High Predicted Binding Affinity Research Articles

Screening from the world's largest TCM database for inhibiting DNA repair protein XRCC4

Human XRCC4 protein is a key component of DNA double-strand break (DSB) repair pathway related to the diseases of stroke and cancer. Cancer cells being treated with the drugs that interfere with DSBs repair mechanism have shown increased radiosensitivity to ionising radiation. Therefore, the development of novel radiosensitiser for radiation therapy becomes important for cancer treatment. We screened from the world's largest traditional Chinese medicine (TCM) database and found potential TCM molecules that can dock at XRCC4 functional site. Among the selected potential TCM compounds, we specifically investigated the top-ranked molecules: glycyrrhizic acid and macedonoside C. The molecular docking and molecular dynamics simulations on these compounds show similar location with high predicted binding affinity. Both compounds form continuous interaction with Lys188 and Arg192 of chain C and Lys187 and Lys190 of chain D. All these protein residues are required to form key hydrophobic interactions with other components participating in DNA repair. We suggested both glycyrrhizic acid and macedonoside C as potential lead compounds for inhibiting XRCC4.

Abstract LB-322: A modified HER2/neu peptide vaccine (2L9V-GP2) demonstrates enhanced CD8+ T cell recognition and stimulation in breast cancer patients at high risk for recurrence

Abstract Introduction: The affinity between tumor-associated antigens and MHC Class I complexes directly affects the degree of CD8+ T cell (CTL) response. GP2 (IISAVVGIL, p654-662) is a HER2/neu-derived peptide, that has poor binding affinity to HLA-A2 (A2) and HLA-A3 (A3). We conducted a preclinical study to determine if amino acid substitutions in the GP2 peptide, predicted to increase binding affinity to A2 and A3, enhance CTL recognition and stimulation. Methods: Based on computer modeling algorithms (BIMAS, SYFPEITHI), two GP2 peptide variants were predicted to have higher binding affinity to A2: SPA-12(2L9V-GP2) and SPA-13(2L5L9V-GP2). Freshly isolated PBMCs were obtained from randomly chosen, disease-free, node positive (NP) and high risk node negative (NN) breast cancer (BCa) patients, who had completed standard adjuvant therapy and are enrolled in the E75 vaccine booster trial. Peptide-specific CD8+ T cells were measured by HLA-A2: IgG dimer (A2 pts) and by interferon-γ ELISPOT (A2, A3 pts) assays. Groups were compared by t-test (dimer) and Mann-Whitney U (ELISPOT) tests. Results: Thirty-five A2 (21 NP, 14 NN) and thirteen A3 (13 NP) samples were included in the study. Among A2 pts, the mean level of SPA-12-specific CTLs was greater than the GP2- (2.8±0.3% vs. 1.7±0.1%, p<0.001) and SPA-13-specific CTLs (2.8±0.3% vs. 1.1±0.1%, p<0.001). The level of SPA-13-specific CTLs was less than GP2-specific CTLs (p<0.001). For both A2 and A3 pts, the median number of IFN- producing cells was greater in response to SPA-12 (144, range 0-799 spots/105 cells) compared to GP2 (88, range 0-432 spots/105 cells, p=0.03) and SPA-13 (0, range 0-344 spots/105 cells, p<0.001). Again, the response to SPA-13 was less than GP2 (p=0.001). In addition, the NP A2 pts demonstrated a more robust CD8+ T cell response to SPA-12 compared to the NNs, both by HLA-A2: IgG dimer (3.2±.5 vs 2.2±0.2, p=0.05) and ELISPOT assay (223, range 0-799 vs 65, range 0-548 p=0.003). Nodal status did not have any significant bearing on the responses to GP2 and SPA-13. Conclusion: The substituted sequences SPA-12 (2L9V-GP2) and SPA-13 (2L5L9V-GP2), both have higher predicted binding affinity to A2 and A3 than GP2. While SPA-12 showed significantly increased recognition and functional CTL responses compared to GP2, SPA-13 demonstrated lower responses. These data suggest that SPA-12 may be a more effective cancer vaccine than GP2, particularly in NP BCa patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-322.

A rationally designed tyrosine hydroxylase DNA vaccine induces specific antineuroblastoma immunity

Therapeutic vaccination against tumor antigens without induction of autoimmunity remains a major challenge in cancer immunotherapy. Here, we show for the first time effective therapeutic vaccination followed by suppression of established spontaneous neuroblastoma metastases using a tyrosine hydroxylase (TH) DNA minigene vaccine. We identified three novel mouse TH (mTH3) derived peptides with high predicted binding affinity to MHC class I antigen H2-K(k) according to the prediction program SYFPEITHI and computer modeling of epitopes into the MHC class I antigen binding groove. Subsequently, a DNA minigene vaccine was generated based on the expression vector pCMV-F3Ub encoding mutated ubiquitin (Gly(76) to Ala(76)) and mTH3. Prophylactic and therapeutic efficacies of this vaccine were established following oral delivery with attenuated Salmonella typhimurium SL7207. Only mice immunized with mTH3 were free of spontaneous liver metastases. This effect was clearly dependent on ubiquitin and high affinity of the mTH epitopes to MHC class I antigens. Specifically, we showed a crucial role for minigene expression as a stable ubiquitin-Ala(76) fusion peptide for vaccine efficacy. The immune response following the mTH3 DNA minigene vaccination was mediated by CD8(+) T cells as indicated by infiltration of primary tumors and TH-specific cytolytic activity in vitro. Importantly, no cell infiltration was detectable in TH-expressing adrenal medulla, indicating the absence of autoimmunity. In summary, we show effective therapeutic vaccination against neuroblastoma with a novel rationally designed TH minigene vaccine without induction of autoimmunity providing an important baseline for future clinical application of this strategy.

Differentiation of AmpC beta-lactamase binders vs. decoys using classification kNN QSAR modeling and application of the QSAR classifier to virtual screening

The use of inaccurate scoring functions in docking algorithms may result in the selection of compounds with high predicted binding affinity that nevertheless are known experimentally not to bind to the target receptor. Such falsely predicted binders have been termed 'binding decoys'. We posed a question as to whether true binders and decoys could be distinguished based only on their structural chemical descriptors using approaches commonly used in ligand based drug design. We have applied the k-Nearest Neighbor (kNN) classification QSAR approach to a dataset of compounds characterized as binders or binding decoys of AmpC beta-lactamase. Models were subjected to rigorous internal and external validation as part of our standard workflow and a special QSAR modeling scheme was employed that took into account the imbalanced ratio of inhibitors to non-binders (1:4) in this dataset. 342 predictive models were obtained with correct classification rate (CCR) for both training and test sets as high as 0.90 or higher. The prediction accuracy was as high as 100% (CCR = 1.00) for the external validation set composed of 10 compounds (5 true binders and 5 decoys) selected randomly from the original dataset. For an additional external set of 50 known non-binders, we have achieved the CCR of 0.87 using very conservative model applicability domain threshold. The validated binary kNN QSAR models were further employed for mining the NCGC AmpC screening dataset (69653 compounds). The consensus prediction of 64 compounds identified as screening hits in the AmpC PubChem assay disagreed with their annotation in PubChem but was in agreement with the results of secondary assays. At the same time, 15 compounds were identified as potential binders contrary to their annotation in PubChem. Five of them were tested experimentally and showed inhibitory activities in millimolar range with the highest binding constant K(i) of 135 microM. Our studies suggest that validated QSAR models could complement structure based docking and scoring approaches in identifying promising hits by virtual screening of molecular libraries.

Identification of potential HIV-1 targets of minocycline

Minocycline, a broad spectrum antibiotic, has been discovered to have inhibitory activity against HIV-1 in vitro, but the targets inhibited are unknown. We used a docking with dynamics protocol developed by us to predict the binding affinities of minocycline against seven active sites of five HIV-1 proteins to putatively identify the potential target(s) of minocycline. The results indicate that minocycline has the highest predicted binding affinity against HIV-1 integrase.

Xenogeneic tyrosine hydroxylase DNA vaccination is effective against neuroblastoma (48.14)

Abstract Neuroblastoma cells are derived from sympathetic neuroblasts and express high amounts of tyrosine hydroxylase (TH), the first step enzyme in catecholamine biosynthesis. We demonstrated that expression profiles of 68 neuroblastoma revealed stable TH gene expression independent of stage and MYCN amplification. Here, we report that a xenogeneic immunization with TH DNA vaccines induces an anti-neuroblastoma immune response in vivo. For this purpose, three DNA vaccines were designed: one encoding for the whole coding sequence of the human TH gene (hTH) (cDNA). The second vaccine was constructed using the epitope prediction program syfpeithi: it comprises of three hTH derived epitopes with high predicted binding affinity to MHC class I with the epitopes separated by an AAY-spacer peptide. In order to amplify the immune response we designed a third DNA vaccine encoding for the hTH gene and single chain (sc)IL-12. The bifunctional characteristics of this construct, antigen expression and IL-12 bioactivity, were demonstrated in vitro. The induction of protective immunity against neuroblastoma was determined in vivo using strain SL7207 of Salmonella typhimurium as an oral vehicle for the DNA vaccines. Interestingly, the hTH cDNA vaccine and the hTH minigene vaccine were equally efficient in inducing T-cell mediated suppression of subcutanous tumor growth and could effectively protect mice from spontaneous liver metastasis. Furthermore, the introduction of scIL-12 did not further amplify the anti-tumor immune response indicating the efficiency of the “danger signal” provided by the bacterial vaccine vehicle. The results indicate that xenogeneic immunization is a promising concept for active immunotherapy in neuroblastoma patients. Supported by DFG (Lo635/2).

Small-molecule-mediated stabilization of familial amyotrophic lateral sclerosis-linked superoxide dismutase mutants against unfolding and aggregation

Familial amyotrophic lateral sclerosis (FALS) is a fatal motor neuron disease that is caused by mutations in the gene encoding superoxide dismutase-type 1 (SOD1). The affected regions of the FALS brain are characterized by aggregated SOD1, and the mutations that destabilize SOD1 appear to promote its aggregation in vitro. Because dissociation of the native SOD1 dimer is required for its in vitro aggregation, we initiated an in silico screening program to find drug-like molecules that would stabilize the SOD1 dimer. A potential binding site for such molecules at the SOD1 dimer interface was identified, and its importance was validated by mutagenesis. About 1.5 million molecules from commercial databases were docked at the dimer interface. Of the 100 molecules with the highest predicted binding affinity, 15 significantly inhibited in vitro aggregation and denaturation of A4V, a FALS-linked variant of SOD1. In the presence of several of these molecules, A4V and other FALS-linked SOD1 mutants such as G93A and G85R behaved similarly to wild-type SOD1, suggesting that these compounds could be leads toward effective therapeutics against FALS.

DNA Minigene Vaccination for Adjuvant Neuroblastoma Therapy

The disruption of self-tolerance against neuroblastoma is the ultimate goal of an effective DNA-vaccine. We demonstrate the induction of protective immunity against syngeneic murine NXS2 neuroblastoma in A/J mice following vaccination with tyrosine hydroxylase (TH)-derived antigens. Oral gene delivery was accomplished using an attenuated strain of Salmonella typhimurium as a carrier harboring vectors encoding for mouse tyrosine hydroxylase (mTH) antigens. Vaccination was effective in protecting animals from a lethal challenge with wild-type NXS2 tumor cells. These findings were extended by comparing efficacy of mTH minigene vaccines with a minigene vaccine comprising three novel epitopes isolated fom NXS2 neuroblastoma cells. For this purpose, MHC class I was immunoprecipitated from NXS2 cell lysates, and peptides were eluted and examined in tandem-mass spectrometry analysis. This led to the identification of three novel natural MHC class I peptide ligands: TEALPVKLI, from ribonucleotide reductase M2; NEYIMSLI, from Ser/Thr protein phosphatase 2A; and FEMVSTLI, of unknown origin. Two minigenes were constructed, one encoding for the three novel epitopes and the second for three known mTH-derived epitopes with high predicted binding affinity to MHC class I, by cloning them into the mammalian expression vector pCMV-3FUB. Immunized mice showed a reduction in primary tumor growth and the absence of spontaneous liver metastasis in the majority of animals. Importantly, there was no significant difference between the two minigenes, suggesting that, compared with tumor peptide isolation, mTH epitope prediction is similarly effective for designing efficient DNA-minigene vaccines. In summary, these findings establish proof of the concept that disruption of self-tolerance against neuroblastoma-associated epitopes may be an effective adjuvant therapeutic strategy.

Vaccination with minigenes encoding for novel ‘self’ antigens are effective in DNA-vaccination against neuroblastoma

The induction of T-cell mediated immunity against neuroblastoma is a challenge due to poor immunogenicity of this malignancy. Here, we demonstrate the induction of protective immunity in a syngeneic murine neuroblastoma model following vaccination with minigenes comprising of three novel natural MHC class I ligands. First, after immunoprecipitation of MHC class I from NXS2 cells, peptides were eluted and examined in tandem-MS analysis which lead to the identification of three novel natural MHC class I peptide ligands, TEALPVKLI from ribonucleotide reductase M2, NEYIMSLI from Ser/Thr protein phosphatase 2A and FEMVSTLI with unknown origin. Second, we constructed two different minigenes, one encoding for the three novel epitopes and the second for three known mTH derived epitopes with high predicted binding affinity to MHC class I by cloning them into the mammalian expression vector pCMV-3FUB. This lead to constructs with an ubiquitin-tag upstream the inserted epitopes in order to facilitate proteasomal degradation. Furthermore the epitopes were separated by a spacer peptide (AAY), which proved to be a preferential proteasome cleavage site. Third, we demonstrate the induction of protective immunity against neuroblastoma using an attenuated strain of Salmonella typhimurium as a carrier harboring pCMV 3FUb vectors encoding for the two minigenes. These findings establish proof of concept that disruption of self tolerance against neuroblastoma associated epitopes may be an effective adjuvant therapeutic strategy.