos and Ikaros and does not promote IL-2 secretion suggesting that NX-5948 does not convey IMiD activity associated with agents such as lenalidomide.We examined the activity of NX-5948 in a mouse CIA model compared to that of the BTK inhibitor ibrutinib or dexamethasone as a positive control. In mice treated with NX-5948, symptoms of arthritis were resolved, and a significant reduction in arthritis clinical score was observed. Treatment with NX-5948 resulted in a reduction in anti-type II collagen titer and serum levels of the pro-inflammatory cytokine IL-6. Treatment with NX-5948 yielded superior anti-inflammatory activity relative to ibrutinib and similar activity to dexamethasone. Treatment with NX-5948 was well-tolerated and, unlike dexamethasone, did not promote body weight loss.Conclusion:Degradation of BTK by NX-5948 shows robust activity in a CIA model compared to existing agents tested as controls. These findings provide support for further investigation of NX-5948 in additional models of autoimmune disease to inform plans for clinical development.

Nx-2127, a Degrader of BTK and IMiD Neosubstrates, for the Treatment of B-Cell Malignancies

Bruton's tyrosine kinase (BTK) plays a key role in cell survival in B cell malignancies, such as chronic lymphocytic leukemia (CLL). Covalent inhibitors of BTK, such as ibrutinib and acalabrutinib, while effective, have been associated with the occurrence of resistance mutations. The most prevalent site of mutation, C481, renders covalent BTK inhibitors unable to form a covalent bond with BTK leading to diminished efficacy and disease progression. Small molecule-induced protein degradation offers a unique approach to target BTK for the treatment of B-cell malignancies. Chimeric Targeting Molecules (CTMs) catalyze ubiquitylation and proteasomal degradation of target proteins and are comprised of a ubiquitin ligase binding element ("harness"), a linker, and a target binding element ("hook"). NX-2127 is a CTM that contains a BTK hook linked to a cereblon (CRBN) harness. NX-2127 degrades 50% of cellular BTK (DC50) at < 5 nM across multiple cancer cell lines and in human PBMCs. BTK CTMs impair viability in the BTK-dependent ABC-DLBCL cell line, TMD8 (EC50: < 15 nM after 72 hours). Importantly, NX-2127 induces degradation of the mutated BTK-C481S in cells and inhibits proliferation of BTK-C481S mutant TMD8 cells more effectively than ibrutinib (NX-2127 EC50 values of < 30 nM versus > 1 μM for ibrutinib). Oral administration of NX-2127 in mice leads to dose-proportional exposure in plasma and BTK degradation to <10% of baseline levels in circulating and splenic B cells. In both WT TMD8 and C481S mutant xenograft models, daily oral administration of NX-2127 resulted in superior tumor growth inhibition (TGI) as compared to ibrutinib. NX-2127 also demonstrates potent degradation of BTK in cynomolgus monkeys with oral administration. Following 14 days of once daily, oral dosing in cynomolgus monkey, BTK levels are suppressed to <10% of baseline levels at doses as low as 1 mg/kg. In addition to potent BTK degradation, NX-2127 possesses IMiD-like properties through the design of the CRBN binding harness that catalyzes the degradation of CRBN neosubstrates Aiolos (IKZF3) and Ikaros (IKZF1). This activity is associated with increased T cell activation and anti-tumor effects of the IMiD drugs lenalidomide and pomalidomide. In primary human T cells, NX-2127 catalyzes the degradation of Aiolos and Ikaros with of 25 nM and 54 nM, respectively, potencies which are similar to those of lenalidomide (20 nM and 343 nM, respectively). Corresponding with such degradation, NX-2127 stimulates T cell activation as measured by increased IL-2 production in primary human T Cells in a manner similar to lenalidomide and pomalidomide. The dual activity of BTK degradation combined with immunomodulation of NX-2127 supports its development for the treatment of B-cell malignancies. Disclosures Robbins: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Kelly:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tan:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. McIntosh:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Wu:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Konst:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Kato:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Peng:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mihalic:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Weiss:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Perez:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tung:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Kolobova:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Borodovsky:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Rountree:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tenn-McClellan:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Noviski:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ye:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Basham:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ingallinera:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. McKinnell:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Karr:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Powers:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Guiducci:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sands:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Prey for the Proteasome: Targeted Protein Degradation-A Medicinal Chemist's Perspective.

Targeted protein degradation (TPD), the ability to control a proteins fate by triggering its degradation in a highly selective and effective manner, has created tremendous excitement in chemical biology and drug discovery within the past decades. The TPD field is spearheaded by small molecule induced protein degradation with molecular glues and proteolysis targeting chimeras (PROTACs) paving the way to expand the druggable space and to create a new paradigm in drug discovery. However, besides the therapeutic angle of TPD a plethora of novel techniques to modulate and control protein levels have been developed. This enables chemical biologists to better understand protein function and to discover and verify new therapeutic targets. This Review gives a comprehensive overview of chemical biology techniques inducing TPD. It explains the strengths and weaknesses of these methods in the context of drug discovery and discusses their future potential from a medicinal chemist's perspective.

Optical Control of Small Molecule-Induced Protein Degradation.

As an emerging approach to protein perturbation, small molecule-induced protein degradation has gained significant attention as both a chemical tool and a potential therapeutic. To enable discrete control over its function, we have developed a broadly applicable approach for the optical activation of small molecule-induced protein degradation. By installing two different photolabile protecting groups, so-called caging groups, onto two different ligands recruiting Von Hippel-Lindau (VHL) and cereblon (CRBN) E3 ubiquitin ligases, our strategy enables light-triggered protein degradation for any small molecule warhead.

Targeted Protein Degradation of BTK As a Unique Therapeutic Approach for B Cell Malignancies

Bruton's tyrosine kinase (BTK) is a key component of B cell receptor signaling and is involved in B cell development and function. BTK plays a crucial role in cell survival in B cell malignancies such as Chronic Lymphocytic Leukemia (CLL), and covalent inhibitors of BTK, such as ibrutinib, have been successful clinically. However, long-term therapy with covalent BTK inhibitors has been shown to generate resistance mutations, which lead to disease progression. New treatments are needed to address this unmet medical need. Small molecule-induced protein degradation offers a unique approach to inhibiting BTK function. Chimeric Targeting Molecules (CTMs) mediate ubiquitylation and proteasomal degradation of specific target proteins. CTMs are comprised of a ubiquitin ligase binding element ("harness"), a chemical linker, and a target binding element ("hook"). Use of CTMs to degrade both WT and ibrutinib-resistant forms of BTK present a novel approach to targeting BTK and could affect both its catalytic and potential scaffolding functions. We have identified multiple CTMs that catalyze BTK degradation in multiple B cell lines; the concentration of one of such CTM, NRX0492, required to degrade 50% BTK (DC50) was < 1 nM after 4 hours. BTK CTMs impair viability in the BTK-dependent ABC-DLBCL cell line, TMD8 (EC50: < 10 nM after 72 hours). These CTMs also induce degradation of the ibrutinib-resistant C481S mutant form of BTK in cells and confer loss of viability in BTKC481S mutant TMD8 cells with EC50 values of < 10 nM compared to > 1 µM for ibrutinib. Oral administration of NRX0492 in mice leads to dose-proportional exposure in plasma and BTK degradation in circulating and splenic B cells: at 6 hours after a single oral dose of NRX0492, 11% BTK remained in mouse splenocytes compared to 100% BTK in mice dosed with vehicle (P < 0.0001). In a WT TMD8 xenograft model, NRX0492 treatment resulted in similar tumor growth inhibition (TGI) as compared to ibrutinib over 23 days of daily oral administration: 54.4% TGI for NRX0492 and 55.8% for Ibrutinib, both as compared to placebo (P = 0.0006 and P = 0.0004, respectively). Notably, in a TMD8 BTKC481S xenograft model, NRX0492 demonstrated superior TGI as compared to ibrutinib: 51.3% versus 15.2%, (P = 0.033). Preclinical safety and toxicity studies for BTK CTMs are ongoing to inform plans for clinical development. CTM-mediated degradation of BTK may provide an alternative therapeutic approach for B cell malignancies, particularly in the ibrutinib-resistant setting. Disclosures Kelly: Nurix Therapeutics: Employment. Robbins:Nurix Therapeutics: Employment. Tan:Nurix Therapeutics: Employment. Tenn-McClellan:Nurix Therapeutics: Employment. McIntosh:Nurix Therapeutics: Employment. Wu:Nurix Therapeutics: Employment. Konst:Nurix Therapeutics: Employment. Kato:Nurix Therapeutics: Employment. Perez:Nurix Therapeutics: Employment. Tung:Nurix Therapeutics: Employment. Kolobova:Nurix Therapeutics: Employment. Ingallinera:Nurix Therapeutics: Employment. McKinnell:Nurix Therapeutics: Employment. Weiss:Nurix Therapeutics: Employment. Noviski:Nurix Therapeutics: Employment. Ye:Nurix Therapeutics: Employment. Peng:Nurix Therapeutics: Employment. Cardozo:Nurix Therapeutics: Employment. Mihalic:Nurix Therapeutics: Employment. Basham:Nurix Therapeutics: Employment. Rountree:Nurix Therapeutics: Employment. Karr:Nurix Therapeutics: Employment. Bence:Nurix Therapeutics: Employment. Zapf:Nurix Therapeutics: Employment. Sands:Nurix Therapeutics: Employment.

Novel class of 7-Oxabicyclo[2.2.1]heptene sulfonamides with long alkyl chains displaying improved estrogen receptor α degradation activity

Hormone therapy is widely used in clinic for breast cancer treatment, such as tamoxifen, but long-term use can cause drug resistance. In this regard, a strategy based on small molecule-induced protein degradation, i.e. selective estrogen receptor downregulator (SERD), might be an effective alternative to hormone therapy for breast cancer. However, most of the SERD candidates involve very limited scaffolds and are still in clinical trials, and none of them has been approved for marketing. In this study, a series of novel 7-oxabicyclo[2.2.1]heptene sulfonamide (OBHSA) derivatives with long alkyl chains were identified as novel SERDs. We found that the position and the length of alkyl side chain have significant effect on the biological activity of the SERD compounds and with the six-carbon side chain was the best. Among them, compounds 23a and 36 displayed potent inhibitory activity against MCF-7 breast cancer cell line with IC50 values of 0.84 μM and 0.77 μM, respectively, as well as excellent ERα degradation activity. Primary mechanism study indicated that the degradation of ERα is mediated through proteasome-mediated process. Flow cytometry analysis of apoptosis of 36 suggested that the effect of this type compounds on MCF-7 cells is associated with apoptosis. As such, these compounds have shown potential to become promising leads for the development of highly efficient SERDs for drug-resistance breast cancer therapies.

HaloPROTACS: Use of Small Molecule PROTACs to Induce Degradation of HaloTag Fusion Proteins.

Small molecule-induced protein degradation is an attractive strategy for the development of chemical probes. One method for inducing targeted protein degradation involves the use of PROTACs, heterobifunctional molecules that can recruit specific E3 ligases to a desired protein of interest. PROTACs have been successfully used to degrade numerous proteins in cells, but the peptidic E3 ligase ligands used in previous PROTACs have hindered their development into more mature chemical probes or therapeutics. We report the design of a novel class of PROTACs that incorporate small molecule VHL ligands to successfully degrade HaloTag7 fusion proteins. These HaloPROTACs will inspire the development of future PROTACs with more drug-like properties. Additionally, these HaloPROTACs are useful chemical genetic tools, due to their ability to chemically knock down widely used HaloTag7 fusion proteins in a general fashion.