Molecular Targets For Drug Development Research Articles

Radionuclide labeling and evaluation of candidate radioligands for PET imaging of histone deacetylase in the brain

Histone deacetylases (HDACs) regulate gene expression by inducing conformational changes in chromatin. Ever since the discovery of a naturally occurring HDAC inhibitor, trichostatin A (TSA) stimulated the recent development of suberoylanilide (SAHA, Zolinza®), HDAC has become an important molecular target for drug development. This has created the need to develop specific in vivo radioligands to study epigenetic regulation and HDAC engagement for drug development for diseases including cancer and psychiatric disorders. 6-([18F]Fluoroacetamido)-1-hexanoicanilide ([18F]FAHA) was recently developed as a HDAC substrate and shows moderate blood–brain barrier (BBB) permeability and specific signal (by metabolic trapping/or deacetylation) but rapid metabolism. Here, we report the radiosynthesis of two carbon-11 labeled candidate radiotracers (substrate- and inhibitor-based radioligand) for HDAC and their evaluation in non-human primate brain. PET studies showed very low brain uptake and rapid metabolism of both labeled compounds but revealed a surprising enhancement of brain penetration by F for H substitution when comparing one of these to [18F]FAHA. Further structural refinement is needed for the development of brain-penetrant, metabolically stable HDAC radiotracers and to understand the role of fluorine substitution on brain penetration.

Molecular docking and simulation studies towards exploring antiviral compounds against envelope protein of Japanese encephalitis virus

Japanese encephalitis (JE) is an acute central nervous system inflammatory disease caused by the Japanese encephalitis virus (JEV). JEV is a small, enveloped, plus-strand RNA virus belonging to the genus Flavivirus. In this study, envelope protein (E) that mediates the entry of JEV into host cell has been preferred as potential molecular target for drug development. The 3-D structure of E protein was designed and validated using modeler9.10 and procheck tool, respectively, and also optimized using molecular dynamics simulation. A number of lead molecules were used for computational virtual screening against JEV E protein. Three top ranked lead molecules with strong binding affinity to JEV E protein were identified based on minimum binding energy. Molecular dynamic simulation was also performed for protein–ligand complex to study the mobility of complex at various time intervals. Drug likeliness and comparative bioactivity analysis for these leads using OSIRIS Property Explorer had shown that these molecules would have the potential to act as better drug. The mycophenolate was found to be most suitable as entry inhibitor therapeutic molecule for JEV E protein, which may be considered as a potential ligand for treatment of Japanese encephalitis.

MicroRNAs in osteosarcoma: diagnostic and therapeutic aspects

MicroRNAs (miRNAs) are small RNA molecules, which can interfere with the expression of several genes and act as gene regulator. miRNAs have been proved as a successful diagnostic and therapeutic tool in several cancers. In this review, the differential expression of miRNAs in osteosarcoma and their possibility to be used as diagnostic and therapeutic tools have been discussed. Osteosarcoma is the most common primary bone tumor that mainly affects children and adolescents. The current treatment of osteosarcoma remains difficult, and osteosarcoma causes many deaths because of its complex pathogenesis and resistance to conventional treatments. Several studies demonstrated that the differential expression patterns of miRNAs are a promising tool for the diagnosis and treatment of osteosarcoma. Although some aspect of the mechanism of action of miRNAs in controlling osteosarcoma has been identified (e.g., targeting the Notch signaling pathway), it is far beyond to the clear understanding of miRNA targets in osteosarcoma. Identification of the specific target of miRNAs may aid molecular targets for drug development and future relief of osteosarcoma.

Current understanding of the molecular mechanisms of kidney cancer: a primer for urologists

Renal cell carcinoma (RCC), the fifth leading malignant condition for men and tenth for women, accounts for 3% of all malignancies in Canada. It is a heterogeneous epithelial malignancy with different subtypes and varied tumour biology. Although most cases of RCC are sporadic, up to 4% of patients have an inherited predisposition for the disease. In this article, we review the current molecular genetics of the different subtypes in hereditary and sporadic RCC. Significant developments in understanding the underlying genetic basis of RCC over the last 2 decades are attributed to intensive research about rare inherited renal cancer syndromes and the identification of the genes responsible for them. Many of these genes are also found in sporadic RCC. Understanding the molecular mechanisms involved in the pathogenesis of RCC has aided the development of molecular-targeted drugs for this disease.

SY2-4 - Training Oncologists in Cross-Specialty Medical Oncology

ABSTRACT In the past, cancer chemotherapy was administered by physicians in organ-specific specialties in Japan. For example, patients with lung cancer were treated by chest physicians, and chemotherapy for patients with gastrointestinal (GI) cancers was administered by gastroenterologists and sometimes even by surgeons. However, this method of treatment of oncology patients produced large numbers of ‘cancer refugees’, and patients with metastasis from other organs were inadequately treated. Furthermore, the recent development of molecular target drugs has made anti-cancer drug therapy complicated and highly specialized, and it is now difficult to engage in chemotherapy as an endoscopist or surgeon. An era has come in which medical oncologists who are trained specifically to administer chemotherapy should provide anticancer drug therapy. Cancer chemotherapy should not be given in an organ-specific medical specialty system. Physicians who are able to treat only lung cancer should be called thoracic oncologists, not medical oncologists. Doctors who treat GI cancers but who cannot treat breast or other cancers are GI oncologists, not medical oncologists. Medical oncologists should be able to treat all cancers and have experience in the treatment of hematological malignancies. In 2013, hematological training will be required for all candidates for the examination for Board-Certified Medical Oncologists of the Japanese Society of Medical Oncology (JSMO). A training program in medical graduate schools, ‘GAN-PRO’, has been implemented for 5 years in Japan to educate medical oncologists who can manage various cancers. The JSMO certifies that kind of oncologists as Board-Certified Medical Oncologists. It is best to train oncologists to manage all types of cancers in one department of a medical school, which may be difficult in some medical schools because cancer patients have been treated in organ-specific medical specialties. Currently, we are in a transition phase from an organ-specific specialty to a general medical oncology system. For this purpose, basically two types of training systems are currently implemented in medical schools. In one system, trainees rotate through departments of a hospital, spending several months in each department. In this case, the policy for treatment may not be identical in all departments. In the other system, trainees can receive all their training in one department. Obviously the latter is a better system. In the Section of Medical Oncology/Hematology at Kobe University, the faculty comprises oncologists who cover all solid tumors and hematologists. They can train graduate students in treating all cancers including hematological malignancies. So far trainees of this system have passed the test for Board-Certified Medical Oncologists with high scores. A training system which enables trainees to experience treatment of all cancers should be implemented in all institutions in the near future.

EL23 - Sarcoma: Molecular-Targeted Therapies

ABSTRACT Sarcomas are rare heterogeneous malignant neoplasms that arise from mesenchymal tissues and are clinically divided into two main categories, that is, bone and soft tissue sarcomas. The overall frequency of sarcomas is ∼1% of all adult solid malignancies, and the incidence of bone sarcomas and soft-tissue sarcomas is estimated to be four patients per one million persons per year and two patients per 100 000 persons per year, respectively. Sarcomas are classified into various tumor types on the basis of histological characteristics, for example, osteosarcoma, Ewing family tumor, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, and clear-cell sarcoma. In the last few decades, cytotoxic agents such as anthracycline agents and ifosfamide have been proven as key drugs for treating sarcomas. However, anthracycline based chemotherapy would not be optimal strategy for all histologic type of sarcomas. Recent advances in genomics, molecular biology, and molecular-targeted drug development have changed the treatment strategies for and prognosis of various cancers and hematological malignancies. Furthermore, the therapeutic developments for gastrointestinal stromal tumors constitute a successful paradigm that has affected the prognosis of such patients. However, dramatic changes in therapeutic strategy have not yet been achieved in other types of sarcomas, and many researchers continue to explore critical molecular targets for breakthroughs in treatment strategy. This educational session will introduce recent translational studies to investigate candidate molecular targets for sarcomas and promising molecular targeted drugs on the basis of the results of international phase III clinical trials on sarcomas. In the case of rare malignancies, the patient enrollment method in phase III clinical trials always has limitations with respect to achieving significant clinical benefits regarding aspects such as overall survival because each type of sarcoma is an essentially different neoplasm.

New Therapeutic Targets in Melanoma

Research into molecular targets for drug development in melanoma is starting to bear fruit. Of the drugs tested to date in patients with metastatic melanoma, those that have yielded the best results are V600E BRAF inhibitors in melanomas carrying the V600E mutation; c-kit tyrosine kinase activity inhibitors in melanomas carrying c-kit mutations; and anti-cytotoxic T lymphocyte antigen 4 (CTLA-4) antibodies, which block the mechanisms involved in immune tolerance. Many problems have yet to be resolved in these areas, however, such as the rapid development of resistance to BRAF and c-kit inhibitors and the lack of biomarkers to predict treatment response in the case of CTLA-4 blockers. We review the results of targeted therapy with these and other drugs in metastatic melanoma and discuss what the future holds for this field.

Acquired resistance mechanisms to tyrosine kinase inhibitors in lung cancer with activating epidermal growth factor receptor mutation—diversity, ductility, and destiny

Lung cancers that harbor somatic activating mutations in the gene for the epidermal growth factor receptor (EGFR) depend on mutant EGFR for their proliferation and survival; therefore, lung cancer patients with EGFR mutations often dramatically respond to orally available EGFR tyrosine kinase inhibitors (TKIs). However, emergence of acquired resistance is virtually inevitable, thus limiting improvement in patient outcomes. To elucidate and overcome this acquired resistance, multidisciplinary basic and clinical investigational approaches have been applied, using in vitro cell line models or samples obtained from lung cancer patients treated with EGFR-TKIs. These efforts have revealed several acquired resistance mechanisms and candidates, including EGFR secondary mutations (T790M and other rare mutations), MET amplification, PTEN downregulation, CRKL amplification, high-level HGF expression, FAS-NFκB pathway activation, epithelial-mesenchymal transition, and conversion to small cell lung cancer. Interestingly, cancer cells harbor potential destiny and ductility together in acquiring resistance to EGFR-TKIs, as shown in in vitro acquired resistance models. Molecular mechanisms of "reversible EGFR-TKI tolerance" that occur in early phase EGFR-TKI exposure have been identified in cell line models. Furthermore, others have reported molecular markers that can predict response to EGFR-TKIs in clinical settings. Deeper understanding of acquired resistance mechanisms to EGFR-TKIs, followed by the development of molecular target drugs that can overcome the resistance, might turn this fatal disease into a chronic disorder.

Structural role of the active‐site metal in the conformation of Trypanosoma brucei phosphoglycerate mutase

Phosphoglycerate mutases (PGAMs) participate in both the glycolytic and the gluconeogenic pathways in reversible isomerization of 3-phosphoglycerate and 2-phosphoglycerate. PGAMs are members of two distinct protein families: enzymes that are dependent on or independent of the 2,3-bisphosphoglycerate cofactor. We determined the X-ray structure of the monomeric Trypanosoma brucei independent PGAM (TbiPGAM) in its apoenzyme form, and confirmed this observation by small angle X-ray scattering data. Comparing the TbiPGAM structure with the Leishmania mexicana independent PGAM structure, previously reported with a phosphoglycerate molecule bound to the active site, revealed the domain movement resulting from active site occupation. The structure reported here shows the interaction between Asp319 and the metal bound to the active site, and its contribution to the domain movement. Substitution of the metal-binding residue Asp319 by Ala resulted in complete loss of independent PGAM activity, and showed for the first time its involvement in the enzyme's function. As TbiPGAM is an attractive molecular target for drug development, the apoenzyme conformation described here provides opportunities for its use in structure-based drug design approaches. Database Structural data for the Trypanosoma brucei 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM) has been deposited with the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank under code 3NVL.

Gene targeting diagnosis and treatment in cancer

With the development and clinical use of the molecular targeted drugs and individualized treatment,cancer research has been focused on gene targeting diagnosis and treatment.Especially for the epidermal growth factor receptor (EGFR) signaling pathway-related genes,DNA replication-related genes,spindle apparatus format-related genes,cell metabolism-related genes and other molecular targeted detection and treatment,the polymorphism of targeting genes/molecules determines the clinical efficiency of the therapies. Key words: Neoplasms; Molecular diagnostic techniques; Antineoplastic agents

EGFR/HER1 : histoire d’une cible thérapeutique

EGFR may be considered as an old target, which can be inhibited both by monoclonal antibodies and tyrosine kinase inhibitors. Those molecular targeted strategies are now approved in a wild range of tumors: colorectal cancer, lung cancer, pancreatic cancer and head and neck cancer. This paper proposes to describe the development of anti-EGFR drugs, highlighting several strategies points. Predicting biomarkers have been extensively studied for these agents, sustaining the hallmarks of the development of molecular targeting drugs.

Nuevas dianas terapéuticas en el melanoma

Research into molecular targets for drug development in melanoma is starting to bear fruit. Of the drugs tested to date in patients with metastatic melanoma, those that have yielded the best results are V600E BRAF inhibitors in melanomas carrying the V600E mutation; c-kit tyrosine kinase activity inhibitors in melanomas carrying c-kit mutations; and anti-cytotoxic T lymphocyte antigen 4 (CTLA-4) antibodies, which block the mechanisms involved in immune tolerance. Many problems have yet to be resolved in these areas, however, such as the rapid development of resistance to BRAF and c-kit inhibitors and the lack of biomarkers to predict treatment response in the case of CTLA-4 blockers. We review the results of targeted therapy with these and other drugs in metastatic melanoma and discuss what the future holds for this field.

GSK-3: A Bifunctional Role in Cell Death Pathways

Although glycogen synthase kinase-3 beta (GSK-3β) was originally named for its ability to phosphorylate glycogen synthase and regulate glucose metabolism, this multifunctional kinase is presently known to be a key regulator of a wide range of cellular functions. GSK-3β is involved in modulating a variety of functions including cell signaling, growth metabolism, and various transcription factors that determine the survival or death of the organism. Secondary to the role of GSK-3β in various diseases including Alzheimer's disease, inflammation, diabetes, and cancer, small molecule inhibitors of GSK-3β are gaining significant attention. This paper is primarily focused on addressing the bifunctional or conflicting roles of GSK-3β in both the promotion of cell survival and of apoptosis. GSK-3β has emerged as an important molecular target for drug development.

TDR Targets: a chemogenomics resource for neglected diseases

The TDR Targets Database (http://tdrtargets.org) has been designed and developed as an online resource to facilitate the rapid identification and prioritization of molecular targets for drug development, focusing on pathogens responsible for neglected human diseases. The database integrates pathogen specific genomic information with functional data (e.g. expression, phylogeny, essentiality) for genes collected from various sources, including literature curation. This information can be browsed and queried using an extensive web interface with functionalities for combining, saving, exporting and sharing the query results. Target genes can be ranked and prioritized using numerical weights assigned to the criteria used for querying. In this report we describe recent updates to the TDR Targets database, including the addition of new genomes (specifically helminths), and integration of chemical structure, property and bioactivity information for biological ligands, drugs and inhibitors and cheminformatic tools for querying and visualizing these chemical data. These changes greatly facilitate exploration of linkages (both known and predicted) between genes and small molecules, yielding insight into whether particular proteins may be druggable, effectively allowing the navigation of chemical space in a genomics context.

Abundant Pleiotropy in Human Complex Diseases and Traits

We present a systematic review of pleiotropy among SNPs and genes reported to show genome-wide association with common complex diseases and traits. We find abundant evidence of pleiotropy; 233 (16.9%) genes and 77 (4.6%) SNPs show pleiotropic effects. SNP pleiotropic status was associated with gene location (p = 0.024; pleiotropic SNPs more often exonic [14.5% versus 4.9% for nonpleiotropic, trait-associated SNPs] and less often intergenic [15.8% versus 23.6%]), "predicted transcript consequence" (p = 0.001; pleiotropic SNPs more often predicted to be structurally deleterious [5% versus 0.4%] but not more often in regulatory sequences), and certain disease classes. We develop a method to calculate the likelihood that pleiotropic links between traits occurred more often than expected and demonstrate that this approach can identify etiological links that are already known (such as between fetal hemoglobin and malaria risk) and those that are not yet established (e.g., between plasma campesterol levels and gallstones risk; and between immunoglobulin A and juvenile idiopathic arthritis). Examples of pleiotropy will accumulate over time, but it is already clear that pleiotropy is a common property of genes and SNPs associated with disease traits, and this will have implications for identification of molecular targets for drug development, future genetic risk-profiling, and classification of diseases.

Roles of Pim‐3, a novel survival kinase, in tumorigenesis

Pim-3 is a member of the Provirus integrating site Moloney murine leukemia virus (Pim) family, which belongs to the Ca(2+) /calmodulin-dependent protein kinase (CaMK) group and exhibits serine/threonine kinase activity. Similar to other members of the Pim family (i.e. Pim-1 and Pim-2), Pim-3 can prevent apoptosis and promote cell survival and protein translation, thereby enhancing cell proliferation of normal and malignant cells. Pim-3 is expressed in vital organs, such as the heart, lung, and brain. However, minimal phenotypic changes in Pim-3-deficient mice suggest that Pim-3 may be physiologically dispensable. Pim-3 expression is enhanced in several cancer tissues, particularly those of endoderm-derived organs, including the liver, pancreas, colon, and stomach. The development of hepatocellular carcinoma is accelerated in mice expressing the Pim-3 gene selectively in the liver only when these mice are treated with a hepatocarcinogen, indicating that Pim-3 can act as a promoter but not as an initiator. Moreover, inhibition of Pim-3 expression can retard in vitro cell proliferation of hepatocellular, pancreatic, and colon carcinoma cell lines by promoting cell apoptosis. Furthermore, a Pim-3 kinase inhibitor has been reported to inhibit cell proliferation in an in vivo xenograft model using a human pancreatic cancer cell line without inducing any major adverse effects. Thus, Pim-3 kinase may be a candidate molecule for the development of molecular targeting drugs against cancer.

Abstract 4081: Sensitivity of gastric cancer cell lines against 2-deoxy-D-glucose is independent of PI3K/mTOR inhibition

Abstract 2-deoxy-D-glucose (2-DG) is currently studied in Phase I clinical trials as a novel therapeutic strategy for anti-cancer therapy. Several preclinical studies have suggested that the anti-proliferative activity of 2-DG might be linked, amongst others, to the inhibition of the PI3K/mTOR signalling pathway. Blocking the PI3K/mTOR pathway has become a major focus for molecular targeted drug development. In the current study we assessed the biological relevance of PI3K/mTOR signalling for the anti-proliferative activity of 2-DG in gastric cancer cells. The gastric cancer cell lines (N87, MKN-28, MKN-45) were treated with 2-DG and evaluated for cell proliferation, apoptosis induction, cell cycle arrest, and PI3K/mTOR signalling. Everolimus, a specific mTORC1 inhibitor, was employed as positive control. Treatment with 2-DG potently suppressed cell proliferation resulting in an up to 300% reduced IC50 in the gastric cancer cell lines. Cell cycle inhibition and apoptosis induction varied substantially among cell lines. However, the distinct 2-DG sensitivity of gastric cancer cell lines was not reflected by any alteration in the PI3K/mTOR signalling pathway. In all cancer cell lines, no major inhibition of pERK and pAKT was observed, whereas both, 2-DG as well as everolimus strongly inhibited phosphorylation of S6. In contrast to 2-DG everolimus induced also a clear inhibition of 4EBP1 phosphorylation. In conclusion, inhibition of PI3K/mTOR signalling pathway by 2-DG differs from inhibition by everolimus and is not linked with 2-DG anti-proliferative sensitivity in gastric cancer cell lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4081. doi:10.1158/1538-7445.AM2011-4081

Shikimate Kinase (EC 2.7.1.71) from Mycobacterium tuberculosis: Kinetics and Structural Dynamics of a Potential Molecular Target for Drug Development

The enzymes of the shikimate pathway represent potential molecular targets for the development of non-toxic antimicrobial agents and anti-parasite drugs. One of the most promising of these enzymes is shikimate kinase (EC 2.7.1.71), which is responsible for the fifth step in the shikimate pathway. This enzyme phosphorylates shikimic acid to yield shikimate-3-phosphate, using ATP as a substrate. In this work, the conformational dynamics of the shikimate kinase from Mycobacterium tuberculosis was investigated in its apostate in solution. For this study, the enzyme was subjected to a gradient of temperatures from 15°C to 45°C in the presence or absence of deuterium oxide, and the amide H/D exchange was monitored using ESI-mass spectrometry. We observed: i) the phosphate binding domain in the apo-enzyme is fairly rigid and largely protected from solvent access, even at relatively high temperatures; ii) the shikimate binding domain is highly flexible, as indicated by the tendency of the apo-enzyme to exhibit large conformational changes to permit LID closure after the shikimate binding; iii) the nucleotide binding domain is initially conformationally rigid, which seems to favour the initial orientation of ADP/ATP, but becomes highly flexible at temperatures above 30°C, which may permit domain rotation; iv) part of the LID domain, including the phosphate binding site, is partially rigid, while another part is highly flexible and accessible to the solvent.

Experimental therapeutics for patients with myeloproliferative neoplasias.

Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms (MPNs) are characterized by stem cell-derived, unrestrained clonal myeloproliferation. The World Health Organization classification system, proposed in 2008, identifies 7 distinct categories of Ph-negative MPNs including essential thrombocythemia (ET); polycythemia vera (PV); primary myelofibrosis (PMF); mastocytosis; chronic eosinophilic leukemia; chronic neutrophilic leukemia; and MPN, unclassifiable. For many years, the treatment of ET, PV, and PMF, the most frequently diagnosed Ph-negative MPNs, has been largely supportive. In recent years, that paradigm has been challenged because of the discovery of a recurrent point mutation in the Janus kinase 2 (JAK2) gene (JAK2(V617F)). This mutation can be detected in the vast majority of patients with PV and approximately half of patients with ET or PMF and serves as both a diagnostic marker as well as representing a putative molecular target for drug development. Several putative targeted agents with significant in vitro JAK2 inhibitory activity and various degrees of JAK2 specificity are currently undergoing clinical evaluation. Furthermore, other investigational non-tyrosine kinase inhibitor approaches such as immunomodulatory agents and pegylated interferon- have also shown promising results in MPNs.

THE FUTURE OF POSTMORTEM RESEARCH

up such an online publically available database, the Stanley Neuropathology Consortium Integrative Database (SNCID; http:// sncid.stanleyresearch.org). The database currently integrates 1,747 datasets from neuropathology studies using 12 different brain regions and genome-wide expression microarray datasets from three independent studies using frontal cortex and cerebellum. All data are derived from the same 60 cases, 15 each with schizophrenia, bipolar disorder, severe depression, and unaffected controls. Additional SNP-array data, microRNA array data, and methylation array data from the same cohort will eventually be included as will similar data on another cohort of 105 brains, 35 each with schizophrenia, bipolar disorder, and unaffected controls. User-friendly statistical tools such as descriptive analysis, variance analysis, and correlation analysis are included. An interface links the SNCID to a functional annotation database, DAVID (http://david. abcc.ncifcrf.gov), that enables users to efficiently explore genes and pathways that underlie the mental disorders. Our initial genomewide correlation study using the integrated database demonstrated that several previously identified candidate genes for schizophrenia were significantly correlated with abnormalities of cytorchitectural traits in the frontal cortex. We also explored the possible genetic causalities that may underlie the cytoarchitectural abnormalities in the prefrontal cortex of patients with schizophrenia by converging results from genome-wide SNP scans for the traits and expression SNP associations. The integrative analysis yielded novel candidate genes for the abnormalities including a glutamine transporter. Thus, such applications of the database could also potentially lead to the identification of molecular targets for drug development.