Cathleen Gonzales

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.

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.

Characterization of transient focal ischemia-induced increases in extracellular glutamate and aspartate in spontaneously hypertensive rats

Using middle cerebral artery occlusion (MCAO) and in vivo microdialysis, we have evaluated the changes in extracellular concentrations of the excitatory amino acids (EAA) glutamate and aspartate during varying periods of MCAO (0, 30, 60 min) in the striatum of spontaneously hypertensive rats (SHR). A positive correlation between occlusion time-dependent elevations in EAAs and the resulting ischemic injury was observed. This is the first demonstration of the temporal profile of EAA efflux during transient focal ischemia in SHRs. Possible sources and mechanisms of ischemia-induced EAA efflux were examined during 60 min of MCAO. Removal of Ca(2+) from the microdialysis infusion media significantly attenuated ischemia-induced increases in both glutamate (from ischemic peak of 4892 +/- 1298 to 1144 +/- 666% of preischemic values) and aspartate (from 2703 +/- 682 to 2090 +/- 599% of preischemic values). Similarly, infusion of the voltage dependent Na(+) channel blocker tetrodotoxin (TTX; 10 microM) significantly attenuated MCAO-induced increases in glutamate (to 1313 +/- 648%) and aspartate (to 359 +/- 114%). Infusion of the GLT-1 selective nontransportable inhibitor, dihydrokainate (DHK; 1 mM) also significantly attenuated the ischemia-induced increases in both EAAs (1285 +/- 508 and 1366 +/- 741% of the preischemic levels, respectively). These results indicate that during transient focal ischemia the increase in extracellular EAAs originates from both the neuronal pool, via conventional exocytotic release, and glial sources via the reversal of the GLT-1 transporter.

Pancortin-2 Interacts with WAVE1 and Bcl-xL in a Mitochondria-Associated Protein Complex That Mediates Ischemic Neuronal Death

The actin-modulating protein Wiskott-Aldrich syndrome protein verprolin homologous-1 (WAVE1) and a novel CNS-specific protein, pancortin, are highly enriched in adult cerebral cortex, but their functions are unknown. Here we show that WAVE1 and pancortin-2 interact in a novel cell death cascade in adult, but not embryonic, cerebral cortical neurons. Focal ischemic stroke induces the formation of a protein complex that includes pancortin-2, WAVE1, and the anti-apoptotic protein Bcl-xL. The three-protein complex is associated with mitochondria resulting in increased association of Bax with mitochondria, cytochrome c release, and neuronal apoptosis. In pancortin null mice generated using a Cre-loxP system, ischemia-induced WAVE1–Bcl-xL interaction is diminished, and cortical neurons in these mice are protected against ischemic injury. Thus, pancortin-2 is a mediator of ischemia-induced apoptosis of neurons in the adult cerebral cortex and functions in a novel mitochondrial/actin-associated protein complex that sequesters Bcl-xL.

Temporal assessment of caspase activation in experimental models of focal and global ischemia

Rodent models of focal and global ischemia were used to examine caspase activation. Several readouts were employed on identical tissue to provide correlative measurement of caspase induction, activation and enzymatic activity. In a rat focal ischemia model, caspase-3 enzymatic activity, as recorded by DEVD-AMC cleavage, peaked in penumbral cortex at 6-12 h following ischemia, correlating with increases in caspase 3-cleaved substrates of PARP and alpha-spectrin and subsequent disappearance of caspase-3 zymogen. Although induction of caspases 8 and 2 proteins was detectable as early as 6 h following ischemia, examination of the same tissues for caspase 8 or 2 enzymatic activities did not show significant modulation up to 12 h after ischemic insult. Caspase 9 induction was evident only after 24 h postischemia and did not correlate with elevated LDHD-AMC cleavage. Following global ischemia in gerbils, levels of caspase-3 enzyme activity peaked at 12 h in hippocampal tissue extracts. Cleaved caspase-3 signal was prominent in NeuN-positive layers in the CA1 region 6-12 h following ischemia. Interestingly, strong caspase-3 immunoreactivity was also detected in the subgranular zone of the dentate gyrus, a known region of ischemia-induced neurogenesis. Caspase-3 activation may be responsible for the loss of these cells, thereby hindering the endogenous recovery process.

Utilizing Structures of CYP2D6 and BACE1 Complexes To Reduce Risk of Drug-Drug Interactions with a Novel Series of Centrally Efficacious BACE1 Inhibitors.

In recent years, the first generation of β-secretase (BACE1) inhibitors advanced into clinical development for the treatment of Alzheimer’s disease (AD). However, the alignment of drug-like properties and selectivity remains a major challenge. Herein, we describe the discovery of a novel class of potent, low clearance, CNS penetrant BACE1 inhibitors represented by thioamidine 5. Further profiling suggested that a high fraction of the metabolism (>95%) was due to CYP2D6, increasing the potential risk for victim-based drug–drug interactions (DDI) and variable exposure in the clinic due to the polymorphic nature of this enzyme. To guide future design, we solved crystal structures of CYP2D6 complexes with substrate 5 and its corresponding metabolic product pyrazole 6, which provided insight into the binding mode and movements between substrate/inhibitor complexes. Guided by the BACE1 and CYP2D6 crystal structures, we designed and synthesized analogues with reduced risk for DDI, central efficacy, and improved hERG therapeutic margins.

Neuroprotective Profile of Novel Src Kinase Inhibitors in Rodent Models of Cerebral Ischemia

Src kinase signaling has been implicated in multiple mechanisms of ischemic injury, including vascular endothelial growth factor (VEGF)-mediated vascular permeability that leads to vasogenic edema, a major clinical complication in stroke and brain trauma. Here we report the effects of two novel Src kinase inhibitors, 4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4-methyl-1-piperazinyl)propoxy]-3-quinolinecarbonitrile (SKI-606) and 4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[4-(4-methypiperazin-1-yl)but-1-ynyl]-3-quinolinecarbonitrile (SKS-927), on ischemia-induced brain infarction and short- and long-term neurological deficits. Two well established transient [transient middle cerebral artery occlusion (tMCAO)] and permanent [permanent middle cerebral artery occlusion (pMCAO)] focal ischemia models in the rat were used with drug treatments initiated up to 6 h after onset of stroke to mimic the clinical scenario. Brain penetration of Src inhibitors, their effect on blood-brain barrier integrity and VEGF signaling in human endothelial cells were also evaluated. Our results demonstrate that both agents potently block VEGF-mediated signaling in human endothelial cells, penetrate rat brain upon systemic administration, and inhibit postischemic Src activation and vascular leakage. Treatment with SKI-606 or SKS-927 (at the doses of 3–30 mg/kg i.v.) resulted in a dose-dependent reduction in infarct volume and robust protection from neurological impairments even when the therapy was initiated up to 4- to 6-h after tMCAO. Src blockade after pMCAO resulted in accelerated improvement in recovery from motor, sensory, and reflex deficits during a long-term (3 weeks) testing period poststroke. These data demonstrate that the novel Src kinase inhibitors provide effective treatment against ischemic conditions within a clinically relevant therapeutic window and may constitute a viable therapy for acute stroke.

Discovery of a Series of Efficient, Centrally Efficacious BACE1 Inhibitors through Structure-Based Drug Design.

The identification of centrally efficacious β-secretase (BACE1) inhibitors for the treatment of Alzheimers disease (AD) has historically been thwarted by an inability to maintain alignment of potency, brain availability, and desired absorption, distribution, metabolism, and excretion (ADME) properties. In this paper, we describe a series of truncated, fused thioamidines that are efficiently selective in garnering BACE1 activity without simultaneously inhibiting the closely related cathepsin D or negatively impacting brain penetration and ADME alignment, as exemplified by 36. Upon oral administration, these inhibitors exhibit robust brain availability and are efficacious in lowering central Amyloid β (Aβ) levels in mouse and dog. In addition, chronic treatment in aged PS1/APP mice effects a decrease in the number and size of Aβ-derived plaques. Most importantly, evaluation of 36 in a 2-week exploratory toxicology study revealed no accumulation of autofluorescent material in retinal pigment epithelium or histology findings in the eye, issues observed with earlier BACE1 inhibitors.

Aminomethyl-Derived Beta Secretase (BACE1) Inhibitors: Engaging Gly230 without an Anilide Functionality.

A growing subset of β-secretase (BACE1) inhibitors for the treatment of Alzheimer’s disease (AD) utilizes an anilide chemotype that engages a key residue (Gly230) in the BACE1 binding site. Although the anilide moiety affords excellent potency, it simultaneously introduces a third hydrogen bond donor that limits brain availability and provides a potential metabolic site leading to the formation of an aniline, a structural motif of prospective safety concern. We report herein an alternative aminomethyl linker that delivers similar potency and improved brain penetration relative to the amide moiety. Optimization of this series identified analogues with an excellent balance of ADME properties and potency; however, potential drug–drug interactions (DDI) were predicted based on CYP 2D6 affinities. Generation and analysis of key BACE1 and CYP 2D6 crystal structures identified strategies to obviate the DDI liability, leading to compound 16, which exhibits robust in vivo efficacy as a BACE1 inhibitor.