Structure-Based Design of First-Generation Small Molecule Inhibitors Targeting the Catalytic Pockets of AID, APOBEC3A, and APOBEC3B

Abstract

Activation-induced cytidine deaminase (AID) initiates antibody diversification by mutating immunoglobulin loci in B lymphocytes. AID and related APOBEC3 (A3) enzymes also induce genome-wide mutations and lesions implicated in tumorigenesis and tumor progression. The most prevalent mutation signatures across diverse tumor genomes are attributable to the mistargeted mutagenic activities of AID/A3s. Thus, inhibiting AID/A3s has been suggested to be of therapeutic benefit. We previously used a computationalbiochemical approach to gain insight into the structure of AID's catalytic pocket, which resulted in the discovery of a novel type of regulatory catalytic pocket closure that regulates AID/A3s that we termed the "Schrodinger's CATalytic pocket". Our findings were subsequently confirmed by direct structural studies. Here, we describe our search for small molecules that target the catalytic pocket of AID. We identified small molecules that inhibit purified AID, AID in cell extracts, and endogenous AID of lymphoma cells. Analogue expansion yielded derivatives with improved potencies. These were found to also inhibit A3A and A3B, the two most tumorigenic siblings of AID. Two compounds exhibit low micromolar IC 50 inhibition of AID and A3A, exhibiting the strongest potency for A3A. Docking suggests key interactions between their warheads and residues lining the catalytic pockets of AID, A3A, and A3B and between the tails and DNAinteracting residues on the surface proximal to the catalytic pocket opening. Accordingly, mutants of these residues decreased inhibition potency. The chemistry and abundance of key stabilizing interactions between the small molecules and residues within and immediately outside the catalytic pockets are promising for therapeutic development.

Design, Synthesis, and Evaluation of a Cross-Linked Oligonucleotide as the First Nanomolar Inhibitor of APOBEC3A

“…As K m (and K i ) depends strongly on the surrounding environment (e.g., buffer composition, pH, and ionic strengthsee Table ), inhibition with even lower K i may be observed in the cellular environment, because K m and K d values in the low μM , and nM range have been reported. During our study, two articles have been published on drug discovery efforts to obtain small molecule inhibitors of A3. , None approaches the potency of our inhibitors. This highlights the interest and need for powerful A3 inhibitors.…”

“…During our study, two articles have been published on drug discovery efforts to obtain small molecule inhibitors of A3. 63,64 None approaches the potency of our inhibitors. This highlights the interest and need for powerful A3 inhibitors.…”

Structure-Guided Design of a Potent and Specific Inhibitor against the Genomic Mutator APOBEC3A

“…To date, efforts to inhibit AID/APOBEC enzymes have primarily focused on high-throughput small-molecule drug discovery or using cytosine analogues incorporated into ssDNA. Small-molecule inhibitors of A3G and AID have been reported; − however, only low- to mid-micromolar levels of inhibition have been achieved. More recent work has leveraged studies on cytidine deaminase (CDA), a member of the wider deaminase family that acts on nucleosides rather than nucleic acids.…”

The current toolbox for APOBEC drug discovery

“…However, to date, hits from these screens have not been pursued owing to issues of cellular crossreactivity [64]. More recently, virtual screening was used to discover inhibitors of AID using the ZINC 'clean leads' subset of 4.6 million compounds [84]. Follow-up computational experiments prioritized 10 compounds that were evaluated in a deaminase activity assay against AID, A3A, A3B, and A3G.…”