Magid Abou-Gharbia

Desvenlafaxine Succinate: A New Serotonin and Norepinephrine Reuptake Inhibitor

The purpose of this study was to characterize a new chemical entity, desvenlafaxine succinate (DVS). DVS is a novel salt form of the isolated major active metabolite of venlafaxine. Competitive radioligand binding assays were performed using cells expressing either the human serotonin (5-HT) transporter (hSERT) or norepinephrine (NE) transporter (hNET) with Ki values for DVS of 40.2 ± 1.6 and 558.4 ± 121.6 nM, respectively. DVS showed weak binding affinity (62% inhibition at 100 μM) at the human dopamine (DA) transporter. Inhibition of [3H]5-HT or [3H]NE uptake by DVS for the hSERT or hNET produced IC50 values of 47.3 ± 19.4 and 531.3 ± 113.0 nM, respectively. DVS (10 μM), examined at a large number of nontransporter targets, showed no significant activity. DVS (30 mg/kg orally) rapidly penetrated the male rat brain and hypothalamus. DVS (30 mg/kg orally) significantly increased extracellular NE levels compared with baseline in the male rat hypothalamus but had no effect on DA levels using microdialysis. To mimic chronic selective serotonin reuptake inhibitor treatment and to block the inhibitory 5-HT1A autoreceptors, a 5-HT1A antagonist, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclo hexanecarboxamide maleate salt (WAY-100635) (0.3 mg/kg s.c.), was administered with DVS (30 mg/kg orally). 5-HT increased 78% compared with baseline with no additional increase in NE or DA levels. In conclusion, DVS is a new 5-HT and NE reuptake inhibitor in vitro and in vivo that demonstrates good brain-to-plasma ratios, suggesting utility in a variety of central nervous system-related disorders.

Begacestat (GSI-953): A Novel, Selective Thiophene Sulfonamide Inhibitor of Amyloid Precursor Protein γ-Secretase for the Treatment of Alzheimer's Disease

The presenilin containing γ-secretase complex is responsible for the regulated intramembraneous proteolysis of the amyloid precursor protein (APP), the Notch receptor, and a multitude of other substrates. γ-Secretase catalyzes the final step in the generation of Aβ40 and Aβ42 peptides from APP. Amyloid β-peptides (Aβ peptides) aggregate to form neurotoxic oligomers, senile plaques, and congophilic angiopathy, some of the cardinal pathologies associated with Alzheimers disease. Although inhibition of this protease acting on APP may result in potentially therapeutic reductions of neurotoxic Aβ peptides, nonselective inhibition of the enzyme may cause severe adverse events as a result of impaired Notch receptor processing. Here, we report the preclinical pharmacological profile of GSI-953 (begacestat), a novel thiophene sulfonamide γ-secretase inhibitor (GSI) that selectively inhibits cleavage of APP over Notch. This GSI inhibits Aβ production with low nanomolar potency in cellular and cell-free assays of γ-secretase function, and displaces a tritiated analog of GSI-953 from enriched γ-secretase enzyme complexes with similar potency. Cellular assays of Notch cleavage reveal that this compound is approximately 16-fold selective for the inhibition of APP cleavage. In the human APP-overexpressing Tg2576 transgenic mouse, treatment with this orally active compound results in a robust reduction in brain, plasma, and cerebral spinal fluid Aβ levels, and a reversal of contextual fear-conditioning deficits that are correlated with Aβ load. In healthy human volunteers, oral administration of a single dose of GSI-953 produces dose-dependent changes in plasma Aβ levels, confirming pharmacodynamic activity of GSI-953 in humans.

Enhanced clearance of Aβ in brain by sustaining the plasmin proteolysis cascade

The amyloid hypothesis states that a variety of neurotoxic β-amyloid (Aβ) species contribute to the pathogenesis of Alzheimers disease. Accordingly, a key determinant of disease onset and progression is the appropriate balance between Aβ production and clearance. Enzymes responsible for the degradation of Aβ are not well understood, and, thus far, it has not been possible to enhance Aβ catabolism by pharmacological manipulation. We provide evidence that Aβ catabolism is increased after inhibition of plasminogen activator inhibitor-1 (PAI-1) and may constitute a viable therapeutic approach for lowering brain Aβ levels. PAI-1 inhibits the activity of tissue plasminogen activator (tPA), an enzyme that cleaves plasminogen to generate plasmin, a protease that degrades Aβ oligomers and monomers. Because tPA, plasminogen and PAI-1 are expressed in the brain, we tested the hypothesis that inhibitors of PAI-1 will enhance the proteolytic clearance of brain Aβ. Our data demonstrate that PAI-1 inhibitors augment the activity of tPA and plasmin in hippocampus, significantly lower plasma and brain Aβ levels, restore long-term potentiation deficits in hippocampal slices from transgenic Aβ-producing mice, and reverse cognitive deficits in these mice.

Binding of rapamycin analogs to calcium channels and FKBP52 contributes to their neuroprotective activities

Rapamycin is an immunosuppressive immunophilin ligand reported as having neurotrophic activity. We show that modification of rapamycin at the mammalian target of rapamycin (mTOR) binding region yields immunophilin ligands, WYE-592 and ILS-920, with potent neurotrophic activities in cortical neuronal cultures, efficacy in a rodent model for ischemic stroke, and significantly reduced immunosuppressive activity. Surprisingly, both compounds showed higher binding selectivity for FKBP52 versus FKBP12, in contrast to previously reported immunophilin ligands. Affinity purification revealed two key binding proteins, the immunophilin FKBP52 and the β1-subunit of L-type voltage-dependent Ca2+ channels (CACNB1). Electrophysiological analysis indicated that both compounds can inhibit L-type Ca2+ channels in rat hippocampal neurons and F-11 dorsal root ganglia (DRG)/neuroblastoma cells. We propose that these immunophilin ligands can protect neurons from Ca2+-induced cell death by modulating Ca2+ channels and promote neurite outgrowth via FKBP52 binding.

Discovery of Begacestat, a Notch-1-Sparing γ-Secretase Inhibitor for the Treatment of Alzheimer's Disease

SAR on HTS hits 1 and 2 led to the potent, Notch-1-sparing GSI 9, which lowered brain Abeta in Tg2576 mice at 100 mg/kg po. Converting the metabolically labile methyl groups in 9 to trifluoromethyl groups afforded the more stable analogue 10, which had improved in vivo potency. Further side chain modification afforded the potent Notch-1-sparing GSI begacestat (5), which was selected for development for the treatment of Alzheimers disease.

Comparison of human and rat uterine leiomyomata: identification of a dysregulated mammalian target of rapamycin pathway.

Uterine leiomyomata, or fibroids, are benign tumors of the uterine myometrium that significantly affect up to 30% of reproductive-age women. Despite being the primary cause of hysterectomy in the United States, accounting for up to 200,000 procedures annually, the etiology of leiomyoma remains largely unknown. As a basis for understanding leiomyoma pathogenesis and identifying targets for pharmacotherapy, we conducted transcriptional profiling of leiomyoma and unaffected myometrium from humans and Eker rats, the best characterized preclinical model of leiomyomata. A global comparison of mRNA from leiomyoma versus myometrium in human and rat identified a highly significant overlap of dysregulated gene expression in leiomyomata. An unbiased pathway analysis using a method of gene-set enrichment based on the sigPathway algorithm detected the mammalian target of rapamycin (mTOR) pathway as one of the most highly up-regulated pathways in both human and rat tumors. To validate this pathway as a therapeutic target for uterine leiomyomata, preclinical studies were conducted in Eker rats. These rats develop uterine leiomyomata as a consequence of loss of Tsc2 function and up-regulation of mTOR signaling. Inhibition of mTOR in female Eker rats with the rapamycin analogue WAY-129327 for 2 weeks decreased mTOR signaling and cell proliferation in tumors, and treatment for 4 months significantly decreased tumor incidence, multiplicity, and size. These results identify dysregulated mTOR signaling as a component of leiomyoma etiology across species and directly show the dependence of uterine leiomyomata with activated mTOR on this signaling pathway for growth.

Dehydroepiandrosterone: An anti‐obesity and anti‐carcinogenic agent

Long-term treatment of female C3H-Avy/A (obese) and C3H-A/A (non-obese) mice with dehydroepiandrosterone, an adrenal steroid found in subnormal levels in women predisposed to develop breast cancer, reduces weight gain without suppressing appetite and significantly inhibits the development of spontaneous breast cancer. This steroid also antagonizes the capacity of the tumor promoter, 12-0-tetradecanoyl-phorbol-13-acetate, to stimulate 3H-thymidine incorporation in mouse epidermis and in a cultured rat kidney epithelial cell line.

Resistance to BET Bromodomain Inhibitors Is Mediated by Kinome Reprogramming in Ovarian Cancer

Small-molecule BET bromodomain inhibitors (BETis) are actively being pursued in clinical trials for the treatment of a variety of cancers, but the mechanisms of resistance to BETis remain poorly understood. Using a mass spectrometry approach that globally measures kinase signaling at the proteomic level, we evaluated the response of the kinome to targeted BETi treatment in a panel of BRD4-dependent ovarian carcinoma (OC) cell lines. Despite initial inhibitory effects of BETi, OC cells acquired resistance following sustained treatment with the BETi JQ1. Through application of multiplexed inhibitor beads (MIBs) and mass spectrometry, we demonstrate that BETi resistance is mediated by adaptive kinome reprogramming, where activation of compensatory pro-survival kinase networks overcomes BET protein inhibition. Furthermore, drug combinations blocking these kinases may prevent or delay the development of drug resistance and enhance the efficacy of BETi therapy.

Estrogen Receptor Antagonists Are Anti-Cryptococcal Agents That Directly Bind EF Hand Proteins and Synergize with Fluconazole In Vivo

ABSTRACT Cryptococcosis is an infectious disease of global significance for which new therapies are needed. Repurposing previously developed drugs for new indications can expedite the translation of new therapies from bench to beside. Here, we characterized the anti-cryptococcal activity and antifungal mechanism of estrogen receptor antagonists related to the breast cancer drugs tamoxifen and toremifene. Tamoxifen and toremifene are fungicidal and synergize with fluconazole and amphotericin B in vitro. In a mouse model of disseminated cryptococcosis, tamoxifen at concentrations achievable in humans combines with fluconazole to decrease brain burden by ~1 log10. In addition, these drugs inhibit the growth of Cryptococcus neoformans within macrophages, a niche not accessible by current antifungal drugs. Toremifene and tamoxifen directly bind to the essential EF hand protein calmodulin, as determined by thermal shift assays with purified C. neoformans calmodulin (Cam1), prevent Cam1 from binding to its well-characterized substrate calcineurin (Cna1), and block Cna1 activation. In whole cells, toremifene and tamoxifen block the calcineurin-dependent nuclear localization of the transcription factor Crz1. A large-scale chemical genetic screen with a library of C. neoformans deletion mutants identified a second EF hand-containing protein, which we have named calmodulin-like protein 1 (CNAG_05655), as a potential target, and further analysis showed that toremifene directly binds Cml1 and modulates its ability to bind and activate Cna1. Importantly, tamoxifen analogs (idoxifene and methylene-idoxifene) with increased calmodulin antagonism display improved anti-cryptococcal activity, indicating that calmodulin inhibition can be used to guide a systematic optimization of the anti-cryptococcal activity of the triphenylethylene scaffold. IMPORTANCE Worldwide, cryptococcosis affects approximately 1 million people annually and kills more HIV/AIDS patients per year than tuberculosis. The gold standard therapy for cryptococcosis is amphotericin B plus 5-flucytosine, but this regimen is not readily available in regions where resources are limited and where the burden of disease is highest. Herein, we show that molecules related to the breast cancer drug tamoxifen are fungicidal for Cryptococcus and display a number of pharmacological properties desirable for an anti-cryptococcal drug, including synergistic fungicidal activity with fluconazole in vitro and in vivo, oral bioavailability, and activity within macrophages. We have also demonstrated that this class of molecules targets calmodulin as part of their mechanism of action and that tamoxifen analogs with increased calmodulin antagonism have improved anti-cryptococcal activity. Taken together, these results indicate that tamoxifen is a pharmacologically attractive scaffold for the development of new anti-cryptococcal drugs and provide a mechanistic basis for its further optimization. Worldwide, cryptococcosis affects approximately 1 million people annually and kills more HIV/AIDS patients per year than tuberculosis. The gold standard therapy for cryptococcosis is amphotericin B plus 5-flucytosine, but this regimen is not readily available in regions where resources are limited and where the burden of disease is highest. Herein, we show that molecules related to the breast cancer drug tamoxifen are fungicidal for Cryptococcus and display a number of pharmacological properties desirable for an anti-cryptococcal drug, including synergistic fungicidal activity with fluconazole in vitro and in vivo, oral bioavailability, and activity within macrophages. We have also demonstrated that this class of molecules targets calmodulin as part of their mechanism of action and that tamoxifen analogs with increased calmodulin antagonism have improved anti-cryptococcal activity. Taken together, these results indicate that tamoxifen is a pharmacologically attractive scaffold for the development of new anti-cryptococcal drugs and provide a mechanistic basis for its further optimization.

β-lactamase inhibitors display anti-seizure properties in an invertebrate assay.

Antibiotics containing a beta-lactam ring (e.g. ceftriaxone) display anti-glutamate effects that underlie their efficacy in animal models of central nervous system (CNS) diseases [Rothstein JD, Patel S, Regan MR, Haenggeli C, Huang YH, Bergles DE, Jin L, Dykes Hoberg M, Vidensky S, Chung DS, Toan SV, Bruijn LI, Su ZZ, Gupta P, Fisher PB (2005) Nature 433:73-77]. We hypothesized that the structurally related beta-lactamase inhibitors (clavulanic acid, tazobactam)--which also contain a beta-lactam ring--will mimic ceftriaxone efficacy in an invertebrate (planarian) assay designed to screen for anti-seizure activity [Rawls SM, Thomas T, Adeola M, Patil T, Raymondi N, Poles A, Loo M, Raffa RB (2009) Pharmacol Biochem Behav 93:363-367]. Glutamate or cocaine administration produced planarian seizure-like activity (pSLA). Glutamate- or cocaine-induced pSLA was inhibited by ceftriaxone, clavulanic acid, or tazobactam, but not by the non-beta-lactam antibiotic vancomyocin. The present findings indicate beta-lactamase inhibitors display efficacy, and mimic ceftriaxone activity, in an invertebrate anti-seizure screen. These results suggest beta-lactamase inhibitors--particularly ones such as clavulanic acid that display enhanced brain penetrability, oral bioavailability, and negligible anti-bacterial activity--might offer an attractive alternative to direct antibiotic therapy for managing CNS diseases caused by increased glutamate transmission and provide a solution to the growing concern that ceftriaxone will be of only limited utility as a CNS-active therapeutic because of its intolerable side effects.