Specific increase in potency via structure-based design of a TCR.

Adoptive immunotherapy with Ag-specific T lymphocytes is a powerful strategy for cancer treatment. However, most tumor Ags are nonreactive "self" proteins, which presents an immunotherapy design challenge. Recent studies have shown that tumor-specific TCRs can be transduced into normal PBLs, which persist after transfer in ∼30% of patients and effectively destroy tumor cells in vivo. Although encouraging, the limited clinical responses underscore the need for enrichment of T cells with desirable antitumor capabilities prior to patient transfer. In this study, we used structure-based design to predict point mutations of a TCR (DMF5) that enhance its binding affinity for an agonist tumor Ag-MHC (peptide-MHC [pMHC]), Mart-1 (27L)-HLA-A2, which elicits full T cell activation to trigger immune responses. We analyzed the effects of selected TCR point mutations on T cell activation potency and analyzed cross-reactivity with related Ags. Our results showed that the mutated TCRs had improved T cell activation potency while retaining a high degree of specificity. Such affinity-optimized TCRs have demonstrated to be very specific for Mart-1 (27L), the epitope for which they were structurally designed. Although of somewhat limited clinical relevance, these studies open the possibility for future structural-based studies that could potentially be used in adoptive immunotherapy to treat melanoma while avoiding adverse autoimmunity-derived effects.

Recovering protein-protein and domain-domain interactions from aggregation of IP-MS proteomics of coregulator complexes.

Coregulator proteins (CoRegs) are part of multi-protein complexes that transiently assemble with transcription factors and chromatin modifiers to regulate gene expression. In this study we analyzed data from 3,290 immuno-precipitations (IP) followed by mass spectrometry (MS) applied to human cell lines aimed at identifying CoRegs complexes. Using the semi-quantitative spectral counts, we scored binary protein-protein and domain-domain associations with several equations. Unlike previous applications, our methods scored prey-prey protein-protein interactions regardless of the baits used. We also predicted domain-domain interactions underlying predicted protein-protein interactions. The quality of predicted protein-protein and domain-domain interactions was evaluated using known binary interactions from the literature, whereas one protein-protein interaction, between STRN and CTTNBP2NL, was validated experimentally; and one domain-domain interaction, between the HEAT domain of PPP2R1A and the Pkinase domain of STK25, was validated using molecular docking simulations. The scoring schemes presented here recovered known, and predicted many new, complexes, protein-protein, and domain-domain interactions. The networks that resulted from the predictions are provided as a web-based interactive application at http://maayanlab.net/HT-IP-MS-2-PPI-DDI/.

Radiofrequency ablation or stripping of large-diameter incompetent great saphenous varicose veins with C2 or C3 disease.

OBJECTIVE: The objectives of this study were to compare the results of radiofrequency ablation (RFA) and stripping for large-diameter varicose target veins for the period of 1 year, based on a composite end point; to analyze the pain severity on a digital rating scale for 7 days after RFA and stripping; and to detect the factors affecting the level of postoperative pain using the cluster analysis. METHODS: This was a multicenter retrospective cohort study. Two groups, stripping ≥14 mm and RFA ≥14 mm, of 129 varicose vein disease patients underwent surgical treatment in three specialized clinics. We eliminated symptomatic pathologic reflux with RFA in 64 patients and with stripping in 65 patients. In the postoperative phase, we evaluated the pain level, subcutaneous hemorrhage, and paresthesia. A composite end point with four components was used to analyze the results. These were three clinical adverse effects of the intervention (pain, hemorrhage, and paresthesia) and the technical outcome 1 year after the surgical intervention. RESULTS: The frequency of favorable outcomes was 20 (30.8%) in the stripping ≥14 mm group and 61 (95.3%) in the RFA ≥14 mm group (P < .0001). The odds ratio for a favorable outcome between the RFA and the stripping groups was 45.8 (95% confidence interval, 44.5-47.0). The pain clusters that were moderate were created by patients after stripping. These clusters show a link between the pain level on the one hand and an increased body mass index and large vein diameter on the other hand. CONCLUSIONS: For large-diameter veins, RFA is superior to stripping in terms of favorable outcomes according to the composite end point chosen. Significant pain after stripping was linked to a large vein diameter and excess weight or adiposis.

Historeceptomic Fingerprints for Drug-Like Compounds.

Most drugs exert their beneficial and adverse effects through their combined action on several different molecular targets (polypharmacology). The true molecular fingerprint of the direct action of a drug has two components: the ensemble of all the receptors upon which a drug acts and their level of expression in organs/tissues. Conversely, the fingerprint of the adverse effects of a drug may derive from its action in bystander tissues. The ensemble of targets is almost always only partially known. Here we describe an approach improving upon and integrating both components: in silico identification of a more comprehensive ensemble of targets for any drug weighted by the expression of those receptors in relevant tissues. Our system combines more than 300,000 experimentally determined bioactivity values from the ChEMBL database and 4.2 billion molecular docking scores. We integrated these scores with gene expression data for human receptors across a panel of human tissues to produce drug-specific tissue-receptor (historeceptomics) scores. A statistical model was designed to identify significant scores, which define an improved fingerprint representing the unique activity of any drug. These multi-dimensional historeceptomic fingerprints describe, in a novel, intuitive, and easy to interpret style, the holistic, in vivo picture of the mechanism of any drug's action. Valuable applications in drug discovery and personalized medicine, including the identification of molecular signatures for drugs with polypharmacologic modes of action, detection of tissue-specific adverse effects of drugs, matching molecular signatures of a disease to drugs, target identification for bioactive compounds with unknown receptors, and hypothesis generation for drug/compound phenotypes may be enabled by this approach. The system has been deployed at drugable.org for access through a user-friendly web site.

Morphologic changes in the vein after different numbers of radiofrequency ablation cycles.

OBJECTIVE: It has not yet been clarified whether it is possible to decrease the percentage of recurrences after radiofrequency (RF) ablation by way of increasing the number of RF ablation cycles. The aim of this study was to assess the morphologic changes in excised vein fragments after different durations of RF ablation exposure. METHODS: In the first part of the study, we performed a morphologic analysis of eight cases of great saphenous vein (GSV) recanalization 6 months after RF ablation. The second part was performed on a suprafascial segment of the GSV with a length of >22 cm and a minimum diameter of 5 mm in 10 patients, who had given their consent to intraoperative excision of suprafascial GSV segments after RF ablation treatment through four 1-cm-long diametrical cuts. Prior ultrasound analysis had shown an average 6.9-mm diameter of the suprafascial segments. The segment was divided into three 7-cm-long subsegments and one control segment. The first, second, and third segments were treated with three, two, and one RF ablation cycles (ClosureFast; Covidien, Mansfield, Mass), respectively; the control segment was not exposed to RF ablation at all. Morphologic study of 160 sections of the vein (five sections of each segment and 10 control specimens) was carried out. The specimens were dyed with hematoxylin and orcein. The ensuing analysis was performed by an experienced expert with the blind study method (the specimens were numbered without any hint as to the quantity of RF ablation cycles performed on them). The intergroup comparison of the depth of venous wall damage was based on comparison of the coefficient of alteration, which is calculated as the relation of damage depth to thickness of the vein. RESULTS: After one RF ablation cycle, the depth of blurring of the structural elements only on some portions reached the middle of the muscle layer of the wall (coefficient of alteration, α = 26%). After two cycles, blurring of the structural elements on some portions extended to the adventitia (α = 53%). After three cycles, uniform blurring of the structural elements of all layers of the venous wall up to the adventitia was seen (α = 92%). The statistically significant difference in the alteration coefficient, depending on the number of cycles of RF ablation (P < .005), was established. CONCLUSIONS: The number of RF ablation cycles has an impact on the depth of vein wall damage. One and two cycles do not cause damage to all layers of the vein wall. Three cycles cause damage to all vein wall layers.

Sequence-conserved and antibody-accessible sites in the V1V2 domain of HIV-1 gp120 envelope protein.

The immune-correlates analysis of the RV144 trial suggested that epitopes targeted by protective antibodies (Abs) reside in the V1V2 domain of gp120. We mapped V1V2 positional sequence variation onto the conserved V1V2 structural fold and showed that while most of the solvent-accessible V1V2 amino acids vary between strains, there are two accessible molecular surface regions that are conserved and also naturally antigenic. These sites may contain epitopes targeted by broadly cross-reactive anti-V1V2 antibodies.

Computational prediction of neutralization epitopes targeted by human anti-V3 HIV monoclonal antibodies.

The extreme diversity of HIV-1 strains presents a formidable challenge for HIV-1 vaccine design. Although antibodies (Abs) can neutralize HIV-1 and potentially protect against infection, antibodies that target the immunogenic viral surface protein gp120 have widely variable and poorly predictable cross-strain reactivity. Here, we developed a novel computational approach, the Method of Dynamic Epitopes, for identification of neutralization epitopes targeted by anti-HIV-1 monoclonal antibodies (mAbs). Our data demonstrate that this approach, based purely on calculated energetics and 3D structural information, accurately predicts the presence of neutralization epitopes targeted by V3-specific mAbs 2219 and 447-52D in any HIV-1 strain. The method was used to calculate the range of conservation of these specific epitopes across all circulating HIV-1 viruses. Accurately identifying an Ab-targeted neutralization epitope in a virus by computational means enables easy prediction of the breadth of reactivity of specific mAbs across the diversity of thousands of different circulating HIV-1 variants and facilitates rational design and selection of immunogens mimicking specific mAb-targeted epitopes in a multivalent HIV-1 vaccine. The defined epitopes can also be used for the purpose of epitope-specific analyses of breakthrough sequences recorded in vaccine clinical trials. Thus, our study is a prototype for a valuable tool for rational HIV-1 vaccine design.

Identification and characterization of small molecules that inhibit nonsense-mediated RNA decay and suppress nonsense p53 mutations.

Many of the gene mutations found in genetic disorders, including cancer, result in premature termination codons (PTC) and the rapid degradation of their mRNAs by nonsense-mediated RNA decay (NMD). We used virtual library screening, targeting a pocket in the SMG7 protein, a key component of the NMD mechanism, to identify compounds that disrupt the SMG7-UPF1 complex and inhibit NMD. Several of these compounds upregulated NMD-targeted mRNAs at nanomolar concentrations, with minimal toxicity in cell-based assays. As expected, pharmacologic NMD inhibition disrupted SMG7-UPF1 interactions. When used in cells with PTC-mutated p53, pharmacologic NMD inhibition combined with a PTC "read-through" drug led to restoration of full-length p53 protein, upregulation of p53 downstream transcripts, and cell death. These studies serve as proof-of-concept that pharmacologic NMD inhibitors can restore mRNA integrity in the presence of PTC and can be used as part of a strategy to restore full-length protein in a variety of genetic diseases.

Can the energy gap in the protein-ligand binding energy landscape be used as a descriptor in virtual ligand screening?

The ranking of scores of individual chemicals within a large screening library is a crucial step in virtual screening (VS) for drug discovery. Previous studies showed that the quality of protein-ligand recognition can be improved using spectrum properties and the shape of the binding energy landscape. Here, we investigate whether the energy gap, defined as the difference between the lowest energy pose generated by a docking experiment and the average energy of all other generated poses and inferred to be a measure of the binding energy landscape sharpness, can improve the separation power between true binders and decoys with respect to the use of the best docking score. We performed retrospective single- and multiple-receptor conformation VS experiments in a diverse benchmark of 40 domains from 38 therapeutically relevant protein targets. Also, we tested the performance of the energy gap on 36 protein targets from the Directory of Useful Decoys (DUD). The results indicate that the energy gap outperforms the best docking score in its ability to discriminate between true binders and decoys, and true binders tend to have larger energy gaps than decoys. Furthermore, we used the energy gap as a descriptor to measure the height of the native binding phase and obtained a significant increase in the success rate of near native binding pose identification when the ligand binding conformations within the boundaries of the native binding phase were considered. The performance of the energy gap was also evaluated on an independent test case of VS-identified PKR-like ER-localized eIF2α kinase (PERK) inhibitors. We found that the energy gap was superior to the best docking score in its ability to more highly rank active compounds from inactive ones. These results suggest that the energy gap of the protein-ligand binding energy landscape is a valuable descriptor for use in VS.

Specific small molecule inhibitors of Skp2-mediated p27 degradation.

In the ubiquitin proteasome system, the E3 ligase SCF-Skp2 and its accessory protein, Cks1, promote proliferation largely by inducing the degradation of the CDK inhibitor p27. Overexpression of Skp2 in human cancers correlates with poor prognosis, and deregulation of SCF-Skp2-Cks1 promotes tumorigenesis in animal models. We identified small molecule inhibitors specific to SCF-Skp2 activity using in silico screens targeted to the binding interface for p27. These compounds selectively inhibited Skp2-mediated p27 degradation by reducing p27 binding through key compound-receptor contacts. In cancer cells, the compounds induced p27 accumulation in a Skp2-dependent manner and promoted cell-type-specific blocks in the G1 or G2/M phases. Designing SCF-Skp2-specific inhibitors may be a novel strategy to treat cancers dependent on the Skp2-p27 axis.

Stacking and hydrogen bonding: DNA cooperativity at melting.

By taking into account base-base stacking interactions we improve the Generalized Model of Polypeptide Chain (GMPC). Based on a one-dimensional Potts-like model with many-particle interactions, the GMPC describes the helix-coil transition in both polypeptides and polynucleotides. In the framework of the GMPC we show that correctly introduced nearest-neighbor stacking interactions against the background of hydrogen bonding lead to increased stability (melting temperature) and, unexpectedly, to decreased cooperativity (maximal correlation length). The increase in stability is explained as due to an additional stabilizing interaction (stacking) and the surprising decrease in cooperativity is seen as a result of mixing of contributions of hydrogen bonding and stacking.