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Programmed cell death protein 1 (PD‐1), an immune checkpoint receptor expressed by activated T, B, and NK cells, is a well‐known target for cancer immunotherapy. Tislelizumab (BGB‐A317) is an anti‐PD‐1 antibody that has recently been approved for treatment of Hodgkin's lymphoma and urothelial carcinoma. Here, we show that tislelizumab displayed remarkable antitumor efficacy in a B16F10/GM‐CSF mouse model. Structural biology and Surface plasmon resonance (SPR) analyses revealed unique epitopes of tislelizumab, and demonstrated that the CC′ loop of PD‐1, a region considered to be essential for binding to PD‐1 ligand 1 (PD‐L1) but not reported as targeted by other therapeutic antibodies, significantly contributes to the binding of tislelizumab. The binding surface of tislelizumab on PD‐1 overlaps largely with that of the PD‐L1. SPR analysis revealed the extremely slow dissociation rate of tislelizumab from PD‐1. Both structural and functional analyses align with the observed ability of tislelizumab to completely block PD‐1/PD‐L1 interaction, broadening our understanding of the mechanism of action of anti‐PD‐1 antibodies., We evaluated the antitumor efficacy of tislelizumab (BGB‐A317), an approved anti‐PD‐1 antibody. Its critical epitopes were investigated by both structural biology and SPR studies, and the CC′ loop of PD‐1 was discovered to be a novel targetable region for anti‐PD‐1 antibodies. The unique epitopes and slow dissociation rate of tislelizumab contribute to its complete blocking activity to PD‐L1.

Hematopoietic progenitor kinase 1 (HPK1), a hematopoietic cell-restricted serine/threonine protein kinase, has been reported to serve as a critical negative feedback regulator of T lymphocytes by phosphorylating the adaptor protein SLP76 in the TCR complex. The negative feedback role of HPK1 in TCR signaling makes it a promising target for immuno-oncology therapy. It has been recently demonstrated that the kinase activity of HPK1 is essential for its antitumor activity, and HPK1 inhibitors can be potentially combined with immune checkpoint inhibitor therapy (such as PD-1 antibody) for effective cancer treatment. Here, we report a potent and selective HPK1 inhibitor BGB-15025 which has been identified through in-house high throughput screening and structure-guided drug design. BGB-15025 potently inhibits HPK1 kinase activity in biochemical assay, with a 50% inhibitory concentration (IC50) of 1.04 nM under Km concentration of ATP. Consistently, BGB-15025 can potently reduce SLP76 phosphorylation and increase downstream ERK phosphorylation in a concentration dependent manner in T cells-based assay. As a consequence, BGB-15025 induces TCR activation and IL-2 production in T cells. Based on in-house profiling, BGB-15025 shows good selectivity profile among MAP4K family and does not affect ZAP70 phosphorylation up to 1 μM. Oral administration of BGB-15025 demonstrates dose-dependent pSLP76 inhibition in splenic T cells and induces serum IL-2 in mouse model. In efficacy studies, BGB-15025 exhibits anti-tumor activity in GL261 tumor model as single agent. Moreover, BGB-15025 demonstrated combination effect with anti-PD-1 antibody in CT26 and EMT-6 syngeneic tumor models. BGB-15025 is currently in phase I clinical trial to treat patients with advanced solid tumor, both as a single agent and in combination with anti PD-1 antibody tislelizumab. Citation Format: Ye Liu, Jing Li, Zhuo Li, Qin Wang, Xing Zhou, Xiaoxin Liu, Bo Zhang, Xitao Wang, Sanjia Xu, Hanzi Sun, Xiaomin Song, Xi Yuan, Zhiwei Wang, Xuesong Liu. BGB-15025, a potent and selective HPK1 inhibitor, is efficacious as a single agent or in combination with PD-1 antibody in multiple tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5541.

Background OX40 is a member of the tumor necrosis factor receptor super family (TNFRSF) primarily expressed on activated CD4+ and CD8+ T cells, as well as natural killer (NK) T and NK cells. It is an immune costimulatory receptor which binds to its ligand OX40L and activates downstream NF-κB pathway to induce immune cell activation, proliferation, and survival.1–3 Current agonistic anti-OX40 antibodies in clinic, which are mostly ligand-competitive antibodies, showed limited clinical responses, mainly at lower doses. Blockade of OX40-OX40L interaction might limit the efficacy of these ligand-competitive antibodies at higher doses, as OX40-OX40L interaction is essential for enhancing effective anti-tumor immunity. Here we report pre-clinical data of BGB-A445, which is a ligand non-blocking agonistic anti-OX40 humanized antibody. Methods Cell-based flow cytometry assay was established to determine whether BGB-A445 interferes with OX40-OX40L interaction. Co-crystal structure of OX40/BGB-A445 Fab was solved to study the molecular binding mechanism. A mixed lymphocyte reaction (MLR) assay was set up to investigate the ability of BGB-A445 to activate CD4+ T-cells. The anti-tumor efficacy of BGB-A445 was evaluated in MC38 colon cancer and CT26WT colon cancer models either as a single agent or in combination with anti-PD-1 antibody. Results The flow cytometry study showed that BGB-A445 did not interfere with the binding of OX40 to OX40L even at high concentrations. In contrast, MOXR0916, an anti-OX40 agonistic antibody developed by Genentech, completely blocked OX40 binding to OX40L. Additionally, the co-crystal structure of OX40/BGB-A445 Fab complex indicated that BGB-A445 interacts with the CRD4 region of OX40 which is distant from OX40L binding region. In the MLR assay, combined with an anti-PD-1 antibody, BGB-A445 co-stimulated CD4+ T-cells to secrete IL-2 dose-dependently, while MOXR0916 did not. In the MC38 colon cancer model in human OX40 knock-in mice, BGB-A445 demonstrated remarkable anti-tumor efficacy in a dose-dependent manner, while MOXR0916 showed a ‘hook effect’ in the same setting. In addition, BGB-A445 exhibited significant anti-tumor activity in the PAN02 pancreatic model which is resistant to anti-PD-1 treatment. Besides, BGB-A445 revealed significant combination effects with anti-PD-1 therapy in both MC38 and CT26WT models. Conclusions In conclusion, differentiated from current clinical stage anti-OX40 antibodies, BGB-A445 is an agonistic antibody that does not block the OX40-OX40L interaction. Both in vitro and in vivo results demonstrated that BGB-A445 has remarkable immune stimulating effect and anti-tumor efficacy either as a single agent or in combination with anti-PD-1 therapy, thus warranting further clinical investigation. References Croft M. Control of immunity by the TNFR-related molecule OX40 (CD134). Annu Rev Immunol 2010;28:57–78. Gramaglia I, et al. Ox-40 ligand: a potent costimulatory molecule for sustaining primary CD4 T cell responses. J Immunol 1998;161:6510–6517. Song J, So T, Croft M. Activation of NF-kappaB1 by OX40 contributes to antigen-driven T cell expansion and survival. J Immunol 2008;180:7240–7248.

Novel “pairs” of drugs possessing pharmacological synergies could be encapsulated into polymeric micelles and exert superb therapeutic effects in vivo upon intravenous administration, with the prerequisite that the micelles remain stable. NADP(H) quinone oxidoreductase 1 (NQO1) inhibitors, such as β-lapachone (LPC) and tanshinone IIA (THA), are structurally and pharmacologically similar molecules that are poorly water-soluble, crystallize extremely fast, and demonstrate synergistic anticancer effect when used together with paclitaxel (PTX). However, when coencapsulated with PTX in poly(ethylene glycol)-b-poly(d,l-lactic acid) (PEG-PLA) micelles, only PTX/LPC but not the PTX/THA pair yields satisfactory colloidal stability. To reveal the molecular mechanism contributing to the colloidal stability of the coencapsulated micelles, we investigated the molecular interactions of PTX/LPC and PTX/THA, through both experimental methods (crystallization kinetics, 13C NMR) and molecular dynamic simulation. We observe...

Aberrant activation of Bruton's tyrosine kinase (BTK) plays an important role in pathogenesis of B-cell lymphomas, suggesting that inhibition of BTK is useful in the treatment of hematological malignancies. The discovery of a more selective on-target covalent BTK inhibitor is of high value. Herein, we disclose the discovery and preclinical characterization of a potent, selective, and irreversible BTK inhibitor as our clinical candidate by using in vitro potency, selectivity, pharmacokinetics (PK), and in vivo pharmacodynamic for prioritizing compounds. Compound BGB-3111 (31a, Zanubrutinib) demonstrates (i) potent activity against BTK and excellent selectivity over other TEC, EGFR and Src family kinases, (ii) desirable ADME, excellent in vivo pharmacodynamic in mice and efficacy in OCI-LY10 xenograft models.

Binding-site water molecules play a crucial role in protein-ligand recognition, either being displaced upon ligand binding or forming water bridges to stabilize the complex. However, rigorously treating explicit binding-site waters is challenging in molecular docking, which requires to fully sample ensembles of waters and to consider the free energy cost of replacing waters. Here, we describe a method to incorporate structural and energetic properties of binding-site waters into molecular docking. We first developed a solvent property analysis (SPA) program to compute the replacement free energies of binding-site water molecules by post-processing molecular dynamics trajectories obtained from ligand-free protein structure simulation in explicit water. Next, we implemented a distance-dependent scoring term into DOCK scoring function to take account of the water replacement free energy cost upon ligand binding. We assessed this approach in protein targets containing important binding-site waters, and we demonstrated that our approach is reliable in reproducing the crystal binding geometries of protein-ligand-water complexes, as well as moderately improving the ligand docking enrichment performance. In addition, SPA program (free available to academic users upon request) may be applied in identifying hot-spot binding-site residues and structure-based lead optimization.

One of the most common secondary resistance mechanisms to ibrutinib (IBR, a first-generation, irreversible Bruton's tyrosine kinase [BTK] inhibitor) in CLL is the development of mutations in BTK involving Cys481, leading to impaired drug binding (Woyach et al, NEJM 2014; Furman et al, NEJM 2014). The mechanisms of resistance to second generation BTK inhibitors are currently unknown. We aimed to assess the spectrum of acquired BTK mutations in patients with CLL progression on zanubrutinib (ZANU), a second-generation, irreversible inhibitor of BTK. We identified 38 CLL patients, treated with ZANU on clinical trials (NCT02343120, NCT02569476, NCT03336333, NCT02795182) at three centres, for whom serial samples were available. Four of 38 patients (10.5%) had CLL progression on ZANU (time to progression 5, 26, 29 and 48 months) and underwent amplicon next generation sequencing (NGS) of BTK (exon 11, 15, 16) and PLCG2 (exon 16, 19-20, 24, 27-28). Remarkably, we detected a BTK kinase domain mutation, BTK Leu528Trp (NM_000061.2:c.1583T>G), in all four patients progressing on ZANU. In addition, all four patients had detectable Cys481 mutations at lower variant allele frequency (VAF) than the BTK Leu528Trp (median BTK Leu528Trp 34.9% vs BTK Cys481 9.1%). Analysis of sequence reads from amplicon NGS and RNA-sequencing data demonstrated that BTK Leu528Trp and BTK Cys481 mutations were present on different alleles. Assessment of the BTK Leu528Trp and BTK Cys481 mutations with high sensitivity droplet digital PCR (ddPCR) confirmed the absence of both mutations prior to ZANU exposure in all patients (sensitivity 0.1% VAF). Longitudinal analysis of the four patients with the BTK Leu528Trp mutation demonstrated the appearance of the Leu528Trp coincident with rising measurable disease and subsequent clinical CLL progression. We then went on to test patients on ZANU without disease progression but with persistent measurable disease (n=34) by ddPCR and detected three further patients harbouring low level BTK Leu528Trp mutations (VAF The BTK Leu528Trp mutation has been described only once previously in a patient in the context of IBR resistance (who transformed with Richter's syndrome) where it co-occurred with Cys481 mutations (Maddocks et al, JAMA Oncol 2015). As the prevalence of BTK Leu528Trp among progressive disease samples in our cohort exceeds all prior reports in IBR-treated patients, we sought to further understand the specificity of BTK Leu528Trp for ZANU progression. Targeted sequencing in a cohort of 49 patients progressing on IBR from the European Research Initiative on CLL (ERIC) did not detect the BTK Leu528Trp in any patients (sensitivity 1% VAF). We went on to perform biochemical and cellular studies on the BTK Leu528Trp mutation. Assessment of enzymatic activity of BTKLeu528Trp demonstrated a significant loss of activity compared to both BTKWT and BTKCys481Ser. This was further confirmed by assessing BTK autophosphorylation in HEK293 cells. Autophosphorylation at BTK Tyr223 was markedly reduced in HEK293 cells stably expressing BTKLeu528Trp compared to both BTKCys481Ser and BTKWT. In addition, a crystal structure of apo-BTKLeu528Trp was solved to understand effects of BTKLeu528Trp on ZANU binding to BTK. The alignment of the crystal structure of apo-BTKLeu528Trp with that of BTKWT-ZANU or the modeled structure of BTK-ATP suggested potential steric clashes between BTKLeu528Trp and ZANU (Figure 1A), as well as BTKLeu528Trp and ATP (Figure 1B). In conclusion, we have described the novel enrichment of BTK Leu528Trp mutations occurring in patients with CLL progressing on ZANU and both structural and experimental data consistent with this mutation resulting in interference with both ATP and ZANU binding to BTK. These findings emphasize the potential for agent-specific resistance mutations with second generation BTK inhibitors and the need to include these mutations in diagnostic screening for BTK resistance in the clinic. SH/CPST co-first authors, CT/PB co-senior authors Disclosures Handunnetti: Abbvie: Other: Travel Grant; Gilead: Honoraria. Zhou:Beigene: Employment. Sun:Beigene: Employment. Xing:Beigene: Employment. Zhang:Beigene: Employment. Guo:Beigene: Employment. Sutton:Abbvie: Honoraria; Gilead: Honoraria; Janssen: Honoraria. Ghia:Dynamo: Consultancy, Honoraria; ArQule: Consultancy, Honoraria; BeiGene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; Acerta/AstraZeneca: Consultancy, Honoraria; Pharmacyclics LLC, an AbbVie Company: Consultancy; Novartis: Research Funding; Juno/Celgene: Consultancy, Honoraria. Scarfo:AstraZeneca: Honoraria; Janssen: Honoraria; AbbVie: Honoraria. Seymour:Takeda: Consultancy; Acerta: Consultancy; Celgene: Consultancy, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Research Funding; Roche: Consultancy, Research Funding, Speakers Bureau. Anderson:Walter and Eliza Hall Institute: Employment, Patents & Royalties: Institute receives royalties for venetoclax, and I receive a fraction of these.. Roberts:AbbVie: Other: Unremunerated speaker for AbbVie, Research Funding; Australasian Leukaemia and Lymphoma Group: Membership on an entity's Board of Directors or advisory committees; Walter and Eliza Hall Institute: Patents & Royalties: Institute receives royalties for venetoclax, and I receive a fraction of these.; Janssen: Research Funding; BeiGene: Research Funding. Huang:Genentech: Patents & Royalties: DCSH is an employee of the Walter and Eliza Hall Institute which receives milestone and royalty payments related to venetoclax. Liu:Beigene: Employment. Cheah:Roche, Janssen, MSD, Gilead, Loxo Oncology, Acerta, BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene, Roche, Abbvie: Research Funding; Roche: Other: Travel expenses. Tam:Janssen: Honoraria, Research Funding; BeiGene: Honoraria; Pharmacyclics LLC, an AbbVie company: Honoraria; Roche: Honoraria; Novartis: Honoraria; AbbVie: Honoraria, Research Funding. Blombery:Janssen: Honoraria; Invivoscribe: Honoraria; Novartis: Consultancy.

Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor expressed by activated T, B, and NK cells, which interacts with its ligand PD-L1/L2 to inhibit T-cell proliferation and effector functions such as tumor cell killing and cytokine production. Two anti-PD-1 antibodies approved by the FDA, pembrolizumab and nivolumab, have shown efficacy in many cancer types, nevertheless there are some indications where limited efficacy is observed. Tislelizumab (BGB-A317), an investigational anti-PD-1 antibody, has demonstrated significant clinical activity (85.7% ORR, including 61.4% CR) in relapsed/refractory classical Hodgkin’s lymphoma (R/R cHL). Additionally, tislelizumab is being studied in global pivotal trials in a number of malignancies, including non-small cell lung cancer, hepatocellular carcinoma, and esophageal squamous cell carcinoma. However, how tislelizumab binds to PD-1 has yet to be shown, particularly in comparison to pembrolizumab and nivolumab. Here we report the co-crystal structure of PD-1 extracellular domain and the Fab of tislelizumab. Tislelizumab interacts with IgV-like domain of PD-1 with an interface area of 1112 Å2. Structure-guided mutagenesis of PD-1 and surface plasmon resonance were performed to compare the binding of tislelizumab, pembrolizumab and nivolumab to mutant and wild type PD-1. The dissociation rate (kd) of tislelizumab from wild type PD-1 is about 100-fold and 50-fold slower than that of pembrolizumab and nivolumab, respectively. Gln75, Thr76, Asp77 and Arg86 on PD-1 are critical epitopes for tislelizumab, but mutation of them showed little effect on binding of PD-1 to pembrolizumab and nivolumab. Both the co-crystal structure and mutagenesis study identified the unique epitopes of tislelizumab that correlate to the extremely slow-off property of tislelizumab after binding to PD-1. In conclusion, we observed that tislelizumab is differentiated from pembrolizumab and nivolumab by its unique binding epitopes as well as binding kinetics. Citation Format: Yingcai Feng, Yuan Hong, Hanzi Sun, Bo Zhang, Hongfu Wu, Kang Li, Xuesong (Mike) Liu, Ye Liu. The molecular binding mechanism of tislelizumab, an investigational anti-PD-1 antibody, is differentiated from pembrolizumab and nivolumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2383.

Inflammatory caspases cleave the gasdermin D (GSDMD) protein to trigger pyroptosis, a lytic form of cell death that is crucial for immune defences and diseases. GSDMD contains a functionally important gasdermin-N domain that is shared in the gasdermin family. The functional mechanism of action of gasdermin proteins is unknown. Here we show that the gasdermin-N domains of the gasdermin proteins GSDMD, GSDMA3 and GSDMA can bind membrane lipids, phosphoinositides and cardiolipin, and exhibit membrane-disrupting cytotoxicity in mammalian cells and artificially transformed bacteria. Gasdermin-N moved to the plasma membrane during pyroptosis. Purified gasdermin-N efficiently lysed phosphoinositide/cardiolipin-containing liposomes and formed pores on membranes made of artificial or natural phospholipid mixtures. Most gasdermin pores had an inner diameter of 10–14 nm and contained 16 symmetric protomers. The crystal structure of GSDMA3 showed an autoinhibited two-domain architecture that is conserved in the gasdermin family. Structure-guided mutagenesis demonstrated that the liposome-leakage and pore-forming activities of the gasdermin-N domain are required for pyroptosis. These findings reveal the mechanism for pyroptosis and provide insights into the roles of the gasdermin family in necrosis, immunity and diseases.

Distal arthrogryposis type 2B (DA2B) is an important genetic disorder in humans. However, the mechanisms governing this disease are not clearly understood. In this study, we generated knock-in mice carrying a DA2B mutation (K175del) in troponin I type 2 (skeletal, fast) (TNNI2), which encodes a fast-twitch skeletal muscle protein. Tnni2K175del mice (referred to as DA2B mice) showed typical DA2B phenotypes, including limb abnormality and small body size. However, the current knowledge concerning TNNI2 could not explain the small body phenotype of DA2B mice. We found that Tnni2 was expressed in the osteoblasts and chondrocytes of long bone growth plates. Expression profile analysis using radii and ulnae demonstrated that Hif3a expression was significantly increased in the Tnni2K175del mice. Chromatin immunoprecipitation assays indicated that both wild-type and mutant tnni2 protein can bind to the Hif3a promoter using mouse primary osteoblasts. Moreover, we showed that the mutant tnni2 protein had a higher capacity to transactivate Hif3a than the wild-type protein. The increased amount of hif3a resulted in impairment of angiogenesis, delay in endochondral ossification, and decrease in chondrocyte differentiation and osteoblast proliferation, suggesting that hif3a counteracted hif1a-induced Vegf expression in DA2B mice. Together, our data indicated that Tnni2K175del mutation led to abnormally increased hif3a and decreased vegf in bone, which explain, at least in part, the small body size of Tnni2K175del mice. Furthermore, our findings revealed a new function of tnni2 in the regulation of bone development, and the study of gain-of-function mutation in Tnni2 in transgenic mice opens a new avenue to understand the pathological mechanism of human DA2B disorder., Author Summary Distal arthrogryposis type 2B (DA2B) is an autosomal dominant genetic disorder. The typical clinical features of DA2B include hand and/or foot contracture and shortness of stature in patients. To date, mutations in TNNI2 can explain approximately 20% of familial incidences of DA2B. TNNI2 encodes a subunit of the Tn complex, which is required for calcium-dependent fast twitch muscle fiber contraction. In the absence of Ca2+ ions, TNNI2 impedes sarcomere contraction. Here, we reported a knock-in mouse carrying a DA2B mutation TNNI2 (K175del) had typical limb abnormality and small body size that observed in human DA2B. However, the small body did not seem to be convincingly explained using the present knowledge of TNNI2 associated skeletal muscle contraction. Our findings showed that the Tnni2K175del mutation impaired bone development of Tnni2K175del mice. Our data further showed that the mutant tnni2 protein had a higher capacity to transactivate Hif3a than the wild-type protein and led to a reduction in Vegf expression in bone of DA2B mice. Taken together, our findings demonstrated that the disease-associated Tnni2K175del mutation caused bone defects, which accounted for, at least in part, the small body size of Tnni2K175del mice. Our data also suggested, for the first time, a novel role of tnni2 in the regulation of bone development of mice by affecting Hif-vegf signaling.

A better understanding of the interaction between extrinsic factors and surface receptors on stem cells will greatly benefit stem cell research and applications. Recently, we showed that several angiopoietin-like proteins (Angptls) bind and activate the immune inhibitory receptor human leukocyte immunoglobulin (Ig)-like receptor B2 (LILRB2) to support ex vivo expansion of hematopoietic stem cells (HSCs) and leukemia development. However, the molecular basis for the interaction between Angptls and LILRB2 was unclear. Here, we demonstrate that Angptl2 expressed in mammalian cells forms high-molecular-weight species and that ligand multimerization is required for activation of LILRB2 for downstream signaling. A novel motif in the first and fourth Ig domains of LILRB2 was identified that is necessary for the receptor to be bound and activated by Angptl2. The binding of Angptl2 to LILRB2 is more potent than and not completely overlapped with the binding of another ligand, HLA-G. Immobilized anti-LILRB2 antibodies induce a more potent activation of LILRB2 than Angptl2, and we developed a serum-free culture containing defined cytokines and immobilized anti-LILRB2 that supports a net expansion of repopulating human cord blood HSCs. Our elucidation of the mode of Angptl binding to LILRB2 enabled the development of a new approach for ex vivo expansion of human HSCs.

Binding-site water molecules play a crucial role in protein-ligand recognition, either being displaced upon ligand binding or forming water bridges to stabilize the complex. However, rigorously treating explicit binding-site waters is challenging in molecular docking, which requires to fully sample ensembles of waters and to consider the free energy cost of replacing waters. Here, we describe a method to incorporate structural and energetic properties of binding-site waters into molecular docking. We first developed a solvent property analysis (SPA) program to compute the replacement free energies of binding-site water molecules by post-processing molecular dynamics trajectories obtained from ligand-free protein structure simulation in explicit water. Next, we implemented a distance-dependent scoring term into DOCK scoring function to take account of the water replacement free energy cost upon ligand binding. We assessed this approach in protein targets containing important binding-site waters, and we demonstrated that our approach is reliable in reproducing the crystal binding geometries of protein-ligand-water complexes, as well as moderately improving the ligand docking enrichment performance. In addition, SPA program (free available to academic users upon request) may be applied in identifying hot-spot binding-site residues and structure-based lead optimization.

The introduction of selenium into DNA in the place of oxygen provides a unique opportunity for studying the fidelity of DNA replication, as well as providing advantages in the growth of DNA crystals and the greater resolution of their structures. However, the atomic mechanisms of the relative stability and base pair recognition of the selenium-modified DNA are poorly understood. In the present study, quantum mechanics calculations were performed on base pairings, base stacking, and base-water interactions for both unmodified thymine and thymine with the 2-exo-oxygen replaced with selenium, and the results were used to develop and validate CHARMM force field parameters for the 2-Se-thymine. Subsequently, molecular dynamics simulations and free-energy perturbation calculations were performed on 11-base DNA sequences containing native thymine and the 2-Se-thymine. The calculated relative free-energy values are in good agreement with experimentally determined relative stability, where the 2-Se-thymine offers similar stability to T-A in cognate DNA, while it dramatically destabilizes the DNA containing the T-G mismatch base pair when 2-Se-thymine is incorporated. Thus, 2-Se-thymine largely increases the native T-A base pair fidelity by discouraging the T-G wobble pair. Insights into the high pairing specificity and the relative stability of selenium-modified DNA were obtained based on detailed structural and energetic analysis of molecular dynamics trajectories. Our studies move one step further toward an understanding of the high base pair fidelity and thermodynamic properties of Se-DNA in solution and in protein-DNA complexes.