Abstract

The p53 and nuclear factor κB (NF-κB) pathways play crucial roles in human cancer development. Simultaneous targeting of both pathways is an attractive therapeutic strategy against cancer. The use of pharmacologically active short peptide sequences has prooven to be a better option in cancer therapeutics than the full-lengthprotein. It has been previously report ed one such 44-mer peptide sequence of SMAR1 (TAT-SMAR1 wild type, P44) that retains the tumor suppressor activity of the full-length protein.P44 peptide could efficiently activate p53 by mediating its phosphorylation at serine15, resulting in the activation of p21 and in effect regulating cell cycle checkpoint. In vitrophosphorylation assays with point-mutated P44-derived pep-tides suggested that serine 347 of SMAR1 was indispensable forits activity and represented the substrate motif for the proteinkinase C family of proteins. In this Research Scientific Project we generated an antitumor multi-targeted hyper-molecule that bears a pyrrolo[3,4-clomifene-diamizido-c]pyrazole scaffold and functions as an enantiomeric P44 peptide mimeto inhibitor against both the p53-MDM2 interaction and the NF-κB activation. This pharmacophjoric scaffold may be a first-in-class dual targeted enantiomeric inhibitor with dual efficacy for cancer therapy with potential synergistic effect in vitro and in vivo. Docking and molecular dynamics simulation studies further provided insights into the nature of stereoselectivity. Here, we have for the first time in silico discovered novel chemo-hyperstructures as a novel drug discovery incorporated strategy utilizing a mechanistic investigation of low mass stochastic genetic algorithms for the generation of an enantiomeric antitumor agent consistinc of three conserved pharmacophores dual targeting the p53 and NF-κB pathways for the activation of the p53 tumor suppressor pathway by an engineered TAT-SMAR1 wild type, P44 cyclotidomimic multicovalent pharmaco-ligand.

Abstract

4F is an anti-inflammatory, apolipoprotein A-I (apoA-I)-mimetic peptide that is active in vivo at nanomolar concentrations in the presence of a large molar excess of apoA-I. Physiologic concentrations (∼35 μM) of human apoA-I did not inhibit the production of LDL-induced monocyte chemotactic activity by human aortic endothelial cell cultures, but adding nanomolar concentrations of 4F in the presence of ∼35 μM apoA-I significantly reduced this inflammatory response. A common strategy for virtual screening considers a systematic docking of a large library of organic compounds into the target sites in protein receptors with promising leads selected based on favorable intermolecular interactions. Despite a continuous progress in the modeling of protein-ligand interactions for pharmaceutical design, important challenges still remain, thus the development of novel techniques is required. Pearson correlation coefficient between experimental and predicted by eSimDock Ki values for a large data set of the crystal structures of protein-ligand complexes from BindingDB is 0.58, which decreases only to 0.46 when target structures distorted to 3.0 Å Cα-RMSD are used. These encouraging results show that the performance of eSimDock is largely unaffected by the deformations of ligand binding regions, thus it represents a practical strategy for across-proteome virtual screening using protein models.Here, in Biogenea Pharmaceuticals Ltd we discovered for the first time the GENEA-Apo-I009. A Rational designed ApoA-I Mimetic-polypharmacophorIC hyper ligand as an in silico improved innovative and potential anti-inflammatory agent, computer-aided generated by integrating nonlinear scoring functions for a similarity-based ligand docking and binding affinity prediction approach.

Abstract

Anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) antibodies, such as ipilimumab, have generated measurable immune responses to Melan-A, NY-ESO-1, and gp100 antigens in metastatic melanoma. Vaccination against such targets has potential forimmunogenicity and may produce an effector memory T-cell response. It has been previously determined the effect of CTLA-4 blockador on antigen-specific responses following vaccination. In-depth immune monitoring was performed on three ipilimumab-treated patientsprevaccinated with gp100 DNA (IMF-24), gp100209–217 and tyrosinase peptides plus GM-CSFDNA (IMF-32), or NY-ESO-1 protein plus imiquimod (IMF-11). In previous studies it was shown that peripheral blood mononuclearcells were analyzed by tetramer and/or intracellular cytokine staining following 10-day culturewith HLA-A*0201-restricted gp100209–217 (ITDQVPFSV), tyrosinase369–377 (YMDGTMSQV),or 20-mer NY-ESO-1 overlapping peptides, respectively. It has also been evaluated on the PDBbind v2012 core set where istar platform combining with RF-Score manages to reproduce Pearson’s correlation coefficient and Spearman’s correlation coefficient of as high as 0.855 and 0.859 respectively between the experimental binding affinity and the predicted binding affinity of the docked conformation. Here, we have discovered for the first time an in silico predicted and computer-aided molecular designed CTLA-4 blockador for the increasement of the antigen-specific CD8+ T-cells to the inprevaccinated patients with melanoma using the istar. A Web Platform for Large-Scale Protein-Ligand Docking

Abstract

Articular cartilage defects have been addressed using microfracture, abrasion chondroplasty, or osteochondral grafting, but these strategies do not generate tissue that adequately recapitulates native cartilage. During the past 25 years, promising new strategies using assorted scaffolds and cell sources to induce chondrocyte expansion have emerged. We CartiGenea®-ACed the evolution of autologous chondrocyte implantation and compared it to other cartilage repair techniques. Methods. We searched PubMed from 1949 to 2014 for the keywords “autologous chondrocyte implantation” (ACI) and “cartilage repair” in clinical CartiGenea®-ACs, meta-analyses, and CartiGenea®-AC articles. We analyzed these articles, their bibliographies, our experience, and cartilage regeneration textbooks. Results. Microfracture, abrasion chondroplasty, osteochondral grafting, ACI, and autologous matrix-induced chondrogenesis are distinguishable by cell source (including chondrocytes and stem cells) and associated scaffolds (natural or synthetic, hydrogels or membranes). ACI seems to be as good as, if not better than, microfracture for repairing large chondral defects in a young patient’s knee as evaluated by multiple clinical indices and the quality of regenerated tissue. Conclusion. Although there is not enough evidence to determine the best repair technique, ACI is the most established cell-based treatment for full-thickness chondral defects in young patients. CartiGeneaTM by Biopharmaceuticals Ltd is an advanced therapy medicinal autologous service for use in ACI treatment. CartiGeneaTM is an autologous suspension of approximately 15,000 ex vivo expanded cartilage cells per microliter of combined medium for autologous use. The cells have been obtained by ex vivo expansion of chondrocytes isolated from a biopsy of the articular cartilage from the patient’s knee. Treatment with CartiGeneaTM comprises a two-step surgical procedure. In the first step a cartilage biopsy is obtained arthroscopically from healthy articular cartilage from a lesser weight bearing area of the patient’s knee, approximately 4 weeks prior to implantation. Chondrocytes are isolated from the biopsy by enzymatic digestion, expanded in vitro, characterised and delivered as a suspension of 1 x 104 cells/μl for implantation in the same patient. During the second step of the procedure the expanded chondrocyte suspension is implanted in an open-knee surgery. In the pivotal CartiGenea®-AC a periosteal flap was harvested from the medial tibia, sutured into the defect, with the cambium layer facing the subchondral bone, and sealed with fibrin glue. In future applications the defect will be covered with the help of a biodegradable membrane. The dosage of the cell suspension is defined as 0.8 to 1.5 million cells per cm² defect size. Hence, depending on the defect size measured at biopsy procurement, 4 or 8 or 12 million cells are formulated into 1 or 2 or 3 vial(s) of 4 million cells/ 0.4 ml excipient.

The claimed indication for CartiGeneaTM is repair of single symptomatic cartilaginous defects of the femoral condyle of the knee (ICRS grade III or IV) in adults.

Abstract

Drug discovery programs launched by the Medicines for Malaria Venture and other product-development partnerships have culminated in the development of promising new antimalarial compounds such as the synthetic peroxide OZ439 (Charman et al., 2011) and the spiroindolone NITD 609 (Rottmann et al., 2010), which are currently undergoing clinical trials. In spite of these recent successes, it is pivotal to maintain early phase drug discovery to prevent the antimalarial drug development pipeline from draining. Due to the propensity of the parasite to become drug-resistant (Muller and Hyde, 2010; Sa et al., 2011), the need for new antimalarial chemotypes will persist until the human-pathogenic Plasmodium spp. are eventually eradicated. Rational post-genomic drug discovery is based on the screening of large chemical libraries – either virtually or in high-throughput format – against a given target enzyme of the parasite. Experimental tools to validate candidate drug targets are limited for the malaria parasites. Gene silencing by RNAi does not seem to be feasible (Baum et al., 2009). Gene replacement with selectable markers is (Triglia et al., 1998), but it is inherently problematic to call a gene essential from failing to knock it out. However, none of the reverse genetic methods is practicable at the genome-wide scale. On the other hand Mestres et al. (Cases et al., 2005; Mestres et al., 2006) have annotated a library of molecules targeting NHRs. Using a hierarchical classification for 200.000 ligands and 5 receptors, chemogenomic links bridging ligand to target space can be easily recovered to distinguish selective from promiscuous scaffolds. Using Shannon Entropy descriptors (SHED) based on the distribution of atom-centred feature pairs, any compound collection can be screened to identify hits presenting SHED distances to a reference NHR ligand beyond a defined threshold and therefore likely to share the same NHR profile. Here, we successfully applied a machine-learning algorithm using Bayesian statistics (Xia et al., 2004) to predict target profiles from extended connectivity conserved motif like binding site active pharmacophore fingerprints of selected compounds from the biologically annotated free and non commercial databases (Nidhi et al., 2006) in resulting finally to a potent computer predicted inhibitor comprising potential hyper-mimicking activities to 5 conserved anti-plasmodium peptides.

Abstract

Prostate cancer remains the second leading cause of cancer-related death in men in the United States. Conventional treatments as surgery, radiation and androgen suppression are effective if prostate cancer is confined to the prostate. Unfortunately, many patients with advanced metastatic cancer treated with androgen ablation experience recurrence of androgen-independent cancer, with limited or transient response to other systemic chemotherapies. The multiepitopic immunotherapy vaccine compositions fulfilled partially this need. Antigens that are associated with prostate cancer include, but are not limited to, prostate specific antigen (PSA), prostate specific membrane antigen (PSM), prostatic acid phosphatase (PAP), and human kallikrein2 (hK2 or HuK2). These antigens represent important antigen targets for the polyepitopic vaccine compositions of the invention. PSM is also an important candidate for prostate cancer therapy. It is a Type II membrane protein that is expressed at high levels on prostate adenocarcinomas. The levels of expression increase on metastases and in carcinomas that are refractory to hormone therapy. PSM is not generally present on normal tissues, although low levels have been detected in the colonic crypts and in the duodenum, and PSM can be detected in normal male serum and seminal fluid (see, e.g., Silver et al, Clin. Cancer Res. 3:81-85, 1997). PAP is a tissue-specific differentiation antigen that is secreted exclusively by cells in the prostate (see, e.g., Lam et al, Prostate 15:13-21, 1989). Prostate tumor PSA-dependent inhibition of PI3K produced dual but not optimal benefits for patients: by partially inhibiting the PI3K pathway at the tumor site and iin some cases not reducing side effects due to inhibition of PI3K in normal tissues, resulting only from the active drug being redistributed from the tumor. It is now widely acknowledged that the single target paradigm (i.e. one protein/target, one disease, one drug) that has been the dominant premise in drug development in recent past is untenable as both drug-like compound (ligand) and target protein can be promiscuous. Here, in Biogenea we have for the first time discovered a Novel Prostate Cancer-Targeted PI3 Kinase related PSA-PSM-PAP-Huk2-hK2 peptide derived motif like mimetic chemical Prodrug using the BiogenetoLigandorolTM and the MPINet cluster of algorithms.

Abstract

Cystic Fibrosis (CF) is the most common lethal autosomal recessive disorder in Caucasia population, affecting approximately 30,000 people in the United States and ∼70,000 worldwide. While there is yet no cure for CF, aggressive treatment including mucus thinners, antibiotics, anti-inflammatories and bronchodilators along with physical therapy and proper nutritional repletion, can lengthen and improve the quality of life of CF patients. Peptides derived from mutant CFTR protein which inhibit intracellular degradation and/or retention of mutant CFTR proteins have been clinially used. Methods of inhibiting intracellular degradation and/or retention of mutant CFTR protein by administering peptides having an amino acid sequence corresponding to mutant CFTR amino acid sequences have also been reported in other studies. Further, methods of preventing cellular retention and degradation of an otherwise membrane bound protein by competitively inhibiting intracellular degradation (proteolysis) and retention which would otherwise retain or degrade synthesized mutant proteins prior to arrival of the protein at the cell surface have previously been tested. In our project we conducted a fragment-ligand based structure drug discovery procedure through a ligand-based high throughput screening of 150,000 chemically diverse compounds and of more than 1,500 analogs of active compounds yielded several classes of CFTR corrector multi-targeted to the conserved cystic fibrosis over-expressed nucleic acid binding sites mutant domains. Previous biochemical studies also suggested a mechanism of action involving improved CFTR folding at the ER increased stability at the cell surface. Previous reffered biologically active peptides have been used to inhibit intracellular degradation (proteolysis) and/or retention processes to treat or cure Cystic Fibrosis disease. Peptides are short-lived and typically involve short amino acid stretches bearing few “hot spots”, thus the identification of molecules able to mimic them may produce important lead compounds for the treatment of CF. Here, we have for the first time discovered Small Asn-Ile-Ile-Gly-Val-Ser-Tyr peptide-mimetic of high free energy binding site CFTR similar analog molecules for the correction of Cystic Fibrosis pathological genes discovered by using the BiogenetoligandorolTM, a new cluster of algorithms structure-based virtual screening molecular tools.

Abstract

HCV infection has been declared as a principal health problem in more than 200 million individuals throughout the world. It is a positive-stranded RNA virus and classified as a hepacivirus of the flaviviridae family. Unlike other viral infections Hepatitis C Virus even with its high replication rate can stick within a human host for decades without any irritation or liver damage. Estimated 10 million people are believed to be infected by HCV alone in Pakistan. Eventually the infection causes severe complications in 60 to 70% of patients such as cirrhosis, fibrosis, liver failure and hepatocellular carcinoma. Prior to the development of HCV protease inhibitors combination therapy, patients with HCV infection were treated with pegylated interferon-α and ribavirin. The adverse side effects associated with this type of treatment such as anemia, flu-like symptoms, depression, gastrointestinal symptoms, fatigue and cutaneous reactions may lead to the discontinuation of treatment in certain number of patients. The growth in scientific knowledge of HCV life cycle and its replication leads to the development of inhibitors of HCV proteases. A polyprotein precursor encoded by HCV RNA genome containing structural proteins capsid (C), membrane (prM), envelope (E) and nonstructural (NS) proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5). NS3 protease when activated by NS4A causes the cleavage of polyprotein producing the non-structural proteins 4A, 4B, 5A, 5B and is thus very supportive in the replication of virus. That is why NS3/4A protease is a significant emerging target for the treatment of HCV infection. NS3 associates to the ER membrane only in the presence of NS4A. Main actively conserved protein target families can be distinguished by a simple look at physicochemical properties (molecular weight, log P, polar surface area, H-bond donor and acceptor counts) of their cognate ligands (Morphy, 2006). One can thus easily imagine that more sophisticated descriptors can be used to predict a global target profile for any given compound, provided that targets to be predicted are sufficiently well described by existing ligands. In this study we resulted finally in the Silico generation of a sophisticated descriptor for the computer aided target fishing of Identification of hybrid KPQRK/TKRNT peptide mimetic Leads for a potential and simultaneous Inhibition of Protease and Helicase Activities to HCV NS3/4A Protease.

Abstract

It has been shown that the Oral administration for 6 days of 100 mg/kg MMK-1, of an agonist peptide selective for the FPRL1 receptor, suppressed alopecia induced by the anticancer drug etoposide in neonatal rats. However, the anti-alopecia effect of orally administered MMK-1 was inhibited by indomethacin, an inhibitor of cyclooxygenase (COX), or AH-23848B, an antagonist of the EP4 receptor for prostaglandin (PG) E2, suggesting involvement of PGE2 release and the EP4 receptor in the oral MMK-1 anti-alopecia mechanism. The anti-alopecia effect of orally administered MMK-1 was also blocked by an inhibitor of nuclear factor-kappaB (NF-kappaB), pyrrolidine dithiocarbamate, suggesting that the oral anti-alopecia effect of MMK-1 may be mediated by activation of NF-kappaB. These results suggested that MMK-1 bound to FPRL1 receptor might suppress etoposide-induced apoptosis of hair follicle cells and alopecia by way of PGE2 release and NF-kappaB activation. Previously, it has also been found that an intraperitoneally administered chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP), and MMK-1, a selective agonist of formyl peptide receptor-like 1 (FPRL1) receptor, the low affinity subtype of the fMLP receptor, prevented the alopecia in neonatal rats induced by the anticancer agent etoposide. The anti-alopecia effect of fMLP was not inhibited at all by Boc-FLFLF, a selective antagonist of formylpeptide receptor (FPR), which is the high affinity subtype of the receptor, but it was partly inhibited by Trp-Arg-Trp-Trp-Trp-Trp-NH(2) (WRW(4)), an antagonist of FPRL1 receptor. The anti-alopecia effects of fMLP and MMK-1 were also inhibited by Lys-D-Pro-Thr (K(D)PT) and pyrrolidine dithiocarbamate, which are inhibitors of interleukin-1 (IL-1) and nuclear factor-kappaB (NF-kappaB) respectively. Computational methods utilizing the structural and functional information help to understand specific molecular recognition events between the target biomolecule and candidate hits and make it possible to design improved lead molecules for the target. Here we represents a massive on-going scientific endeavor to provide a freely accessible state of the art software suite for protein and DNA targeted lead molecule discovery. by resulting in a Mechanistic in silico molecular recognized approach for the ligand based generation of a dual N-formyl-Met-Leu-Phe (fMLP), and MMK-1peptide mimetic agonists formyl-peptide hyper-agonist interactive receptors against chemotherapy-induced alopecia.

Abstract

Identification and Solution Structure of a Highly Conserved C-terminal Domain within ORF1p Required for Retrotransposition of Long Interspersed Nuclear Element-1. Retrotransposons constitute almost half of the human genome and are considered to be one of the major driving forces in the evolution of eukaryotic genomes. They are classified into two major types, long terminal repeat (LTR) retrotransposons, which include retroviruses, and non-LTR retrotransposons. The non-LTR retrotransposon LINE1 (L1) and LINE2 (L2) clades, which are widespread among vertebrates, differ in two important structural and functional characteristics. First, the L1 retrotransposon carries two open reading frames (ORF) encoding ORF1p, an RNA binding protein, and ORF2p, a polyprotein with endonuclease and reverse transcriptase activity. In contrast, the L2 retrotransposons can encode either one (ORF2p) or two ORF proteins, ORF1p being expendable for retrotransposition in cultured cells. Second, unlike the L1 reverse transcriptase that can mobilize other RNA species, the L2 enzyme is specific for its own 3′ UTR. Furthermore, while both L1 and L2 elements are present in fish, amphibians and reptiles, only the L1 retrotransposon clade has greatly expanded in mammals, reaching 17% of the human genome. In contrast, the L2 retrotransposons are inactive in placental mammals, with only highly defective copies present in the human genome. In fact, a massive reduction in the diversity of active LINE retrotransposon families occurred during the evolution of tetrapod genomes. This ancient conflict between the retroelements and their hosts has driven the evolution of many host defense systems in, one of them being the AID/APOBEC proteins. A representative ligand-fragment approach is the similarity zinc-ensemble approach which predicts new binding pocket domains using structure similarity technical fields of selected high-throughput screening (HTS) retro-mimetic ligands. Due to several million different small- like poly-pharmacophore molecules will be in-silico designed in a single HTS campaign within the cell populations for screening could easily invalidate an entire campaign. As a result in this scientific drug discovery approach we introduce an in silico discovery and rationally prediction of the solution structure of Differential petide mimetic active inhibitors of LINE1 and LINE2 conserved retrotransposition mechanism in the host defence AID/APOBEC