Lisa Hunihan

RATIONAL DESIGN OF POTENT CARBOXYLIC ACID BASED BISUBSTRATE INHIBITORS OF RAS FARNESYL PROTEIN TRANSFERASE

Abstract Bisubstrate analog inhibitors in which a substrate mimetic tripeptide is attached to a homologated farnesyl carboxylic acid were synthesized and evaluated for in vitro inhibition versus ras farnesyl protein transferase (FPT). Our results demonstrate that such bisubstrate analogs are potent inhibitors of FPT.

Reversal of the Swedish familial Alzheimer's disease mutant phenotype in cultured cells treated with phorbol 12,13-dibutyrate.

Protein phosphorylation mediated by phorbol ester stimulates secretion of the beta-amyloid precursor protein (beta-APP) in the cell culture. This increase in secretion is produced by a transient increase in cleavage to produce non-amyloidogenic protease nexin II products mediated by the alpha-secretase activity, and a concomitant decrease in beta-protein production. Cells expressing the Swedish familial Alzheimers disease (FAD) variant of beta-APP produce more beta-protein and potentially amyloidogenic fragments than cells expressing wild-type protein; furthermore, cleavage shifts from the alpha- to the beta-secretase cleavage site of the precursor. We show that treatment with phorbol 12,13-dibutyrate (PDBu) of cells expressing the Swedish FAD reverses the mutant phenotype to wild-type. The alpha-secretase cleavage increases with a concomitant loss of beta-protein and other beta-secretase cleaved products. These results show that modulating beta-secretase cleavage directly affects beta-protein production. It suggests that activating protein kinase C through, for example, muscarinic receptor agonists could reduce amyloidosis by modulating the level of beta-protein produced.

Molecular characterization and identification of surrogate substrates for diacylglycerol lipase α

Diacylglycerol lipase α is the key enzyme in the formation of the most prevalent endocannabinoid, 2-arachidonoylglycerol in the brain. In this study we identified the catalytic triad of diacylglycerol lipase α, consisting of serine 472, aspartate 524 and histidine 650. A truncated version of diacylglycerol lipase α, spanning residues 1-687 retains complete catalytic activity suggesting that the C-terminal domain is not required for catalysis. We also report the discovery and the characterization of fluorogenic and chromogenic substrates for diacylglycerol lipase α. Assays performed with these substrates demonstrate equipotent inhibition of diacylglycerol lipase α by tetrahydrolipastatin and RHC-20867 as compared to reactions performed with the native diacylglycerol substrate. Thus, confirming the utility of assays using these substrates for identification and kinetic characterization of inhibitors from pharmaceutical collections.

Discovery of D1 Dopamine Receptor Positive Allosteric Modulators: Characterization of Pharmacology and Identification of Residues that Regulate Species Selectivity

The present studies represent the first published report of a dopamine D1 positive allosteric modulator (PAM). D1 receptors have been proposed as a therapeutic target for the treatment of cognitive deficits associated with schizophrenia. However, the clinical utility of orthosteric agonist compounds is limited by cardiovascular side effects, poor pharmacokinetics, lack of D1 selectivity, and an inverted dose response. A number of these challenges may be overcome by utilization of a selective D1 PAM. The current studies describe two chemically distinct D1 PAMs: Compound A [1-((rel-1S,3R,6R)-6-(benzo[d][1,3]dioxol-5-yl)bicyclo[4.1.0]heptan-3-yl)-4-(2-bromo-5-chlorobenzyl)piperazine] and Compound B [rel-(9R,10R,12S)-N-(2,6-dichloro-3-methylphenyl)-12-methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxamide]. Compound A shows pure PAM activity, with an EC50 of 230 nM and agonist activity at the D2 receptor in D2-expressing human embryonic kidney cells. Compound B shows superior potency (EC50 of 43 nM) and selectivity for D1 versus D2 dopamine receptors. Unlike Compound A, Compound B is selective for human and nonhuman primate D1 receptors, but lacks activity at the rodent (rat and mouse) D1 receptors. Using molecular biology techniques, a single amino acid was identified at position 130, which mediates the species selectivity of Compound B. These data represent the first described D1-selective PAMs and define critical amino acids that regulate species selectivity.

In vitro Characterization of a small molecule inhibitor of the alanine serine cysteine transporter -1 (SLC7A10).

NMDA receptor hypofunction is hypothesized to contribute to cognitive deficits associated with schizophrenia. Since direct activation of NMDA receptors is associated with serious adverse effects, modulation of the NMDA co‐agonists, glycine or D‐serine, represents a viable alternative therapeutic approach. Indeed, clinical trials with glycine and D‐serine have shown positive results, although concerns over toxicity related to the high‐doses required for efficacy remain. Synaptic concentrations of D‐serine and glycine are regulated by the amino acid transporter alanine serine cysteine transporter‐1 (asc‐1). Inhibition of asc‐1 would increase synaptic D‐serine and possibly glycine, eliminating the need for high‐dose systemic D‐serine or glycine treatment. In this manuscript, we characterize Compound 1 (BMS‐466442), the first known small molecule inhibitor of asc‐1. Compound 1 selectively inhibited asc‐1 mediated D‐serine uptake with nanomolar potency in multiple cellular systems. Moreover, Compound 1 inhibited asc‐1 but was not a competitive substrate for this transporter. Compound 1 is the first reported selective inhibitor of the asc‐1 transporter and may provide a new path for the development of asc‐1 inhibitors for the treatment of schizophrenia.

Generation of a clonal induced pluripotent stem cell (iPSC) line expressing the mutant MECP2 allele from a Rett Syndrome patient fibroblast line

Human fibroblast cells collected from a 3-year old, female Rett Syndrome patient with a 32bp deletion in the X-linked MECP2 gene were obtained from the Coriell Institute. Fibroblasts were reprogrammed to iPSC cells using a Sendai-virus delivery system expressing human KOSM transcription factors. Cell-line pluripotency was demonstrated by gene expression, immunocytochemistry, in-vitro differentiation trilineage capacity and was of normal karyotype. Interestingly, subsequent clones retained the epigenetic memory of the parent fibroblasts allowing for the segregation of wild-type and mutant expressing clones. This MECP2 mutant expressing clone may serve as a model for investigating MECP2 reactivation in Retts Syndrome.

Transgenic Rat and In-Vitro Studies of β-Amyloid Precursor Protein Processing

The brains of individuals afflicted with Alzheimer’s Disease (AD) are characterized pathologically by cortical atrophy, deposition of senile plaques (i.e. s-amyloid protein), and the formation of neurofibrillary tangles. The etiology of AD is complex and multifactorial; including evidence for genetic heterogeneity. However, genetic studies have clearly shown that several different mutations in the s-APP gene on chromosome 21 are unambiguously pathogenic for AD in a subset of early-onset families.1–9 The mechanism by which s-protein is generated and formed into amyloid in-vivo have yet to be defined. Aside from the genetic data, putative pathological mechanisms may include overexpression of s-APP, as suggested by trisomy 21 or Down’s Syndrome individuals; or alterations in the proteolytic processing pathways, currently being defined in-vitro by a number of laboratories. To date no animal model exists that can recapitulate the pathological cascade of AD.