Biology subjects

Myint, W.

Publications and source records attributed to Myint, W..

4 recordsLinked to original sources

bioRxiv2024

Next generation APOBEC3 inhibitors: Optimally designed for potency and nuclease stability

APOBEC3 (or A3) enzymes have emerged as potential therapeutic targets due to their role in introducing heterogeneity in viruses and cancer, often leading to drug resistance. Inhibiting these enzymes has remained elusive as initial phosphodiester (PO) linked DNA based inhibitors lack stability and potency. We have enhanced both potency and nuclease stability, of 2'-deoxy-zebularine (dZ), substrate-based oligonucleotide inhibitors for two critical A3s: A3A and A3G. While replacing the phosphate backbone with phosphorothioate (PS) linkages increased nuclease stability, fully PS-modified inhibitors lost potency (1.4-3.7 fold) due to the structural constraints of the active site. For both enzymes, mixed PO/PS backbones enhanced potency (2.3-9.2 fold), while also vastly improving nuclease resistance. We also strategically introduced 2'-fluoro sugar modifications, creating the first nanomolar inhibitor of A3G-CTD2. With hairpin-structured inhibitors containing optimized PS patterns and LNA sugar modifications, we characterize the first single-digit nanomolar inhibitor targeting A3A. These extremely potent A3A inhibitors, were highly resistant to nuclease degradation in serum stability assays. Overall, our optimally designed A3 oligonucleotide inhibitors show improved potency and stability, compared to previous attempts to inhibit these critical enzymes, opening the door to realize the therapeutic potential of A3 inhibition.

biochemistry↗

bioRxiv2022

Discovery of E6AP AZUL binding to UBQLN1/2 in cells, phase-separated droplets, and an AlphaFold-NMR integrated structure

The E3 ligase E6AP/UBE3A has a dedicated binding site in the 26S proteasome provided by the RAZUL domain of substrate receptor hRpn10/S5a/PSMD4. Guided by RAZUL sequence similarity, we test and demonstrate here that the E6AP AZUL binds transiently to the UBA of proteasomal shuttle factor UBQLN1/2. Despite a weak binding affinity, E6AP AZUL is recruited to UBQLN2 phase-separated droplets and E6AP interacts with UBQLN1/2 in cells. Steady-state and transfer NOE experiments indicate direct interaction of AZUL with the UBQLN1 UBA domain. Intermolecular contacts identified by NOESY data were combined with AlphaFold2-Multimer predictions to yield an AZUL:UBA model structure. We also identify a concentration-dependent oligomerization domain directly adjacent to UBQLN1/2 UBA (UBA-adjacent, UBAA) that is -helical and allosterically reconfigured by AZUL binding to UBA. These data lead to a model of E6AP recruitment to UBQLN1/2 by AZUL:UBA interaction and provide fundamental information on binding requirements for interactions in droplets and cells.

biophysics↗

bioRxiv2022

Structure of the catalytically active APOBEC3G bound to a DNA oligonucleotide inhibitor reveals tetrahedral geometry of the transition state

APOBEC3 proteins (A3s) are enzymes that catalyze deamination of cytidine to uridine in single-stranded DNA (ssDNA) substrates, thus playing a key role in innate antiviral immunity. However, APOBEC3 family has also been linked to many mutational signatures in cancer cells, which has led to intense interest to develop inhibitors of A3s catalytic activity as therapeutics as well as tools to study A3s biochemistry, structure and cellular function. Recent studies have shown that ssDNA containing 2'-deoxy-zebularine (dZ-ssDNA) is an inhibitor of A3s such as A3A, A3B and A3G, although atomic determinants of this activity remained unknown. To fill this knowledge gap, we determined a 1.5 [A] resolution structure of a dZ-ssDNA inhibitor bound to active A3G. The crystal structure revealed that the activated dZ/H2O mimics the transition state by coordinating the active site Zn2+ and engaging in additional stabilizing interactions, such as the one with the catalytic residues E259. Therefore, this structure allowed us to capture the first snapshot of the A3s transition state, and suggests that developing transition-state mimicking inhibitors may provide a new opportunity to design more targeted molecules for A3s in the future.

biochemistry↗

bioRxiv2017

Substrate sequence selectivity of APOBEC3A implicates intra-DNA interactions

The APOBEC3 (A3) family of human cytidine deaminases is renowned for providing a first line of defense against many exogenous and endogenous retroviruses. However, the ability of these proteins to deaminate deoxycytidines in ssDNA makes A3s a double-edged sword. When overexpressed, A3s can mutate endogenous genomic DNA resulting in a variety of cancers. Although the sequence context for mutating DNA varies among A3s, the mechanism for substrate sequence specificity is not well understood. To characterize substrate specificity of A3A, a systematic approach was used to quantify the affinity for substrate as a function of sequence context, length, substrate secondary structure, and pH. We identified the A3A ssDNA binding motif as (T/C)TC(A/G), and found that A3A binds RNA in a sequence specific manner. Furthermore, A3A bound tighter to its substrate binding motif when in a loop compared to linear oligonucleotide. Our results suggest that the A3A affinity and preference for substrate is modulated by the structure of DNA, and not just its chemical identity. Analysis of previously published co-crystal structures of A3A bound to ssDNA in light of the above findings directed the proposal of a new model for the molecular mechanism underlying A3A sequence preference. On a broader scale, the results of this work not only provide key insights into the mechanism of A3s beneficial roles in the cell, especially in viral restriction, but also into A3s deleterious activity such as in the development of cancer.

biochemistry↗