Covalent Ligand Discovery against Druggable Hotspots Targeted by Anti-cancer Natural Products

Elizabeth A. Grossman, Carl C. Ward, Jessica N. Spradlin, Leslie A. Bateman, Tucker R. Huffman, David K. Miyamoto, Jordan I. Kleinman, Daniel K. Nomura

Cell Chemical Biology, 2017
doi: 10.1016/j.chembiol.2017.08.013

Many natural products that show therapeutic activities are often difficult to synthesize or isolate and have unknown targets, hindering their development as drugs. Identifying druggable hotspots targeted by covalently acting anti-cancer natural products can enable pharmacological interrogation of these sites with more synthetically tractable compounds. Here, we used chemoproteomic platforms to discover that the anti-cancer natural product withaferin A targets C377 on the regulatory subunit PPP2R1A of the tumor-suppressor protein phosphatase 2A (PP2A) complex leading to activation of PP2A activity, inactivation of AKT, and impaired breast cancer cell proliferation. We developed a more synthetically tractable cysteine-reactive covalent ligand, JNS 1-40, that selectively targets C377 of PPP2R1A to impair breast cancer signaling, proliferation, and in vivo tumor growth. Our study highlights the utility of using chemoproteomics to map druggable hotspots targeted by complex natural products and subsequently interrogating these sites with more synthetically tractable covalent ligands for cancer therapy.

Can Relative Binding Free Energy Predict Selectivity of Reversible Covalent Inhibitors?

Payal Chatterjee, Wesley M. Botello-Smith, Han Zhang, Li Qian, Abdelaziz Alsamarah, David Kent, Jerome J. Lacroix, Michel Baudry, and Yun Luo

J. Am. Chem. Soc., 2017
doi: 10.1021/jacs.7b08938

Reversible covalent inhibitors have many clinical advantages over noncovalent or covalent drugs. However, apart from selecting a warhead, substantial efforts in design and synthesis are needed to optimize noncovalent interactions to improve target-selective binding. Computational prediction of binding affinity for reversible covalent inhibitors presents a unique challenge since the binding process consists of multiple steps, which are not necessarily independent of each other. In this study, we lay out the relation between relative binding free energy and the overall reversible covalent binding affinity using a two-state binding model. To prove the concept, we employed free energy perturbation (FEP) coupled with λ-exchange molecular dynamics method to calculate the binding free energy of a series of α-ketoamide analogs relative to a common warhead scaffold, in both noncovalent and covalent bond states, and for two highly homologous proteases, calpain-1 and calpain-2. We conclude that covalent binding affinity alone, in general, can be used to predict reversible covalent binding selectivity. However, exceptions may exist. Therefore, we also discuss the conditions under which the noncovalent binding step is no longer negligible and propose a novel approach that combines the relative FEP calculations with a single QM/MM calculation of warhead to predict the binding affinity and binding kinetics for a large number of reversible covalent inhibitors. Our FEP calculations also revealed that covalent and noncovalent states of an inhibitor do not necessarily exhibit the same selectivity. Thus, investigating both binding states, as well as the kinetics will provide extremely useful information for optimizing reversible covalent inhibitors.

Development of 5N-Bicalutamide, a High-Affinity Reversible Covalent Antiandrogen

Felipe de Jesus Cortez, Phuong Nguyen, Charles Truillet, Boxue Tian, Kristopher M. Kuchenbecker, Michael J. Evans, Paul Webb, Matthew P. Jacobson , Robert J. Fletterick, and Pamela M. England

ACS Chem. Biol., 2017
DOI: 10.1021/acschembio.7b00702

Resistance to clinical antiandrogens has plagued the evolution of effective therapeutics for advanced prostate cancer. As with the first-line therapeutic bicalutamide (Casodex), resistance to newer antiandrogens (enzalutamide, ARN-509) develops quickly in patients, despite the fact that these drugs have ∼10-fold better affinity for the androgen receptor than bicalutamide. Improving affinity alone is often not sufficient to prevent resistance, and alternative strategies are needed to improve antiandrogen efficacy. Covalent and reversible covalent drugs are being used to thwart drug resistance in other contexts, and activated aryl nitriles are among the moieties being exploited for this purpose. We capitalized on the presence of an aryl nitrile in bicalutamide, and the existence of a native cysteine residue (Cys784) in the androgen receptor ligand binding pocket, to develop 5N-bicalutamide, a cysteine-reactive antiandrogen. 5N-bicalutamide exhibits a 150-fold improvement in Ki and 20-fold improvement in IC50 over the parent compound. We attribute the marked improvement in affinity and activity to the formation of a covalent adduct with Cys784, a residue that is not among the more than 160 androgen receptor point mutations associated with prostate cancer. Increasing the residence time of bound antiandrogen via formation of a covalent adduct may forestall the drug resistance seen with current clinical antiandrogens.