Combinatorial Chemistry & High Throughput Screening - Volume 9, Issue 3, 2006

Although discovered a few decades ago, growth hormone - releasing hormone (GHRH) was thought until recently to have only a single function, namely the regulation of growth hormone release from the pituitary. However, new evidence has provided strong support to the notion that additional physiological and patho-physiological processes, and including carcinogenesis, might be regulated by GHRH. Recently, interest in this small neuropeptide has been increasing steadily and has resulted in the development of GHRH analogs that modulate its activity and potentially might be used as agents that permit clinical intervention. In this special issue of Combinatorial Chemistry & High Throughput Screening an attempt was made to provide the state of the art with respect to information in this field. Contributions include both review and original research papers related to chemical and biological synthesis of GHRH analogs, as well as the biological consequenses of such compounds. Articles on GH secretagogues were also included in this effort considering that the complexity of growth hormone regulation has only recently begun to unravel. It is the contributors' and the special editor's hope that this attempt will stimulate further interest regarding the role and use of GH regulatory agents in health and disease.

The development of antagonists of growth hormone (GH) - releasing hormone (GH-RH) is reviewed. GH-RH antagonists bind with a high affinity to pituitary receptors for GH-RH and inhibit the release of GH in vitro and in vivo. The main applications of GH-RH antagonists would be for tumor therapy. The antitumor effects of GH-RH antagonists are exerted in part indirectly through the inhibition of the secretion of pituitary GH and the reduction in the levels of hepatic insulin like growth factor (IGF-I). However, principal effects of the GH-RH antagonists are exerted directly on tumors. Antagonists of GH-RH inhibit the proliferation of various cancer cell lines in vitro and suppress in vivo the levels and the expression of mRNA for IGF-I and IGF-II in tumors. In many human cancers, the effects of GH-RH antagonists appear to be due to the blockade of the action of tumoral GH-RH. GH-RH ligand is present in various human cancers indicating that it may be an autocrine/paracrine growth factor. Splice variants (SVs) of GH-RH receptors and pituitary type of GH-RH receptors that might mediate effects of tumoral GH-RH and of GH-RH antagonists were demonstrated in many human cancers. This suggests the presence of a stimulatory loop based on GH-RH and SVs or pituitary type of GHRH receptors in diverse tumors. It was shown that GH-RH antagonists inhibited the growth of various human cancer lines xenografted into nude mice including mammary, ovarian, endometrial and prostate cancers, small cell lung carcinomas (SCLC) and non-SCLC, renal, pancreatic, gastric and colorectal carcinomas, malignant gliomas, osteosarcomas and Non- Hodgkin's lymphomas. Further development of GH-RH antagonists should lead to potential therapeutic agents for various cancers.

Growth hormone-releasing factor was discovered in 1982 by Guillemin and has been subjected to intense investigations because of its huge potential applications. The major concerns encountered with the native molecules were their short half-lives in vivo in many species including man, precluding the practical use of these peptides for medical or production purposes. Many efforts to produce analogs of shorter length, more resistant to degradation and having higher affinity to the receptors have been made during the last decades. The present paper presents a quick review of the work done to produce such analogs.

Growth hormone secretagogues (GHSs) are synthetic molecules that stimulate and amplify pulsatile pituitary growth hormone release, via a separate pathway distinct from GH releasing hormone/somatostatin. The activity of GHSs is not fully specific for GH secretion; some GHSs also have slight releasing activity on other pituitary hormones and mediate GH independent biological activities. The first GHSs were discovered in 1977. Since then, an intensive research to synthesize a potent oral GHSs has been undertaken. Although the potential applications of GHSs are numerous, long term trials are needed to evaluate the clinical efficacy and safety of these substances. In the present article we review the historic background of GHSs, their potential clinical uses, the types and the main GHSs that have been synthesized hitherto.

Novel DNA-based technologies were recently introduced for various purposes, such as screening of targets identified from genomic projects, shuffled molecules for vaccination, or to direct the in vivo production of hormones and other peptides for therapeutic or preventative applications. We have used a plasmid-based technology to deliver growth hormone releasing hormone (GHRH) to various animal species for screening, toxicology and therapy. A single intramuscular injection of a low dose of plasmid followed by electroporation can ensure that the target species will produce physiological levels of GHRH for extended periods of time, which would replace costly, frequent injections of the recombinant hormone and improve the quality of life and compliance of patients. This therapeutic modality is of particular importance in circumstances requiring long-term administration of small molecules with naturally short half-life (e.g. treatment of anemia and cachexia associated with renal failure, cancer or other chronic disability). A similar technique was used to create, test and validate protease-resistant analogs of GHRH with significantly longer half-life. Analysis of the characteristics of each of the plasmid components and tissue-specific transcription factors and the choice of target tissue is imperative when designing plasmids for therapeutic applications. Using the species-specific sequences of GHRH or other molecule along with the appropriate choice of plasmid backbone and expression cassette components can result in long and steady expression of the transgene product.

Hypothalamic hormones physiologically regulate pulsatile release of growth hormone (GH) from the anterior pituitary gland. Since the discovery of these hormones in the 1970s, several new chemically synthesized peptidyl and nonpeptidyl derivatives have been proved to stimulate and amplify GH secretion, and this series of molecules has been named the growth hormone secretagogues (GHSs). One of these compounds led to the discovery of a GPCR-type receptor for GHSs (GHS-R), and subsequently the endogenous ligand for the receptor has been identified, and is referred to as ghrelin. The identification of GHSs as physiological regulators of GH secretion encouraged us to examine our GHSs pharmacologically. We previously reported that novel oxindole derivatives have been identified as GHS-R agonists from our internal chemical library. Among these derivatives, (+)-6-carbamoyl-3-(2-chlorophenyl)-(2-diethylaminoethyl)-4- trifluoromethyloxindole (SM-130686, 37S) was found to have potent activity in vitro with a good pharmacokinetic profile in rats (bioavailability of 28%). In this article, we review the synthesis and pharmacological evaluation of SM-130686. SM-130686 binds specifically to GHS-R and increases the Ca2+ concentration in Chinese hamster ovary cells expressing recombinant GHS-R. Maximal enhancement of the intracellular Ca2+ concentration induced by SM-130686 treatment was approximately 55% that induced by ghrelin, suggesting that SM-130686 may be a partial GHS-R agonist. Also, in in vivo studies, oral administration of SM-130686 increased body length and fat-free mass gain. We compare the pharmacological profile of SM-130686 with other GHSs, including GHRH and ghrelin, and discuss the therapeutic usefulness of GHSs against several disorders, as well as for treatment of GH deficiency.

Growth hormone releasing hormone is one of the hormones secreted from the hypothalamus. Because of its potential applications in agriculture and medicine, its short half-life and its expensive chemical synthesis, an analog with high GHRH activity and prolonged half-life was sought after. The fusion partner gene with 127 amino acid residues of the C-terminus from L-asparaginase was recombined with asp-pro-hGHRH(1-44) gene synthesized by PCR method to form one kind of fusion protein with unique acid labile linker Asp-Pro. The Pro-hGHRH(1-44) peptide was purified to homogeneity by means of cell disruption, washing, ethanol precipitation, acid hydrolysis, SP-Sephadex C-25, and Sephadex G-10 column chromatography. The peptide's molecular weight of 5,139 Da as measured by EIS-MS was coincident with the actual values. In the study of the activity, the doses of peptide were 0.1, 1.0, and 10 g/μl for rat pituitary and 5 g/μl for human pituitary. The peptide increased GH releases from rat pituitary in a concentrationdependent manner (P<0.05; P<0.01). At 1.0 g/ml, there was a significant difference between Pro-Pro-hGHRH(1-44)- Gly-Gly-Cys and Pro-hGHRH(1-44) or Pro-Pro-hGHRH(1-44) (P<0.05), whereas the standard hGHRH(1-40) showed no measured rGH release. For human fetal pituitary, the Pro-hGHRH(1-44) peptides showed good GH-releasing activity, but there were no significant differences between them. The structure-activity relationship showed that for both rat and human fetal pituitary, the net GH-releasing activity of the Pro-hGHRH(1-44) analog was more than that of Pro-Pro-hGHRH(1- 44). The results of the other hormones from human pituitary showed that the analog had good function-selectivity and species specificity.

The advent of microarray technologies has dramatically accelerated the functional study of proteins, including enzymes (catalomics) in a proteome. Herein, we review recent advances and exciting new developments of microarrays in high-throughput functional proteomics.

Virtual filtering and screening of combinatorial libraries have recently gained attention as methods complementing the high-throughput screening and combinatorial chemistry. These chemoinformatic techniques rely heavily on quantitative structure-activity relationship (QSAR) analysis, a field with established methodology and successful history. In this review, we discuss the computational methods for building QSAR models. We start with outlining their usefulness in high-throughput screening and identifying the general scheme of a QSAR model. Following, we focus on the methodologies in constructing three main components of QSAR model, namely the methods for describing the molecular structure of compounds, for selection of informative descriptors and for activity prediction. We present both the well-established methods as well as techniques recently introduced into the QSAR domain.

A new strategy to obtain pure ionic liquid support with a high loading (3 mmol/g) of primary amino groups is reported. The compatibility of the imidazolium unit was demonstrated with DIC or DCC coupling reactions of protected amino acids and with classical cleavage of BOC or Fmoc protecting groups. In addition, this is the first report of a traceless ionic liquid support based on a silicon linker with a high loading (3 mmol/g) of aryl bromide reactive groups used in a palladium-catalyzed carbon-carbon coupling reaction.

Dr. Hippokratis Kiaris received his B.Sc. in 1993 from the Department of Biology at the University of Athens and his Ph.D. in 1996 from the University of Crete, Greece. His post doctoral training was accomplished in the laboratories of the Nobel Laureate, Professor Andrew V. Schally at Tulane University, New Orleans, LA and Professor Spyros Artavanis-Tsakonas, Harvard Medical School, Boston, MA, USA. Since 2003 he joined the faculty of the Department of Biological Chemistry at the University of Athens Medical School. His research interests focus on the extrapituitary effects of growth hormone - releasing hormone and the role of tumor-stroma interactions in carcinogenesis. He has authored more than 50 original research papers and his work has received more than 1000 citations. SELECTED PUBLICATIONS [1] Kiaris, H.; Schally, A.V.; Nagy, A.; Sun, B.; Armatis, P.; Szepeshazi, K. Br. J. Cancer, 1999, 81, 966-971. [2] Kiaris, H.; Schally, A.V.; Sun, B.; Armatis, P.; Groot, K. Oncogene, 1999, 18, 7168-7173. [3] Kiaris, H.; Schally, A.V.; Varga, J.; Armatis, P.; Groot, K. Proc. Nat. Acad. Sci. USA, 1999, 96, 14894-14898. Commentary appeared in the Proc. Nat. Acad. Sci. USA, 2000, 97, 532-534. [4] Kiaris, H.; Schally, A.V.; Nagy, A.; Sun, B.; Szepeshazi, K.; Halmos, G. Clin. Cancer Res., 2000, 6, 709-717. [5] Kiaris, H.; Schally, A.V.; Varga, J. Neoplasia, 2000, 2, 242-250. [6] Plonowski, A.; Schally, A.V.; Nagy, A.; Kiaris, H.; Hebert, F.; Halmos, G. Cancer Res., 2000, 60, 2996-3001. [7] Kiaris, H.; Schally, A.V.; Armatis, P. Regulatory Peptides, 2001, 96, 119-124. [8] Kiaris, H.; Schally, A.V.; Varga, J.L.; Armatis, P. Cancer Lett., 2000, 161, 149-155. [9] Kiaris, H.; Schally, A.V.; Nagy, A.; Szepeshazi, K.; Halmos, G. Eur. J. Cancer, 2001, 37, 620-628. [10] Kiaris, H.; Schally, A.V.; Busto, R.; Halmos, G.; Artavanis-Tsakonas, S.; Varga, J.L. Proc. Nat. Acad. Sci. USA, 2002, 99, 196-200. [11] Chatzistamou, I.; Schally, A.V.; Pafiti, A.; Kiaris, H.; Koutselini, H. Eur. J. Endocrinol., 2002, 147, 381-386. [12] Kiaris, H.; Chatzistamou, I.; Schally, A.V.; Halmos, G.; Varga, J.L.; Koutselini, H.; Kalofoutis, A. Proc. Nat. Acad. Sci. USA, 2003, 100, 9512-9517. [13] Kiaris, H.; Koutsilieris, M.; Kalofoutis, A.; Schally, A.V. Exp. Opinion Investigational Drugs, 2003, 12, 1385-1394. [14] Kiaris, H.; Chatzistamou, I.; Kalofoutis, C.H.; Koutselini, H.; Piperi, C. Kalofoutis, A. Mol. Cell Biochem., 2004, 261, 117-122. [15] Kiaris, H.; Politi, K.; Grimm, L.M.; Szabolch, M.; Fisher, P.; Efstratiadis, A.; Artavanis-Tsakonas, S. Am. J. Pathol., 2004, 165, 695-705. [16] Kiaris, H.; Schally, A.V.; Kalofoutis, A. (Invited Review) Vitamins & Hormones, 2005, 70, 1-24. [17] Kiaris, H.; Chatzistamou, I.; Trimis, G.; Frangou-Plemmenou, M.; Pafiti, A.; Kalofoutis, A. (Priority Report) Cancer Res., 2005, 65, 1627-1630.