Elucidation of novel TRAP1-Selective inhibitors that regulate mitochondrial processes.

Hsp90 isoform-selective inhibitors represent a new paradigm for novel anti-cancer drugs as each of the four isoforms have specific cellular localization, function, and client proteins. The mitochondrial isoform, TRAP1, is the least understood member of the Hsp90 family due to the lack of small molecule tools to study its biological function. Herein, we report novel TRAP1-selective inhibitors used to interrogate TRAP1's biological function along with co-crystal structures of such compounds bound to the N-terminus of TRAP1. Solution of the co-crystal structure allowed for a structure-based approach that resulted in compound 36, which is a 40 nM inhibitor with >250-fold TRAP1 selectivity over Grp94, the isoform with the highest structural similarity to TRAP1 within the N-terminal ATP binding site. Lead compounds 35 and 36 were found to selectively induce TRAP1 client protein degradation without inducing the heat shock response or disrupting Hsp90-cytosolic clients. They were also shown to inhibit OXPHOS, alter cellular metabolism towards glycolysis, disrupt TRAP1 tetramer stability, and disrupt the mitochondrial membrane potential.

A Split Renilla Luciferase Complementation Assay for the Evaluation of Hsp90/Aha1 Complex Disruptors and Their Activity at the Aha1 C-Terminal Domain.

Disruption of interactions between Hsp90 and the cochaperone protein, Aha1, has emerged as a therapeutic strategy to inhibit Aha1-driven cancer metastasis and tau aggregation in models of tauopathy. A combination of split Renilla luciferase assays was developed to screen and quantify the ability of small molecules to disrupt interactions between Hsp90 and both full length Aha1 protein (Aha1-FL) and the Aha1 C-terminal domain (Aha1-CTD). This luminescence-based approach was used to identify withaferin A and gedunin as disruptors of Hsp90/Aha1 interactions and provided insight into the binding regions for gambogic acid and gedunin on the Hsp90 homodimer. All compounds tested that disrupted Hsp90/Aha1-CTD interactions were found to disrupt interactions between Hsp90 and Aha1-FL, suggesting that interactions between Hsp90 and the Aha1-CTD play a key role in the stability of Hsp90/Aha1 complexes.

Synthesis and Evaluation of Small Molecule Disruptors of the Aha1/Hsp90 Complex for the Reduction of Tau Aggregation.

KU-177 was recently shown to disrupt interactions between Hsp90 and Aha1 in vitro. Subsequent studies in recombinant thioflavin T (ThT) assays demonstrated that KU-177 ablates Aha1-driven enhancement of Hsp90-dependent tau aggregation, which was confirmed by TEM. Using KU-177 as a lead compound, derivatives of KU-177 were synthesized and evaluated for their ability to disrupt Aha1/Hsp90 interactions and inhibit P301L tau aggregation. Preliminary structure-activity relationships were revealed, which led to the identification of a new lead compound that contains a cis-like amide bond. The new lead compounds retain the ability to disrupt Aha1/Hsp90 interactions in SH-SY5Y and SK-BR-3 cells without direct inhibition of Hsp90, providing a new scaffold for subsequent drug discovery efforts.

Chronic phenmetrazine treatment promotes D2 dopaminergic and α2-adrenergic receptor desensitization and alters phosphorylation of signaling proteins and local cerebral glucose metabolism in the rat brain.

Phenmetrazine (PHEN) is a putative treatment for cocaine and psychostimulant recidivism; however, neurochemical changes underlying its activity have not been fully elucidated. We sought to characterize brain homeostatic adaptations to chronic PHEN, specifically on functional brain activity (local cerebral glucose utilization), G-Protein Coupled Receptor-stimulated G-protein activation, and phosphorylation of ERK1/2Thr202/Tyr204, GSK3ßTyr216, and DARPP-32Thr34. Male Sprague-Dawley rats were implanted with sub-cutaneous minipumps delivering either saline (vehicle), acute (2-day) or chronic (14-day) low dose (25 mg/kg/day) or high dose (50 mg/kg/day) PHEN. Acute administration of high dose PHEN increased local cerebral glucose utilization measured by 2-[14C]-deoxyglucose uptake in basal ganglia and motor-related regions of the rat brain. However, chronically treated animals developed tolerance to these effects. To identify the neurochemical changes associated with PHEN's activity, we performed [35S]GTPγS binding assays on unfixed and immunohistochemistry on fixed coronal brain sections. Chronic PHEN treatment dose-dependently attenuated D2 dopamine and α2-adrenergic, but not 5-HT1A, receptor-mediated G-protein activation. Two distinct patterns of effects on pERK1/2 and pDARPP-32 were observed: 1) chronic low dose PHEN decreased pERK1/2, and also significantly increased pDARPP-32 levels in some regions; 2) acute and chronic PHEN increased pERK1/2, but chronic high dose PHEN treatment tended to decrease pDARPP-32. Chronic low dose, but not high dose, PHEN significantly reduced pGSK3ß levels in several regions. Our study provides definitive evidence that extended length PHEN dosage schedules elicit distinct modes of neuronal acclimatization in cellular signaling. These pharmacodynamic modifications should be considered in drug development for chronic use.

The role and therapeutic potential of Hsp90, Hsp70, and smaller heat shock proteins in peripheral and central neuropathies.

Heat shock proteins (Hsps) are molecular chaperones that also play important roles in the activation of the heat shock response (HSR). The HSR is an evolutionary conserved and protective mechanism that is used to counter abnormal physiological conditions, stressors, and disease states, such as those exemplified in cancer and/or neurodegeneration. In normal cells, heat shock factor-1 (HSF-1), the transcription factor that regulates the HSR, remains in a dormant multiprotein complex that is formed upon association with chaperones (Hsp90, Hsp70, etc.), co-chaperones, and client proteins. However, under cellular stress, HSF-1 dissociates from Hsp90 and induces the transcriptional upregulation of Hsp70 to afford protection against the encountered cellular stress. As a consequence of both peripheral and central neuropathies, cellular stress occurs and results in the accumulation of unfolded and/or misfolded proteins, which can be counterbalanced by activation of the HSR. Since Hsp90 is the primary regulator of the HSR, modulation of Hsp90 by small molecules represents an attractive therapeutic approach against both peripheral and central neuropathies.

Assay design and development strategies for finding Hsp90 inhibitors and their role in human diseases.

Heat shock protein 90 (Hsp90) is a molecular chaperone that facilitates the maturation of its client proteins including protein kinases, transcription factors, and steroid hormone receptors which are structurally and functionally diverse. These client proteins are involved in various cellular signaling pathways, and Hsp90 is implicated in various human diseases including cancer, inflammation, and diseases associated with protein misfolding; thus making Hsp90 a promising target for drug discovery. Some of its client proteins are well-known cancer targets. Instead of targeting these client proteins individually, however, targeting Hsp90 is more practical for cancer drug development. Efforts have been invested in recognizing potential drugs for clinical use that inhibit Hsp90 activity and result in the prevention of Hsp90 client maturation and dampening of subsequent signaling cascades. Here, we discuss current assays and technologies used to find and characterize Hsp90 inhibitors that include biophysical, biochemical, cell-based assays and computational modeling. This review highlights recent discoveries that N-terminal isoform-selective compounds and inhibitors that target the Hsp90 C-terminus that may offer the potential to overcome some of the detriments observed with pan Hsp90 inhibitors. The tools and assays summarized in this review should be used to develop Hsp90-targeting drugs with high specificity, potency, and drug-like properties that may prove immensely useful in the clinic.

Discovery of Novel Hsp90 C-Terminal Inhibitors Using 3D-Pharmacophores Derived from Molecular Dynamics Simulations.

Hsp90 C-terminal domain (CTD) inhibitors are promising novel agents for cancer treatment, as they do not induce the heat shock response associated with Hsp90 N-terminal inhibitors. One challenge associated with CTD inhibitors is the lack of a co-crystallized complex, requiring the use of predicted allosteric apo pocket, limiting structure-based (SB) design approaches. To address this, a unique approach that enables the derivation and analysis of interactions between ligands and proteins from molecular dynamics (MD) trajectories was used to derive pharmacophore models for virtual screening (VS) and identify suitable binding sites for SB design. Furthermore, ligand-based (LB) pharmacophores were developed using a set of CTD inhibitors to compare VS performance with the MD derived models. Virtual hits identified by VS with both SB and LB models were tested for antiproliferative activity. Compounds 9 and 11 displayed antiproliferative activities in MCF-7 and Hep G2 cancer cell lines. Compound 11 inhibited Hsp90-dependent refolding of denatured luciferase and induced the degradation of Hsp90 clients without the concomitant induction of Hsp70 levels. Furthermore, compound 11 offers a unique scaffold that is promising for the further synthetic optimization and development of molecules needed for the evaluation of the Hsp90 CTD as a target for the development of anticancer drugs.

An Isoform-Selective PTP1B Inhibitor Derived from Nitrogen-Atom Augmentation of Radicicol.

A library of natural products and their derivatives was screened for inhibition of protein tyrosine phosphatase (PTP) 1B, which is a validated drug target for the treatment of obesity and type II diabetes. Of those active in the preliminary assay, the most promising was compound 2 containing a novel pyrrolopyrazoloisoquinolone scaffold derived by treating radicicol (1) with hydrazine. This nitrogen-atom augmented radicicol derivative was found to be PTP1B selective relative to other highly homologous nonreceptor PTPs. Biochemical evaluation, molecular docking, and mutagenesis revealed 2 to be an allosteric inhibitor of PTP1B with a submicromolar Ki. Cellular analyses using C2C12 myoblasts indicated that 2 restored insulin signaling and increased glucose uptake.

Cannabinoid Receptor Interacting Protein 1a Competition with ß-Arrestin for CB1 Receptor Binding Sites.

Cannabinoid receptor interacting protein 1a (CRIP1a) is a CB1 receptor (CB1R) distal C-terminal-associated protein that alters CB1R interactions with G-proteins. We tested the hypothesis that CRIP1a is capable of also altering CB1R interactions with ß-arrestin proteins that interact with the CB1R at the C-terminus. Coimmunoprecipitation studies indicated that CB1R associates in complexes with either CRIP1a or ß-arrestin, but CRIP1a and ß-arrestin fail to coimmunoprecipitate with each other. This suggests a competition for CRIP1a and ß-arrestin binding to the CB1R, which we hypothesized could attenuate the action of ß-arrestin to mediate CB1R internalization. We determined that agonist-mediated reduction of the density of cell surface endogenously expressed CB1Rs was clathrin and dynamin dependent and could be modeled as agonist-induced aggregation of transiently expressed GFP-CB1R. CRIP1a overexpression attenuated CP55940-mediated GFP-CB1R as well as endogenous ß-arrestin redistribution to punctae, and conversely, CRIP1a knockdown augmented ß-arrestin redistribution to punctae. Peptides mimicking the CB1R C-terminus could bind to both CRIP1a in cell extracts as well as purified recombinant CRIP1a. Affinity pull-down studies revealed that phosphorylation at threonine-468 of a CB1R distal C-terminus 14-mer peptide reduced CB1R-CRIP1a association. Coimmunoprecipitation of CB1R protein complexes demonstrated that central or distal C-terminal peptides competed for the CB1R association with CRIP1a, but that a phosphorylated central C-terminal peptide competed for association with ß-arrestin 1, and phosphorylated central or distal C-terminal peptides competed for association with ß-arrestin 2. Thus, CRIP1a can compete with ß-arrestins for interaction with C-terminal CB1R domains that could affect agonist-driven, ß-arrestin-mediated internalization of the CB1R.

Chronic baclofen desensitizes GABA(B)-mediated G-protein activation and stimulates phosphorylation of kinases in mesocorticolimbic rat brain.

The GABAB receptor is a therapeutic target for CNS and neuropathic disorders; however, few preclinical studies have explored effects of chronic stimulation. This study evaluated acute and chronic baclofen treatments on GABAB-activated G-proteins and signaling protein phosphorylation as indicators of GABAB signaling capacity. Brain sections from rats acutely administered baclofen (5 mg/kg, i.p.) showed no significant differences from controls in GABAB-stimulated GTPγS binding in any brain region, but displayed significantly greater phosphorylation/activation of focal adhesion kinase (pFAK(Tyr397)) in mesocorticolimbic regions (caudate putamen, cortex, hippocampus, thalamus) and elevated phosphorylated/activated glycogen synthase kinase 3-ß (pGSK3ß(Tyr216)) in the prefrontal cortex, cerebral cortex, caudate putamen, nucleus accumbens, thalamus, septum, and globus pallidus. In rats administered chronic baclofen (5 mg/kg, t.i.d. for five days), GABAB-stimulated GTPγS binding was significantly diminished in the prefrontal cortex, septum, amygdala, and parabrachial nucleus compared to controls. This effect was specific to GABAB receptors: there was no effect of chronic baclofen treatment on adenosine A1-stimulated GTPγS binding in any region. Chronically-treated rats also exhibited increases in pFAK(Tyr397) and pGSK3ß(Tyr216) compared to controls, and displayed wide-spread elevations in phosphorylated dopamine- and cAMP-regulated phosphoprotein-32 (pDARPP-32(Thr34)) compared to acutely-treated or control rats. We postulate that those neuroadaptive effects of GABAB stimulation mediated by G-proteins and their sequelae correlate with tolerance to several of baclofen's effects, whereas sustained signaling via kinase cascades points to cross-talk between GABAB receptors and alternative mechanisms that are resistant to desensitization. Both desensitized and sustained signaling pathways should be considered in the development of pharmacotherapies targeting the GABA system.