Rational Design, Synthesis, and Pharmacological Characterization of Novel Ghrelin Receptor Inverse Agonists as Potential Treatment against Obesity-Related Metabolic Diseases.

A new chemotype of ghrelin inverse agonists was discovered through chimeric design based on molecular scaffolds known as growth-hormone secretagogue receptor (GHSR) modulators but with divergent pharmacodynamic and pharmacokinetic properties. The structure-activities/properties exploration led to compound 47, which displayed potent human GHSR antagonism and inverse agonism in cellular assays (IC50 = 68 nM, EC50 = 29 nM), moderate oral bioavailability, and notable brain penetration in rat ( F = 27%, B/ P ratio = 1.9). First in vivo studies demonstrated effective reduction of food intake after oral or parenteral administration to mouse (78% at 1 h and 38% at 8 h, respectively). Further preclinical studies are needed to evaluate the most suited mode of administration with the aim of promoting a first central-acting ghrelin inverse agonist molecule to development, which would represent a significant step toward therapeutic agents to treat metabolic disorders related to obesity, such as type 2 diabetes mellitus.

Synthesis of structurally diverse major groove DNA interstrand crosslinks using three different aldehyde precursors.

DNA interstrand crosslinks (ICLs) are extremely cytotoxic lesions that block essential cellular processes, such as replication and transcription. Crosslinking agents are widely used in cancer chemotherapy and form an array of structurally diverse ICLs. Despite the clinical success of these agents, resistance of tumors to crosslinking agents, for example, through repair of these lesions by the cellular machinery remains a problem. We have previously reported the synthesis of site-specific ICLs mimicking those formed by nitrogen mustards to facilitate the studies of cellular responses to ICL formation. Here we extend these efforts and report the synthesis of structurally diverse major groove ICLs that induce severe, little or no distortion in the DNA. Our approach employs the incorporation of aldehyde precursors of different lengths into complementary strands and ICL formation using a double reductive amination with a variety of amines. Our studies provide insight into the structure and reactivity parameters of ICL formation by double reductive amination and yield a set of diverse ICLs that will be invaluable for exploring structure-activity relationships in ICL repair.

Selective bypass of a lagging strand roadblock by the eukaryotic replicative DNA helicase.

The eukaryotic replicative DNA helicase, CMG, unwinds DNA by an unknown mechanism. In some models, CMG encircles and translocates along one strand of DNA while excluding the other strand. In others, CMG encircles and translocates along duplex DNA. To distinguish between these models, replisomes were confronted with strand-specific DNA roadblocks in Xenopus egg extracts. An ssDNA translocase should stall at an obstruction on the translocation strand but not the excluded strand, whereas a dsDNA translocase should stall at obstructions on either strand. We found that replisomes bypass large roadblocks on the lagging strand template much more readily than on the leading strand template. Our results indicate that CMG is a 3' to 5' ssDNA translocase, consistent with unwinding via "steric exclusion." Given that MCM2-7 encircles dsDNA in G1, the data imply that formation of CMG in S phase involves remodeling of MCM2-7 from a dsDNA to a ssDNA binding mode.

Structure-dependent bypass of DNA interstrand crosslinks by translesion synthesis polymerases.

DNA interstrand crosslinks (ICLs), inhibit DNA metabolism by covalently linking two strands of DNA and are formed by antitumor agents such as cisplatin and nitrogen mustards. Multiple complex repair pathways of ICLs exist in humans that share translesion synthesis (TLS) past a partially processed ICL as a common step. We have generated site-specific major groove ICLs and studied the ability of Y-family polymerases and Pol ζ to bypass ICLs that induce different degrees of distortion in DNA. Two main factors influenced the efficiency of ICL bypass: the length of the dsDNA flanking the ICL and the length of the crosslink bridging two bases. Our study shows that ICLs can readily be bypassed by TLS polymerases if they are appropriately processed and that the structure of the ICL influences which polymerases are able to read through it.

Using synthetic DNA interstrand crosslinks to elucidate repair pathways and identify new therapeutic targets for cancer chemotherapy.

Many cancer chemotherapeutic agents form DNA interstrand crosslinks (ICLs), extremely cytotoxic lesions that form covalent bonds between two opposing DNA strands, blocking DNA replication and transcription. However, cellular responses triggered by ICLs can cause resistance in tumor cells, limiting the efficacy of such treatment. Here we discuss recent advances in our understanding of the mechanisms of ICL repair that cause this resistance. The recent development of strategies for the synthesis of site-specific ICLs greatly contributed to these insights. Key features of repair are similar for all ICLs, but there is increasing evidence that the specifics of lesion recognition and synthesis past ICLs by DNA polymerases are dependent upon the structure of ICLs. These new insights provide a basis for the improvement of antitumor therapy by targeting DNA repair pathways that lead to resistance to treatment with crosslinking agents.

Generation of DNA interstrand cross-links by post-synthetic reductive amination.

DNA interstrand cross-links (ICLs) are the clinically most relevant adducts formed by many antitumor agents. To facilitate the study of biological responses triggered by ICLs, we developed a new approach toward the synthesis of mimics of nitrogen mustard ICLs. 7-Deazaguanine residues bearing acetaldehyde groups were incorporated into complementary strands of DNA and cross-link formation induced by double reductive amination. Our strategy enables the synthesis of major groove cross-links in high yields and purity.