Paclitaxel Enhances Oncolytic Potential of Herpes Simplex Virus Type-1 in Cancer Cells

Taxanes are spindle poisons that bind to and stabilize microtubules resulting in mitotic arrest. Herpes simplex Typ-1 (HSV-1) virions utilize the microtubular network for intracellular transport during both virus entry and virus egress from infected cells. It has been reported previously that taxanes may synergize with oncolytic herpes simplex viruses in the treatment of experimental prostate and breast tumors in mice. Other reports have indicated that taxanes may inhibit viral replication in infected cells. In this study the previously characterized Oncolytic Herpes Simplex Virus type 1 (OSVP), which was constructed in Kousoulas lab was used in conjugation with paclitaxel (taxol) in 4T1 mouse mammary tumor and RM9 mouse prostate cancer cell lines. The first goal of this study is to examine the effects of taxol on OSVP life cycle. The effect of taxol on the extent of virus entry, replication and number of infectious virus production were measured. The results showed that virus life cycle is not affected by the presence of taxol in these cell lines despite significant stabilization and reorganization of the microtubular network; visual evidence was also provided by immune fluorescence confocal microscopy reflecting that the virus entry was efficiently followed by intracellular transport and finally infection and spread of the virus to the adjacent cells leading to syncytium formation. The second goal of this study is to investigate the potency of the constructed OSVP virus in combination with taxol in cancer cell cultures. In various combinations of virus multiplicities of infection and taxol concentrations, enhanced cell killing effects were observed. Moreover, a few of the combination ratios lead to a more pronounced effect characterized as synergistic. Overall, results confirm that OSVP infection in RM9 and 4T1 cell lines are unaffected by taxol. Moreover, the efficacy and potency of OSVP and taxol are improved when combined. Interestingly, intracellular taxol concentrations and cell cycle analysis results were also affected by virus infection. It is anticipated that taxol-induced leakage of tumors will facilitate spread of OSVP virus within the tumor, producing enhanced tumor destruction and anti-tumor immune responses in addition to the enhanced cellular effect shown in this study

eModel-BDB: a database of comparative structure models of drug-target interactions from the Binding Database

Background: The structural information on proteins in their ligand-bound conformational state is invaluable for protein function studies and rational drug design. Compared to the number of available sequences, not only is the repertoire of the experimentally determined structures of holo-proteins limited, these structures do not always include pharmacologically relevant compounds at their binding sites. In addition, binding affinity databases provide vast quantities of information on interactions between drug-like molecules and their targets, however, often lacking structural data. On that account, there is a need for computational methods to complement existing repositories by constructing the atomic-level models of drug-protein assemblies that will not be determined experimentally in the near future. Results: We created eModel-BDB, a database of  200,005 comparative models of drug-bound proteins based on   1,391,403 interaction data obtained from the Binding Database and the PDB library of 31 January 2017. Complex models in eModel-BDB were generated with a collection of the state-of-the-art techniques, including protein meta-threading, template-based structure modeling, refinement and binding site detection, and ligand similarity-based docking. In addition to a rigorous quality control maintained during dataset generation, a subset of weakly homologous models was selected for the retrospective validation against experimental structural data recently deposited to the Protein Data Bank. Validation results indicate that eModel-BDB contains models that are accurate not only at the global protein structure level but also with respect to the atomic details of bound ligands. Conclusions: Freely available eModel-BDB can be used to support structure-based drug discovery and repositioning, drug target identification, and protein structure determination

eRepo-ORP: Exploring the Opportunity Space to Combat Orphan Diseases with Existing Drugs

© 2017 About 7000 rare, or orphan, diseases affect more than 350 million people worldwide. Although these conditions collectively pose significant health care problems, drug companies seldom develop drugs for orphan diseases due to extremely limited individual markets. Consequently, developing new treatments for often life-threatening orphan diseases is primarily contingent on financial incentives from governments, special research grants, and private philanthropy. Computer-aided drug repositioning is a cheaper and faster alternative to traditional drug discovery offering a promising venue for orphan drug research. Here, we present eRepo-ORP, a comprehensive resource constructed by a large-scale repositioning of existing drugs to orphan diseases with a collection of structural bioinformatics tools, including eThread, eFindSite, and eMatchSite. Specifically, a systematic exploration of 320,856 possible links between known drugs in DrugBank and orphan proteins obtained from Orphanet reveals as many as 18,145 candidates for repurposing. In order to illustrate how potential therapeutics for rare diseases can be identified with eRepo-ORP, we discuss the repositioning of a kinase inhibitor for Ras-associated autoimmune leukoproliferative disease. The eRepo-ORP data set is available through the Open Science Framework at https://osf.io/qdjup/

A graph-based approach to construct target-focused libraries for virtual screening

© 2016 Naderi et al. Background: Due to exorbitant costs of high-throughput screening, many drug discovery projects commonly employ inexpensive virtual screening to support experimental efforts. However, the vast majority of compounds in widely used screening libraries, such as the ZINC database, will have a very low probability to exhibit the desired bioactivity for a given protein. Although combinatorial chemistry methods can be used to augment existing compound libraries with novel drug-like compounds, the broad chemical space is often too large to be explored. Consequently, the trend in library design has shifted to produce screening collections specifically tailored to modulate the function of a particular target or a protein family. Methods: Assuming that organic compounds are composed of sets of rigid fragments connected by flexible linkers, a molecule can be decomposed into its building blocks tracking their atomic connectivity. On this account, we developed eSynth, an exhaustive graph-based search algorithm to computationally synthesize new compounds by reconnecting these building blocks following their connectivity patterns. Results: We conducted a series of benchmarking calculations against the Directory of Useful Decoys, Enhanced database. First, in a self-benchmarking test, the correctness of the algorithm is validated with the objective to recover a molecule from its building blocks. Encouragingly, eSynth can efficiently rebuild more than 80 % of active molecules from their fragment components. Next, the capability to discover novel scaffolds is assessed in a cross-benchmarking test, where eSynth successfully reconstructed 40 % of the target molecules using fragments extracted from chemically distinct compounds. Despite an enormous chemical space to be explored, eSynth is computationally efficient; half of the molecules are rebuilt in less than a second, whereas 90 % take only about a minute to be generated. Conclusions: eSynth can successfully reconstruct chemically feasible molecules from molecular fragments. Furthermore, in a procedure mimicking the real application, where one expects to discover novel compounds based on a small set of already developed bioactives, eSynth is capable of generating diverse collections of molecules with the desired activity profiles. Thus, we are very optimistic that our effort will contribute to targeted drug discovery. eSynth is freely available to the academic community at www.brylinski.org/content/molecular-synthesis. Graphical abstract Assuming that organic compounds are composed of sets of rigid fragments connected by flexible linkers, a molecule can be decomposed into its building blocks tracking their atomic connectivity. Here, we developed eSynth, an automated method to synthesize new compounds by reconnecting these building blocks following the connectivity patterns via an exhaustive graph-based search algorithm. eSynth opens up a possibility to rapidly construct virtual screening libraries for targeted drug discovery

Large-scale computational drug repositioning to find treatments for rare diseases

© 2018, The Author(s). Rare, or orphan, diseases are conditions afflicting a small subset of people in a population. Although these disorders collectively pose significant health care problems, drug companies require government incentives to develop drugs for rare diseases due to extremely limited individual markets. Computer-aided drug repositioning, i.e., finding new indications for existing drugs, is a cheaper and faster alternative to traditional drug discovery offering a promising venue for orphan drug research. Structure-based matching of drug-binding pockets is among the most promising computational techniques to inform drug repositioning. In order to find new targets for known drugs ultimately leading to drug repositioning, we recently developed eMatchSite, a new computer program to compare drug-binding sites. In this study, eMatchSite is combined with virtual screening to systematically explore opportunities to reposition known drugs to proteins associated with rare diseases. The effectiveness of this integrated approach is demonstrated for a kinase inhibitor, which is a confirmed candidate for repositioning to synapsin Ia. The resulting dataset comprises 31,142 putative drug-target complexes linked to 980 orphan diseases. The modeling accuracy is evaluated against the structural data recently released for tyrosine-protein kinase HCK. To illustrate how potential therapeutics for rare diseases can be identified, we discuss a possibility to repurpose a steroidal aromatase inhibitor to treat Niemann-Pick disease type C. Overall, the exhaustive exploration of the drug repositioning space exposes new opportunities to combat orphan diseases with existing drugs. DrugBank/Orphanet repositioning data are freely available to research community at https://osf.io/qdjup/

Hinge Region in DNA Packaging Terminase pUL15 of Herpes Simplex Virus: A Potential Allosteric Target for Antiviral Drugs

Approximately 80% of adults are infected with a member of the herpesviridae family. Herpesviruses establish life-long latent infections within neurons, which may reactivate into lytic infections due to stress or immune suppression. There are nine human herpesviruses (HHV) posing health concerns from benign conditions to life threatening encephalitis, including cancers associated with viral infections. The current treatment options for most HHV conditions mainly include several nucleoside and nucleotide analogs targeting viral DNA polymerase. Although these drugs help manage infections, their common mechanism of action may lead to the development of drug resistance, which is particularly devastating in immunocompromised patients. Therefore, new classes of drugs directed against novel targets in HHVs are necessary to alleviate this issue. We analyzed the conservation rates of all proteins in herpes simplex virus 1 (HHV-1), a representative of the HHV family and one of the most common viruses infecting the human population. Furthermore, we generated a full-length structure model of the most conserved HHV-1 protein, the DNA packaging terminase pUL15. A series of computational analyses were performed on the model to identify ATP and DNA binding sites and characterize the dynamics of the protein. Our study indicates that proteins involved in HHV-1 DNA packaging and cleavage are amongst the most conserved gene products of HHVs. Since the packaging protein pUL15 is the most conserved among all HHV-1 gene products, the virus will have a lower chance of developing resistance to small molecules targeting pUL15. A subsequent analysis of the structure of pUL15 revealed distinct ATP and DNA binding domains and the elastic network model identifies a functionally important hinge region between the two domains of pUL15. The atomic information on the active and allosteric sites in the ATP- and DNA-bound model of pUL15 presented in this study can inform the structure-based drug discovery of a new class of drugs to treat a wide range of HHVs

Effect of Acidic Environment on the Push-Out Bond Strength of Calcium-Enriched Mixture Cement

Introduction: This laboratory study was performed to evaluate the effect of different acidic pH values on the push-out bond strength of calcium-enriched mixture (CEM) cement. Methods and Materials: Forty-eight root dentin slices were obtained from freshly extracted single rooted human teeth and their lumen were instrumented to achieve a diameter of 1.3 mm. Then, CEM cement was mixed according to manufacturers’ instruction and placed in the lumens with minimal pressure. The specimens were randomly divided into four groups (n=12) which were wrapped in pieces of gauze soaked in either synthetic tissue fluid (STF) (pH=7.4) or butyric acid which was buffered at pH values of 4.4, 5.4 and 6.4. They were then incubated for 4 days at 37°C. The push-out test was performed by means of the universal testing machine. Specimens were then examined under a digital light microscope at 20× magnification to determine the nature of the bond failure. The data were analyzed using the Kruskal-Wallis test followed by Dunn’s test for pair-wise comparisons. Results: The highest push-out bond strength (10.19±4.39) was seen in the pH level of 6.4, which was significantly different from the other groups (P<0.05). The values decreased to 2.42±2.25 MPa after exposure to pH value of 4.4. Conclusion: Lower pH value of highly acidic environments (pH=4.4), adversely affects the force needed for displacement of CEM cement; while in higher pH values (pH=6.4) the bond-strength was not affected. CEM cement is recommended in clinical situations where exposure to acidic environment is unavoidable

Hinge Region in DNA Packaging Terminase pUL15 of Herpes Simplex Virus: A Potential Allosteric Target for Antiviral Drugs

Approximately 80% of adults are infected with a member of the herpesviridae family. Herpesviruses establish life-long latent infections within neurons, which may reactivate into lytic infections due to stress or immune suppression. There are nine human herpesviruses (HHV) posing health concerns from benign conditions to life threatening encephalitis, including cancers associated with viral infections. The current treatment options for most HHV conditions mainly include several nucleoside and nucleotide analogs targeting viral DNA polymerase. Although these drugs help manage infections, their common mechanism of action may lead to the development of drug resistance, which is particularly devastating in immunocompromised patients. Therefore, new classes of drugs directed against novel targets in HHVs are necessary to alleviate this issue. We analyzed the conservation rates of all proteins in herpes simplex virus 1 (HHV-1), a representative of the HHV family and one of the most common viruses infecting the human population. Furthermore, we generated a full-length structure model of the most conserved HHV-1 protein, the DNA packaging terminase pUL15. A series of computational analyses were performed on the model to identify ATP and DNA binding sites and characterize the dynamics of the protein. Our study indicates that proteins involved in HHV-1 DNA packaging and cleavage are amongst the most conserved gene products of HHVs. Since the packaging protein pUL15 is the most conserved among all HHV-1 gene products, the virus will have a lower chance of developing resistance to small molecules targeting pUL15. A subsequent analysis of the structure of pUL15 revealed distinct ATP and DNA binding domains and the elastic network model identifies a functionally important hinge region between the two domains of pUL15. The atomic information on the active and allosteric sites in the ATP- and DNA-bound model of pUL15 presented in this study can inform the structure-based drug discovery of a new class of drugs to treat a wide range of HHVs

Cysteines and N-glycosylation sites conserved among all alphaherpesviruses regulate membrane fusion in herpes simplex virus 1 infection

© 2017 American Society for Microbiology. Neurotropism is a defining characteristic of alphaherpesvirus pathogenicity. Glycoprotein K (gK) is a conserved virion glycoprotein of all alphaherpesviruses that is not found in other herpesvirus subfamilies. The extracellular amino terminus of gK has been shown to be important to the ability of the prototypic alphaherpesvirus herpes simplex virus 1 (HSV-1) to enter neurons via axonal termini. Here, we determined the role of the two conserved N-linked glycosylation (N48 and N58) sites of gK in virus-induced cell fusion and replication. We found that N-linked glycosylation is important to the regulation of HSV-1-induced membrane fusion since mutating N58 to alanine (N58A) caused extensive virus-induced cell fusion. Due to the known contributions of N-linked glycosylation to protein processing and correct disulfide bond formation, we investigated whether the conserved extracellular cysteine residues within the amino terminus of gK contributed to the regulation of HSV-1-induced membrane fusion. We found that mutation of C37 and C114 residues led to a gK-null phenotype characterized by very small plaque formation and drastic reduction in infectious virus production, while mutation of C82 and C243 caused extensive virus-induced cell fusion. Comparison of N-linked glycosylation and cysteine mutant replication kinetics identified disparate effects on infectious virion egress from infected cells. Specifically, cysteine mutations caused defects in the accumulation of infectious virus in both the cellular and supernatant fractions, while glycosylation site mutants did not adversely affect virion egress from infected cells. These results demonstrate a critical role for the N glycosylation sites and cysteines for the structure and function of the amino terminus of gK