First-in-class small molecule calmodulin pathway modulators attenuate excess p53 activity and correct erythropoietic defects in models of diamond-blackfan anemia (DBA) Free

Diamond-Blackfan Anemia (DBA) is a rare congenital bone marrow failure disorder with patients manifesting macrocytic anemia in infancy (Da Costa, et al. Blood 2020; Da Costa, et al. F1000Res. 2018). DBA is caused by germline heterozygous loss-of-function mutations in one of the twenty small- or large-subunit ribosomal protein (RP) genes. Current therapies for DBA include chronic red blood cell transfusions, glucocorticoid treatment, and allogeneic hematopoietic stem cell transplantation, all of which are associated with severe toxicities.

The mutations in RP genes cause defects in ribosomes, leading to ribosomal stress and aberrant p53 activation. Over-activation of p53 is a crucial mediator of DBA-associated hematopoietic defects, including erythroid failure and anemia. Previous studies (Taylor, et al. Sci Transl Med. 2020; Taylor, et al. Exp Hematol. 2012) have demonstrated that calmodulin (CaM) inhibitors, including the FDA-approved anti-psychotic trifluoperazine (TFP), improve anemia in multiple models of DBA by reducing activation of p53 targets. However, TFP is associated with serious side effects, including neurotoxicity attributed to its high brain penetrance and interaction with dopamine D2 receptors (DRD2) in the central nervous system (CNS).

We therefore embarked on a drug discovery program to identify novel CaM inhibitors with improved potency compared to TFP, but with activity against DRD2 eliminated and CNS penetrance attenuated. This effort resulted in the discovery of two novel small molecules, FTX-1 and FTX-2. Relative to TFP, FTX-1 and FTX-2 showed superior activity in reducing aberrant p53 activity in in vitro CD34+ human hematopoietic stem and progenitor cell (HSPC) models designed to mimic DBA via the introduction of ribosomal mutations in RPS19, RPL5, or RPL11 with CRISPR-Cas9 editing. Activation of p53 was assessed by monitoring the mRNA levels of CDKN1A/p21, a direct transcriptional target of p53. Compared to DMSO, 1 µM FTX-2 significantly attenuated p53 activity in the RPS19 model (p < 0.0001). This rescue was significantly greater than the positive control TFP, which rescued p53 activity at 3 µM (p < 0.01- P < 0.0001). FTX-2 was also significantly active in reducing p53 activity at 1 µM in the RPL5 and RPL11 models (p < 0.05 - < 0.0001, depending upon the donor and the experimental replicate), while 3 µM TFP was only active in the RPL5 model (p < 0.05-0.0001) but not in the RPL11 model.

FTX-1 and FTX-2 were also effective in improving anemia in a rps29^-/- zebrafish model of DBA. This model recapitulates the maturation arrest of red blood cells, growth retardation, decreased globin synthesis, and elevated p53 activity commonly observed in DBA patients. FTX-1 and FTX-2 significantly rescued hemoglobin levels at doses of 0.1 µM and 0.001 µM (p < 0.05 - <0.0001), respectively, compared to the positive control fluphenazine at 10 µM (p < 0.05 - <0.01). Lastly, 1 µM FTX-1 was effective in restoring erythroid development by increasing the BFU-E and CFU-E colonies in RPS19 DBA patient-derived HSPCs compared to vehicle (adjusted p-value < 0.05) as assessed using a colony formation assay. FTX-2 is currently being explored in toxicology and safety pharmacology studies in support of an Investigational New Drug (IND) application, with first-in-human studies anticipated in 2026.