Activation of Drug and Biologic Conjugates by Click Chemistry and their Tumor-selective Targeting for Prostate Cancer Suppression

A critical unmet need in prostate cancer management is to prevent disease advance to a castration-resistant and
chemotherapy-inert lethal stage. We will explore a novel anti-cancer approach against castration-resistant
prostate cancer (CRPC) that entails activation of a pro-drug conjugate specifically in tumor tissue leading to
cancer cell ablation without imposing off-targets toxicity. The approach is based on a 2021 report by Tanaka &
colleagues that a drug-conjugated aryl azide reacts with endogenous acrolein molecules that are abundant in
cancer but not normal tissue. This bioorthogonal reaction in cancer cells, based on click chemistry, leads to
rearrangement of the reaction product and drug release. We will utilize this drug release principle in a preclinical
study to target CRPC cells by BO-264 and its more potent derivatives, which are small-molecule anti-cancer
agents & drug candidates, and by docetaxel & cabazitaxel - the chemotherapy drugs for clinical CRPC. Acrolein,
a 3-carbon unsaturated aldehyde, is copiously produced in cancer cells due to abnormal metabolism and
oxidative stress. BO-264, identified from a chemical library screen, inhibits TACC3, a centrosome-associated
protein which regulates chromosome segregation. TACC3 is elevated in prostate cancer and other solid tumors.
This project is a collaboration of PI’s lab with Co-PI’s synthetic chemistry lab. Novel aryl azide conjugates of
biologics and chemotherapeutics, synthesized by the Co-PI’s lab, will be examined for impacts on CRPC in vitro
and in vivo in mouse xenograft & allograft in PI’s lab. Prelim data shows dose-dependent increasing susceptibility
of the PC3 CRPC cells to the azide conjugate of BO-264, synthesized in-house. Non-cancer RWPE-1 prostate
cells were unaffected. PC3 cells showed ~ 7-fold higher acrolein than RWPE-1. Sp. Aim 1: Synthesize at high
yields aryl azide conjugates of the above-noted anti-cancer biologics & taxanes using novel synthesis strategies
and characterize the end products. The products will be characterized by NMR, IR, HRMS, X-ray. Derivatives
of BO-264, not available commercially, will be synthesized. Sp. Aim 2: Investigate activated drugs/biologics,
released from conjugates, for effects on human & mouse CRPC cells, and on CRPC xenograft/allograft tumors
in mice. Conjugate-treated cells in culture and tumors will be assayed for proliferation, apoptosis, migration,
invasion, EMT, AR, and for AKT & ERK activation. For androgen receptor (AR)-positive CRPC, combined effects
of enzalutamide (AR inhibitor) and an azide conjugate will be probed. Tumor volume and metastasis will be
examined and correlated with tumor acrolein levels. Methods: Engineering of cells to stably express
AkaLuciferase reporter; bioluminescence imaging to assess tumor volume, metastasis; NMR, IR, HRMS, X-ray;
Cell invasion, migration; Western; IHC; single-cell RT-qPCR; Acrolein assay; FACS; Statistics. Clinical impact:
1) High clinical translation potential; 2) Novel azide conjugates of anti-cancer biologics & CRPC drugs; 3) Tumor-
selective release & activation of drugs in a bioorthogonal reaction based on click chemistry is applicable to other
cancers; 4) Drug release & activation in an immune-intact allograft tumor microenvironment is clinically relevant.

Grant Number: 5R21CA289167-02
NIH Institute/Center: NIH
Principal Investigator: BANDANA CHATTERJEE