The project started with the development of a synthesis protocol for the RECOBIN-PROTACs to study the proof-of-concept for efficient degradation of target protein in AML cell lines. There are four key building blocks in RECOBIN-PROTACs (E3 ligase ligand, target protein ligand, linkers, and chemoselective functional group). More importantly, the reversible covalent binder (RECOBIN) plays a vital role in the successful implementation of the method. Initially, benzaldehyde was chosen as a reversible covalent binder because of its excellent chemoselectivity with amines. The BET bromodomain degrader, dBET1 consists of cereblon E3 ubiquitin ligase ligand and BRD4 ligand connected with a linker. After careful observation of the crystal structure of the JQ1-BRD4, we have decided to target the BRD4 protein with RECOBIN-PROTACs. There are two proximal lysine residues (K91 and K141 - near the tertiary butyloxy group of JQ1 binding) and the tertiary butyloxy group in JQ1 is exposed out of the surface. So these two parameters allowed us to connect the aldehyde group near to JQ1 which can serve as a covalent anchor for imine formation with the aldehyde group of RECOBIN-PROTAC. The synthesis of the RECOBIN-PROTACs was disintegrated into four parts. The four building blocks, thalidomide ligand, (+) JQ1-acid, the connecting linkers, and benzaldehyde derivatives (part d) were synthesized separately from their respective starting materials. Next, we started connecting these building blocks to achieve the final RECOBIN-PROTAC which can be compared with dBET1. In the penultimate step, alkylation of a primary amine with iodoethoxy benzaldehyde was not proceeded to get the secondary amine-containing compound which will be coupled with JQ1-acid to get the final RECOBIN-PROTAC. However, an alternative approach was developed to overcome this synthesis hurdle to get the secondary amine. Now we are completing the final steps of the RECOBIN-PROTACs. These synthesized RECOBIN-PROTACs will be tested for target protein BRD4 degradation in cancer cell lines and compared its degradation efficacy with dBET1.
During the entire course, another complementary method to RECOBIN-PROTACs was also developed for the degradation of cysteine targeting proteins. This approach doesn’t require the target protein-ligand but enables the degradation of the cysteine-containing proteins. The selectivity is governed by the protein-protein interactions and excellent chemoselectivity of the cysteine reacting groups. For this purpose, we have chosen two classes of cysteine reactive warheads, benzyloxy pyridinium, and carbonylacrylic derivatives. The former reactive warhead serves as a nucleophilic substitution reaction with the thiol group of the cysteine residues whereas the latter one is Michael acceptor. Initially, benzyloxy pyridinium was tested for its chemoselectivity with peptide (glutathione) and protein (ubiquitin). It rendered excellent chemoselectivity for cysteine residue over other nucleophilic residues presented. This was further supported by a control experiment where in absence of Cys in ubiquitin gave no observable labeled protein. Next, we have demonstrated the methodology for showing its excellent chemoselectivity with a structurally diverse set of proteins (HSA and Annexin V). The reactivity of the benzyloxy pyridinium derivatives was not compromised which allows derivatizing with cereblon E3 ligase ligand. The attributes of this novel warhead, chemoselectivity, stability in the aqueous buffer as well as the stability of formed conjugates have inspired us to its selection for developing covalent PROTACs. Secondly, the chemoselectivity of the carbonylacrylic (CAA) derivatives was demonstrated by the host group as mentioned in the proposal. So after having such chemoselective reactive warheads, we have designed covalent electrophilic PROTAC by integrating these warheads with cereblon E3 ligase ligand. The initially synthesized model CAA-PROTAC is being explored for its degradation efficacy towards new cysteine targets in various cancer cell lines including AML cell lines (HL-60).
