Current Medicinal Chemistry - Volume 10, Issue 22, 2003

Histone Deacetylases (HDACs) represent a family of enzymes that compete with histone acetyltranferases (HATs) to modulate chromatin structure and regulate transcriptional activity via changes in the acetylation status of nucleosomal histones. The opposing functions of HATs and HDACs in both activating and repressing transcription by controlling the tightness of nucleosomal integrity, reflect the regulatory processes that are involved in turning genes on or off. Several forms of cancer are characterized by an altered expression or mutation of genes that encode HATs or HDACs. Similarly, aberrant repression of genes mediated by HDACs, is associated with the pathogenesis of various types of cancer, including acute leukemia and non-Hodgkin's lymphoma. The resulting alterations in chromatin structure can impringe on normal cellular differentiation and result in tumor onset and progression. Furthermore, HDAC inhibitors from various structural families induce histone hyperacetylation, reactivate suppressed genes and consequently, inhibit the cell cycle, activate differentiation programmes or induce apoptosis. During the past decade, the potent inhibition of tumor progression in rodent models has generated much interest and excitement, but lately the field has come of age, with preliminary successes of HDAC inhibitors in preclinical trials, and even several compounds in Phase I and II clinical trials. In conclusion, HDAC inhibitors are exciting new anti-cancer agents and despite the fact that there is still much to learn about the exact molecular events that underlie their effects, the remarkable tumor specificity of these compounds, and their potency in vitro and in vivo, underscore the potential of HDAC inhibition as a promising strategy for targeted cancer therapy. This Hot Topic issue addresses various approaches towards the identification of potent, potentially subtype specific HDAC inhibitors. Whereas the first generation of HDAC inhibitors has provided much insight in the specific functions of HDACs, the herein reported drug discovery programs focus on second generation HDAC inhibitors, some of which have reached clinical evaluation. Various strategies have been employed by the different research groups, such as co-crystallization, Structure Based Drug Design, peptide chemistry, synthesis of small molecule inhibitors through combinatorial and directed medicinal chemistry. It is hoped that with this selection of topics, the reader is provided with a perspective on some of the most advanced developments of HDAC inhibitors and a better understanding of the potential of HDAC inhibition in the treatment of cancer.

Histone deacetylases (HDACs) are key enzymes in the regulation of gene expression. By maintaining the dynamic equilibrium of the acetylation status of highly conserved lysine residues on histones, they regulate chromatin remodeling and gene expression. A link between aberrant HDAC activity and cancer has been widely reported and HDAC inhibitors have been shown to inhibit the proliferation of human tumor cell lines in vitro. Furthermore, several HDAC inhibitors have exhibited potent anti-tumor activity in human xenograft models, suggesting this class of compounds to be promising novel cancer therapeutic agents. This review provides an update on the current knowledge of HDAC inhibition with a focus on the most recent progress of HDAC inhibitors in clinical development.

Trichostatin A (TSA) is a Streptomyces metabolite that causes differentiation of murine erythroleukemia cells as well as specific inhibition of the cell cycle of some lower eukaryotes and mammalian cells. The targeted molecule of TSA has been shown by genetic and biochemical analyses to be histone deacetylases (HDACs). Histone acetylation is a key modification to control transcription, and HDACs are profoundly involved in pathogenesis of cancer through removing acetyl groups from histones and other transcriptional regulators. Trapoxin (TPX) and FK228 (also known as FR901228 and depsipeptide because FK228 = FR901228 = depsipeptide), structurally unrelated microbial metabolites, were also shown to inhibit HDACs. These HDAC inhibitors cause cell cycle arrest, differentiation and / or apoptosis of many tumors, suggesting their usefulness for chemotherapy and differentiation therapy. In addition, HDAC inhibitors play important roles in identifying the specific function of the enzymes. Indeed, we identified tubulin as one of the substrates of HDAC6 by means of differential sensitivity to HDAC inhibitors. Since recent studies have revealed that HDACs are structurally and functionally diverse, it should be important to develop inhibitors specific to individual enzymes as more promising agents for cancer therapy. We have synthesized novel TSA / TPX hybrids, which will serve as a basis for developing enzyme-specific HDAC inhibitors.

There is a currently growing interest in the development of histone deacetylase inhibitors (HDACs) as anticancer agents. Histone deacetylases are critically important in the functional regulation of gene transcription as well as in chromatin structure remodeling. A number of small molecule inhibitors of HDAC, such as the naturally occurring trichostatin A (TSA), as well as synthetic compounds, such as suberoylanilide hydroxamic acid (SAHA), scriptaid, oxamflatin or MS-275, have been reported to induce differentiation of several cancer cell lines and suppress cell proliferation. This article will review the recent progress being made in our laboratories in the development of two new families of potent HDAC inhibitors: sulfonamide hydroxamic acids and anilides, as well as TSA-like straight chain derivatives. Some of these compounds inhibit partially purified recombinant human HDAC enzymes with IC50's in the micromolar to low nanomolar range and can induce hyperacetylation of histones in human cancer cells. These compounds significantly inhibit proliferation, induce expression of p21WAF1 / Cip1, and cause cell cycle arrest in various human cancer cells. The lead candidates were screened in a panel of human tumor and normal cell lines. The inhibition of HDAC activity represents a novel approach for intervening in cell cycle regulation and may be used in future cancer therapies. The structure-activity relationships, the antiproliferative activity and the in vivo efficacy are discussed.

Histone deacetylase inhibitors have generated significant interest as anti-cancer agents due to their ability to cause growth arrest, terminal differentiation and / or apoptosis in carcinoma cells. Abbott entered this area after the serendipitous discovery of the biaryl hydroxamate A-161906 in a TGFβ mimetic screen and the subsequent identification of this compound as an inhibitor of selected HDACs. The complex biology of these enzymes became evident when cloning and expression of the HDACs demonstrated that they were present as multiprotein and, in some cases, multi-HDAC containing complexes in their active forms. This discovery suggested that any selectivity determinations would have to be considered in the context of these multi-protein / HDAC complexes. However, siRNA gene knockdown studies did demonstrate that reduction of the Class I HDACs resulted in a phenotype similar to that observed with small molecule HDAC inhibitors. Evaluation of the Abbott small molecule HDAC inhibitors utilized a Class I HDAC (HDAC 1 / 2) preparation and antiproliferation assays using HT1080 fibrosarcoma and MDA435 breast carcinoma cells. Characterization of several series of hydroxamic acids indicated that while many of these analogs possessed potent enzymatic and cellular activity, in general these compounds had unacceptable pharmacokinetic profiles and marginal antitumor effects. Replacement of the potentially labile hydroxamic acid moiety with a trifluoromethyl ketone or a ketooxazole gave measurable HDAC potency but only modest cellular and in vivo activity. However, hydroxamate replacement with an α-ketoamide moiety provided potent HDAC inhibitors (IC50 values as low as 3 nM) with excellent cellular activity (IC50 values < 0.2 μM) and measurable anti-tumor activity in a flank tumor growth model.

The natural products trapoxin B and trichostatin A, as well as the novel marine natural product psammaplin A (PSMA) were found in a cell-based screen for compounds that induced the expression of the cyclin dependent kinase inhibitor p21waf1. The mechanism of p21waf1 induction for these compounds was via histone deacetylase (HDAC) inhibition. Of these compounds, PSMA was of interest because of its novel structure, but the physiological stability of it, and its analogs was poor. Thus, a directed medicinal chemistry effort was undertaken to prepare analogs of the simple HDAC inhibitor dimethylaminobenzamidylcaprylic hydroxamate (DBCH), which led to chromenone amide 9. This compound was efficacious in the HCT116 colon xenograft assay, but was difficult to formulate in pharmaceutically acceptable vehicles. In parallel with these efforts, a screen of the Novartis compound archive for novel HDAC inhibitors uncovered the cinnamyl hydroxamic acid NVP-LAK974. This compound had good enzyme and cellular potency, but poor efficacy in vivo. A systematic structural exploration of cinnamyl hydroxamates based on NVP-LAK974 was undertaken with the goal of finding a novel, well-tolerated and efficacious HDAC inhibitor. Several derivatives were found to be efficacious in the xenograft assay. Of those compounds, NVP-LAQ824 distinguished itself due to its tolerability, efficacy and potency. Based, in part, on these properties, NVP-LAQ824 is currently undergoing human clinical trials as a novel anti-cancer agent.

Lead identification and optimization is always a challenge to the medicinal chemists in drug discovery. Numbers of simple to complex and smaller to bigger organic compounds are prepared to meet the screening purpose of biological targets. Conventional solution phase synthetic methodologies are lacking the speed to run along with the need of medicinally interesting compounds due to their long reaction time, tedious work-up and purification problems. Alternatively opted polymersupported synthesis of combinatorial libraries has been emerged as a promising tool in generating large numbers of structurally diverse molecules in a manner rapidand parallel. Microwave-assisted solid / liquid phase combinatorial synthetic techniques have been proved efficient in reducing the reaction time from days & hours to minutes & seconds and more promisingly to produce improved yields with high purities. This review briefs about the theory behind microwave chemical technology and glimpses of recent advancements in its application on polymer supported combinatorial synthesis.

Since their introduction as bilayer-forming synthetic compounds in the eighties, dioctadecyldimethylammonium (DODA) and dihexadecylphosphate (DHP) salts have found many uses in strategic, applied areas. In particular, DODA chloride or bromide vesicles interacted with negatively charged prokaryotic or eukaryotic cells, yielding adsorption isotherms of high affinity for the cell surface, causing cell adhesion and flocculation, changing the cell surface charge from negative to positive, and causing loss of cell viability over DODA concentration ranges that depended on the cell type being tested. This work reviews data on DODA effects on cell viability (bacteria, fungus and cultured mammalian cells) to propose DODA salts as effective anti-microbial agents that exhibit differential cytotoxicity in vitro and, therefore, deserve to be investigated as potential drugs. The full utility of these inexpensive synthetic bilayers and bilayer fragments able to act as drugs themselves and, simultaneously, as drug, gene or vaccine carriers remains hitherto unexplored.

Specific targeting of radionuclides is a promising approach to improve diagnosis and treatment of tumors. Targeting vectors may be monoclonal antibodies directed toward tumour-specific antigens or regulatory peptides binding to receptors overexpressed on or by malignant cells. Depending on the aim of the procedure and the biokinetics of the targeting vectors, radionuclides with different nuclear properties (decay scheme, half-life, etc.) must be applied. Halogen radioisotopes are attractive since they exhibit a variety of nuclear properties suitable for various applications. At the same time, their chemistry shows great similarities, which enables the use of similar labelling procedures for different nuclides. A problem in using radiohalogens for labelling of tumour-targeting proteins and peptides is that the commonly used radiohalogenation methods provide labels, which, after internalisation and lysosomal digestion, rapidly “leak” from malignant cells as radiohalogenated degradation products. The main reason for such leakage is free diffusion of the radiometabolites through lysosomal and cellular membranes. This review describes current approaches in molecular design to improve cellular retention of radiohalogen labels. These approaches include the use of prosthetic groups for the attachment of radiohalogens to targeting vectors of bulky hydrophilic non-charged molecules, molecules positively charged at lysosomal pH and negatively charged molecules. The emphasis in this paper is on labelling chemistry and the results of the biological testing of labelled compounds.

The von Hippel-Lindau (VHL) protein is able to suppress tumor growth and to down-regulate many angiogenic factors, and is ubiquitously detected in adult and fetal tissues. This makes VHL an excellent target for therapeutic intervention. Observation of VHL alterations in sporadic tumors has been increasing as a result of examination of abnormalities other than intragenic mutations. These abnormalities include loss of chromosome 3p25, changes in the promoter, down-regulation of transcript, and changes in protein level. This article also presents the finding of differential expression of two common VHL proteins among rat tissues, suggesting tissue- and development-dependent functional difference between these two isoforms. Molecular pathways linking VHL to angiogenesis have been extensively characterized and mechanisms have been proposed to explain how altered VHL leads to tumorigenesis. VHL functions in the presence of oxygen and / or oxygen species. Two strategies are proposed here for anti-tumor and anti-angiogenic treatments of VHL-deficient tumors and those without detectable VHL intragenic mutations. One is to restore wild-type VHL function in VHL-deficient tumors and the other is to enhance wild-type VHL expression and activity in tumors under hypoxic conditions.

Glucagon-like peptide-1-(7-36)-amide (GLP-1) is a potent blood glucose-lowering hormone now under investigation for use as a therapeutic agent in the treatment of type 2 (adult onset) diabetes mellitus. GLP-1 binds with high affinity to G protein-coupled receptors (GPCRs) located on pancreatic β-cells, and it exerts insulinotropic actions that include the stimulation of insulin gene transcription, insulin biosynthesis, and insulin secretion. The beneficial therapeutic action of GLP-1 also includes its ability to act as a growth factor, stimulating formation of new pancreatic islets (neogenesis) while slowing β-cell death (apoptosis). GLP- 1 belongs to a large family of structurally-related hormones and neuropeptides that include glucagon, secretin, GIP, PACAP, and VIP. Biosynthesis of GLP-1 occurs in the enteroendocrine L-cells of the distal intestine, and the release of GLP-1 into the systemic circulation accompanies ingestion of a meal. Although GLP-1 is inactivated rapidly by dipeptidyl peptidase IV (DDP-IV), synthetic analogs of GLP-1 exist, and efforts have been directed at engineering these peptides so that they are resistant to enzymatic hydrolysis. Additional modifications of GLP-1 incorporate fatty acylation and drug affinity complex (DAC) technology to improve serum albumin binding, thereby slowing renal clearance of the peptides. NN2211, LY315902, LY307161, and CJC-1131 are GLP-1 synthetic analogs that reproduce many of the biological actions of GLP-1, but with a prolonged duration of action. AC2993 (Exendin-4) is a naturally occurring peptide isolated from the lizard Heloderma, and it acts as a high affinity agonist at the GLP-1 receptor. This review summarizes structural features and signal transduction properties of GLP-1 and its cognate β-cell GPCR. The usefulness of synthetic GLP-1 analogs as blood glucose-lowering agents is discussed, and the applicability of GLP-1 as a therapeutic agent for treatment of type 2 diabetes is highlighted.