Our laboratory recently reported that treatment with the d-amino acid containing peptide HYD1 induces necrotic cell death in multiple myeloma (MM) cell lines. Due to the intriguing biological activity and promising in vivo activity of HYD1, we pursued strategies for increasing the therapeutic efficacy of the parent linear peptide. These efforts led to the development of a cyclized peptidomimetic, MTI-101, with increased in vitro activity and robust in vivo activity as a single agent using two myeloma models that consider the bone marrow microenvironment. MTI-101 treatment resulted in mechanistically similar hallmarks of HYD1 induced cell death, namely the generation of ROS, depletion of ATP levels, and failure to activate caspase-3. Moreover, MTI-101 was shown to be cross-resistant in the HYD1 acquired resistant H929-60 cell line that was previously developed in our laboratory. In the present study, we pursued an unbiased chemical biology approach using biotinylated peptide affinity purification and LC-MS/MS analysis to identify binding partners of MTI-101. Using this approach, CD44 was identified as a predominant binding partner. Using an ELISA based assay, we showed that biotinylated peptide bound to full length recombinant human CD44 in a concentration dependent manner. Reducing the cell surface expression of CD44 was accompanied by the activation of caspase-3 and cell death was observed in the NCI-H929 and U266 MM cell lines, indicating that MM cells require CD44 expression for survival. Ectopic expression of CD44s correlated with increased binding of the FAM-conjugated peptide in the 8226 MM cell line, and this was further corroborated using CD44 knockout mice which also showed less peptide binding compared to wild-type. However, ectopic expression of CD44s was not sufficient to increase the sensitivity to MTI-101 induced cell death. Mechanistically, we show that MTI-101 induced a pro-survival signal through the activation of Erk1/2 and that CD44 formed a complex with Pyk2. These data corroborate with that of which was previously observed with the parental peptide being a partial agonist and inducing an autophagic survival signal. With respect to cell death, we showed that CD44 forms a complex with known death inducing proteins caspase-8, caspase-10, Rip1, Rip3, Drp1, TNFAIP8, and PGAM5. Furthermore, we demonstrated that MTI-101 induced mitochondrial fission which may be modulated by a Rip1, Rip3 or Drp1 dependent and independent pathway. Finally, we show that MTI-101 has robust activity as a single agent in the SCID-Hu bone implant and 5TGM1 in vivo model of multiple myeloma. Together these data continue to support the further development of this class of compounds as well as identify CD44 as a therapeutic target for the treatment of MM.

In recent studies it was reported that the D-amino acid containing peptide HYD1 was used in the treatment of necrotic cell death in multiple myeloma cell lines and showed promising biological activity and in vivo activity. It was meaningful to explore strategies for increasing the therapeutic efficacy of HYD1, a linear peptide. These efforts led to the development of MT1-101 (cyclized peptidomimetics), a lead compound that showed increased in vitro activity and in vivo activity. MTI-101 was found to bind the cell adhesion molecule CD44 and induce programmed necrosis in myeloma cell lines. It was important to improve on the binding efficiency of the MTI-101 to this target and explore more cost effective ways to synthesize this peptide. This lead to developing Cyclic beta-hairpin-like peptoid scaffolds, which introduced diverse families of random peptoid-body libraries that will be screened to find small stable scaffolds that compete with and can replace antibodies as cell-surface targeting reagents. The synthesis of peptoids on solid-support can be more cost effective and a large library can be developed using a diverse library of primary amines. This initiated this thesis project to develop a generalized scheme for the synthesis of TRP-like primary amine peptoid side chains used in the cyclic beta-hairpin - Like scaffolds.