ER-resident chaperon protein disulfide isomerase PDIA1 inhibition restricts and eliminate aberrant plasma cells

Abstract One of the major hallmarks of plasma cell dyscrasias (PCDs) is dysregulated proteostasis. PCDs are a group of heterogeneous diseases characterized by infiltration of monoclonal plasma cells in the bone marrow and high immunoglobulin in the serum. Primary light chain amyloidosis (AL), monoclonal gammopathy of undetermined significance (MGUS), smoldering myeloma (SMM), Multiple Myeloma (MM), plasma cell leukemia (PCL) and plasmacytoma. The clonal plasma cells often secrete disulfide bond-rich antibody light chains along with cytokines and chemokines. The extra secretary burden along with mutations in several key enzymes of proteasomal pathway, create heightened endoplasmic reticulum (ER) stress response. Recently we reported that dysregulated proteostasis can be exploited as therapeutic vulnerability in PCDs. Due to the decoupling of proteosome from ER stress response the clonal plasma cells depend on ER-resident chaperons. Most prominent among them are protein disulfide isomerases family of enzymes, particularly PDIA1, for correct folding and structural integrity of secretory proteins, including light chain immunoglobins thereby avoiding unresolvable ER-stress. Previously, using HTS screening of small molecule libraries with malignant plasma cell-based cytotoxic assays we discovered mechanistically unbiased first-in-class irreversible inhibitor of PDIA1 named CCF642 ((Z)-3-(4-methoxyphenyl)-5-((5-nitrothiophen-2-yl) methylene)-2-thioxothiazolidin-4-one) as a Bonafide 'hit'1, 2. Our initial 'hit' compound has several shortcomings due to poor solubility in biological buffers, selectivity and bioavailability. We pursued a structure-guided design coupled with synthetic medicinal chemistry and significantly improved potency, selectivity, bioavailability and ‘therapeutic index’ of ‘hit’ compound CCF642 to a ‘lead’ candidate CCF642-34. We establish the optimal linker length and functionally relevant derivatives by structure-activity relationships (SARs) approach keeping the selectivity and of the target and potency of pharmacophore. The wide structural variation, as well as the open ends of acid and amine, make it a better choice for synthetic chemistry. Rhodanine presence in pharmacophore for the peptide coupling was a real challenge due to instability of rhodanine ring in alkaline medium. Various coupling agents were evaluated, and only 1,1'-Carbonyldiimidazole (CDI) works without using any base. We evaluated nitro-thiophene by synthetic approach to challenge its PAINS properties by changing the nitro group on thiophene with other electron withdrawing groups. This structural variation and its activity changes helps us to understand the drug molecular mechanism of action. To identify the binding pattern of our hit and lead molecules, we synthesized biotin conjugates. They irreversibly bind to lysine in the PDIA1 active motif CGHCK. These results were confirmed by western blot analysis with PDIA1 antibodies and streptavidin pull down coupled with tandem mass spectroscopy of all the proteins. To further establish the specificity and target engagement we developed PROTAC (Proteolysis Targeting Chimera) with our lead compound and confirmed target engagement. We demonstrate that the lead compound CCF642-34 is orally bioavailable selective PDIA1 inhibitor that induced unresolvable ER stress in clonal plasma cells and induced programmed cell death as evidenced by PARP1 and caspase cleavage. The lead compound was safe at nearly 10-fold the therapeutic dose in normal CD43+hematopoietic stem and progenitor. The lead compound is effective in restricting evolution of clonal plasma cells in vivo in two different model systems2,3. Reference. Vatolin S., et al., Novel Protein Disulfide Isomerase Inhibitor with Anticancer Activity in Multiple Myeloma. Cancer Res. 2016, 76, 3340–3350. Hasipek, M. et al., Therapeutic Targeting of Protein Disulfide Isomerase PDIA1 in Multiple Myeloma. Cancers 2021, 13, 2649. Dima D., et al., Multiple Myeloma Therapy: Emerging Trends and Challenges. Cancers (Basel)2022 Aug 23;14(17):4082.