Overexpression of CD73-F417V/F500V mutant in CD73 null-HEKs indicated stable surface expression but complete loss of Ecto-nucleotidase activity, thus providing a tool to dissect activity-dependent and independent functions of NT5E.
Abstract
NT5E, a 5' Ectonucleotidase, is an emerging hotspot for cancer immunotherapies. However, available enzymatic inhibitors have shown limited promise, indicating activity-independent roles. Delineating activity-dependent and independent roles of this protein with precision requisites identification of critical residues impacting activity without hampering surface expression/stability. Conserved Phenylalanine (F) at 417 and 500 positions, which form a π stack with substrate-AMP, was chosen as the region of interest for in silico mutagenesis. F417 and F500 were mutated to Valine (V) individually using Missense3D to obviate broad structural damage. CD73-F417V/F500V dual mutant was then subjected to Molecular Dynamics simulations for prediction of stability and structural variations in the protein over time in comparison to wild-type crystal structure. Gibbs Free energy indicated a significant decrease in ligand binding affinity of CD73-F417V/F500V. RMSD and RMSF analysis of MD trajectories indicated increased mobility, suggesting weaker ligand binding affinity within the catalytic site of the mutant potentially impacting functional dynamics and protein-ligand contacts. Overexpression of CD73-F417V/F500V mutant in CD73 null-HEKs indicated stable surface expression but complete loss of Ecto-nucleotidase activity, thus providing a tool to dissect activity-dependent and independent functions of NT5E.
The Cluster of Differentiation 47 (CD47)–signal regulatory protein alpha (SIRPα) immune checkpoint is a key regulator of tumor immune evasion and a promising target in cancer immunotherapy. To overcome the limitations of monoclonal antibodies, this study aimed to identify high-affinity nucleic acid aptamers targeting CD47. A systematic single-nucleotide mutagenesis workflow was performed on a known CD47-binding DNA aptamer to generate 270 single-point variants, enabling unbiased evaluation of each nucleotide position. The variants were first screened for structural stability, yielding 81 structurally stable candidates. These candidates were then subjected to molecular docking against CD47, and 45 variants showed improved docking scores compared with the native aptamer. The docking score improved from −226.07 for the native aptamer to −301.94 for the best-performing variant. Based on structural stability and docking performance, the ten top-ranked candidates were selected for molecular dynamics simulations. These variants exhibited improved conformational stability, as reflected by lower root-mean-square deviation (RMSD) and root-mean-square fluctuation (RMSF) values and increased hydrogen bonding, with Seq198 and Seq262 showing the most stable profiles. Binding free energy calculations confirmed improved affinity. The native aptamer exhibited a ΔTOTAL of −97.16 kcal/mol, whereas Seq198 (−173.13 kcal/mol), Seq244 (−152.35 kcal/mol), and Seq112 (−147.19 kcal/mol) showed markedly stronger binding. Seq198 emerged as the most promising candidate. These findings demonstrate that systematic computational mutagenesis is an effective strategy for optimizing aptamer performance and identifying high-affinity CD47-targeting candidates.
Sumeyye Altunok, Emre Can Buluz, Ehed Muhammed Aymaz· Journal of the Turkish Chemi...· 0 citations
Human alkyladenine DNA glycosylase (AAG) initiates base excision repair of various alkylated and deaminated purines. Single nucleotide polymorphisms (SNPs) in the AAG gene occur frequently in populations and tumors, but the functional impact of most variants remains unknown. Previously, we identified three SNPs—P94L, V158M, and E293K—which have been predicted to have a high deleterious potential. This study aimed to characterize their biochemical properties and structural consequences. Using a combination of biochemical assays and molecular dynamics simulations, we assessed the thermal stability, DNA binding affinity, and catalytic activity of these mutants on two structurally distinct substrates: 1, N6-ethenoadenosine (εA) and hypoxanthine (Hx). All three mutants exhibited reduced melting temperatures, indicating pronounced destabilization. Despite this, their DNA-binding affinities remained close to WT AAG. Strikingly, the mutants displayed differential loss of catalytic activity: P94L was inactive against both εA and Hx; V158M retained activity against εA but lost activity against Hx; and E293K was active against Hx but inactive against εA. MD simulations revealed that P94L alters the flexible R138–T143 loop, V158M narrows the active site cleft, and E293K disrupts a C-terminal salt bridge while increasing DNA engagement by the positively charged tail. These findings demonstrate that non-active-site SNPs can qualitatively reprogram the substrate specificity of AAG. These variants could be considered as potential functional biomarkers for cancer risk and response to alkylating chemotherapy.
O. A. Kladova, T. E. Tyugashev, A. S. Bakman et al.· International Journal of Mol...· 0 citations
The interaction between c-Myb and the CBP/p300 KIX domain is a critical transcriptional regulatory event and an attractive target for the development of candidate disruptors of the recombinant c-Myb-KIX interaction. In this study, we used an integrated computational and experimental strategy to identify new small molecules capable of disrupting this protein-protein interaction. A focused Umbelliferyl phosphate scaffold library was subjected to stepwise virtual screening via drug-likeness assessment and docking to the c-Myb-binding region of the KIX domain and short molecular dynamics refinement. Selected compounds were then evaluated by 500 ns molecular dynamics simulations, MM/PBSA analysis, free energy landscape (FEL) mapping, and finally by microscale thermophoresis (MST) assay. Computational analyses showed that stable ligand binding did not necessarily translate into disruption of the c-Myb-KIX interface, allowing separation of compounds that stabilized the complex from those predicted to weaken it. Consistent with this distinction, ΔΔGPPIanalysis identified only MUP and Naphthol AS-BI phosphate as protein-protein interaction-weakening ligands, with Naphthol AS-BI phosphate showing the strongest predicted disruptive effect (ΔΔGPPI=+3.25 kcal/mol), whereas DiFMUP and Naphthol AS-D phosphate were predicted to stabilize the complex. Among the tested molecules, Naphthol AS-BI phosphate showed the clearest disruption-like behavior in silico and was the most potent inhibitor in vitro, with an IC₅₀ of 18.9 ± 0.6 μM. Importantly, MUP emerged as the most promising umbelliferyl phosphate-derived hit, displaying measurable inhibitory activity (IC₅₀ = 33.5 ± 0.3 μM) comparable to the reference Naphthol AS-E phosphate (IC₅₀ = 31.2 ± 1.3 μM) and a more favorable predicted ADMET profile. Overall, this work identifies new chemical starting points for targeting the c-Myb-CBP/p300 KIX interaction and supports MUP as an attractive scaffold for further optimization.
Emadeldin M. Kamel, H. Rudayni, A. A. Allam et al.· Biophysical Chemistry· 0 citations
HDAC6-selective hydroxamate inhibitors, including ricolinostat and citarinostat, have advanced clinically in multiple myeloma and other malignancies, yet resistance mechanisms remain structurally unresolved. We hypothesized that evolutionarily tolerated mutations identified by a protein language model selectively perturb inhibitor binding through access to alternative conformational states. Per-position log-likelihoods from the ESM2 protein language model across the HDAC6 CD2 domain nominated ten candidate resistance mutations. Each mutant and the wild-type tandem was simulated in complex with five hydroxamate inhibitors across three docking-derived poses (159 × 100-ns all-atom MD trajectories; GROMACS; ff14SB/GAFF2; bonded Zn2+). Paired within-system analysis of matched receptor–ligand conditions isolated state-dependent effects. We identified a kinetically stable alternative bound state in which the catalytic histidine H651 is displaced from canonical Zn2+ coordination. The H651 ND1–Zn2+ distance is bimodal (2.16–2.56 Å vs. 2.83–3.34 Å) with no interconversion on the 100-ns timescale, indicating a high barrier and early pose-dependent state selection. The displaced state involves primarily whole-residue backbone translation (0.6–1.8 Å Cα shift) with partial imidazole counter-rotation, not a simple rotamer flip. It is accessed by both active-site-proximal and distal mutations up to 23 Å from H651, demonstrating long-range structural coupling and indicating that mutations shift access probability rather than state identity. The displaced conformation consistently rewires protein–ligand interactions, replacing canonical active-site hydrogen bonds with alternative contacts in the L1 and L7 loops. Critically, bulky-cap hydroxamates (cay10603, citarinostat, ricolinostat) preferentially occupy the displaced state (11 of 12 trajectories), whereas smaller-cap inhibitors largely do not, implicating cap geometry as a determinant of conformational basin selection. These findings reveal a cryptic, mutation-accessible conformational state of HDAC6 that fundamentally alters inhibitor binding. Resistance emerges not from discrete structural disruptions but from shifts in the probability of accessing alternative binding geometries. This mechanism challenges single-structure drug design paradigms and suggests that HDAC6 inhibitor optimization—and resistance prediction—must explicitly account for multiple bound states. The integrated language-model/MD framework provides a general strategy for identifying resistance-relevant conformational landscapes in Zn2+-dependent oncology targets. AI Disclosure: Generative AI assisted in drafting and editing the text of this abstract.
Paul Fong, Fan Yuan. A cryptic alternative bound state of HDAC6 drives mutation- and ligand-dependent remodeling of inhibitor interaction [abstract]. In: Proceedings of AACR Drug Discovery and Development (AACR D3) Conference; 2026 Jul 21-24; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(14_Suppl):Abstract nr B088.
Paula Fong, Fan Yuan· Clinical Cancer Research· 0 citations
The mechanisms of nuclease activation are explored by solving seven ternary cryo-electron mi-croscopy structures of wild-type Cas13d in complex with matched and mismatched targets and an active site loop in the HEPN domains that regulates substrate accessibility is identified.
Chia-Wei Chou, Selma Sinan, Hung-Che Kuo et al.· bioRxiv· 0 citations
The restoration of mutant p53 stability is a highly sought-after strategy in targeted cancer therapy. This study presents a structure-based virtual screening and molecular dynamics approach to nominate FDA-approved drugs as candidate stabilizers of mutant p53 for downstream experimental validation. A virtual screening library of FDA-approved compounds was docked against three representative p53 mutants (7DHY, 7DHZ, and 7V97) to evaluate their binding potential. The prioritized candidates demonstrated consistent, multi-conformer binding affinities. Protein–ligand interaction profiling revealed that the candidate DB09280 possesses a highly dense interaction network, particularly against the V272M and R249S variants. Residue-level analysis of the G245S structural mutant showed that DB09280 uniquely engages His19, a crucial residue for zinc coordination, and forms stabilizing contacts with adjacent flexible loop residues, including ASN35 and PRO32. Subsequent 500 ns molecular dynamics simulations were consistent with DB09280 acting as a putative conformational clamp on the timescale sampled. The ligand-bound (holo) system exhibited substantially reduced global structural drift (RMSD) and attenuated local residue fluctuation (RMSF) within the core domain compared to the highly unstable apo state. Principal component analysis further indicated that DB09280 restricts the broad conformational sampling of the mutant into a stable, dominant basin within the sampled trajectory. Together, these computational findings nominate DB09280 as a promising candidate structural stabilizer of mutant p53 worthy of experimental follow-up. We emphasize that the in silico stabilization observed here is not equivalent to functional rescue of p53 transcriptional activity; biochemical, biophysical, and cell-based assays will be required to establish whether DB09280 restores wild-type-like DNA binding or tumor-suppressor function in mutant p53 contexts.
Mena Abdelsayed, Y. Boulaamane· International Journal of Mol...· 0 citations