Periodic Reporting for period 1 - RECOBIN-PROTACs (Reversible Covalently Binding PROTACs Technology for Protein Degradation in Cancer Therapy)
Okres sprawozdawczy: 2020-04-01 do 2022-03-31
Chemical tools to selectively target cancer cells over healthy cells play a vital role in today’s modern medicine. Over the decades, cancer research focuses on the development of small molecules to inhibit the activity of proteins promoting cell proliferation. Acute myeloid leukemia (AML) is an aggressive cancer of the myeloid blood cell lineage and the treatment for AML involves the use of small molecules or monoclonal antibodies that inhibit the activity of proteins promoting leukemia cell proliferation. The drug dose required for efficient inhibition often leads to off-target effects. Cancerous cells can develop resistance to such drugs by developing new mutations in the target protein. In recent years, proteolysis targeting chimeras (PROTACs) technique receives much attention for therapeutic intervention by degradation of disease-causing proteins. The aim of the project was to develop new degradation approaches by incorporating new modalities on PROTACs to address the challenges associated with stabilization of ternary complex formation to reduce the degradation concentration and differentiation of isomers. The reversible covalently binding PROTACs (RECOBIN-PROTACs) consist of a target protein-ligand (JQ1 for BRD4), cereblon E3 ligase ligand (thalidomide), and a chemoselective functional group (benzaldehyde) connected through flexible linkers. The benzaldehyde group of RECOBIN-PROTAC forms a reversible covalent modification with proximal Lys residue (K91/K141) of BET protein BRD4 (near to JQ1 binding pocket) to enable the efficient degradation of BRD4 protein. The building blocks for RECOBIN-PROTAC were synthesized and the final RECOBIN-PROTACs will be explored for demonstration of their therapeutic efficacy in AML cells. Besides, another covalent electrophilic PROTAC method was developed for degradation of the cysteine-containing proteins. The irreversible covalent PROTAC consists of cereblon E3 ligase ligand and chemoselective warhead (benzyloxy pyridinium and carbonylacrylic derivatives) connected through a linker. The ultimate goal of developing these two reversible and irreversible PROTAC approaches is to meet the broad targeting of druggable as well as undruggable proteomes.
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).
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).
During this project, we synthesized all the required building blocks for RECOBIN-PROTACs. Even though we faced unanticipated problems in the penultimate step of the overall synthesis, finally we have developed the synthesis protocol to get RECOBIN-PROTACs. These molecules will be tested for their efficacy in the target degradation of BRD4 protein. The efficacy of final RECOBIN-PROTAC vs dBET1 PROTAC for BRD4 degradation will be compared in AML cell lines. Besides, we have developed another covalent electrophilic PROTAC with benzyloxy pyridinium and carbonylacrylic derivatives for cysteine targets. The synthesized caa-PROTAC is being explored for the degradation of the cysteine-containing proteins in various cancer cell lines. Although the duration of the fellowship is completed, we are committed to pursuing these two new approaches. Both methods are unique in their mode of action for protein degradation and complementary to each other and will be a great addition to the target protein degradation field.