Dissociating movement from movement timing in the rat primary motor cortex.

Neural encoding of the passage of time to produce temporally precise movements remains an open question. Neurons in several brain regions across different experimental contexts encode estimates of temporal intervals by scaling their activity in proportion to the interval duration. In motor cortex the degree to which this scaled activity relies upon afferent feedback and is guided by motor output remains unclear. Using a neural reward paradigm to dissociate neural activity from motor output before and after complete spinal transection, we show that temporally scaled activity occurs in the rat hindlimb motor cortex in the absence of motor output and after transection. Context-dependent changes in the encoding are plastic, reversible, and re-established following injury. Therefore, in the absence of motor output and despite a loss of afferent feedback, thought necessary for timed movements, the rat motor cortex displays scaled activity during a broad range of temporally demanding tasks similar to that identified in other brain regions.

HSF1 Pathway Inhibitor Clinical Candidate (CCT361814/NXP800) Developed from a Phenotypic Screen as a Potential Treatment for Refractory Ovarian Cancer and Other Malignancies.

CCT251236 1, a potent chemical probe, was previously developed from a cell-based phenotypic high-throughput screen (HTS) to discover inhibitors of transcription mediated by HSF1, a transcription factor that supports malignancy. Owing to its activity against models of refractory human ovarian cancer, 1 was progressed into lead optimization. The reduction of P-glycoprotein efflux became a focus of early compound optimization; central ring halogen substitution was demonstrated by matched molecular pair analysis to be an effective strategy to mitigate this liability. Further multiparameter optimization led to the design of the clinical candidate, CCT361814/NXP800 22, a potent and orally bioavailable fluorobisamide, which caused tumor regression in a human ovarian adenocarcinoma xenograft model with on-pathway biomarker modulation and a clean in vitro safety profile. Following its favorable dose prediction to human, 22 has now progressed to phase 1 clinical trial as a potential future treatment for refractory ovarian cancer and other malignancies.

Gene and protein expression profiling of human ovarian cancer cells treated with the heat shock protein 90 inhibitor 17-allylamino-17-demethoxygeldanamycin.

The promising antitumor activity of 17-allylamino-17-demethoxygeldanamycin (17AAG) results from inhibition of the molecular chaperone heat shock protein 90 (HSP90) and subsequent degradation of multiple oncogenic client proteins. Gene expression microarray and proteomic analysis were used to profile molecular changes in the A2780 human ovarian cancer cell line treated with 17AAG. Comparison of results with an inactive analogue and an alternative HSP90 inhibitor radicicol indicated that increased expression of HSP72, HSC70, HSP27, HSP47, and HSP90beta at the mRNA level were on-target effects of 17AAG. HSP27 protein levels were increased in tumor biopsies following treatment of patients with 17AAG. A group of MYC-regulated mRNAs was decreased by 17AAG. Of particular interest and novelty were changes in expression of chromatin-associated proteins. Expression of the heterochromatin protein 1 was increased, and expression of the histone acetyltransferase 1 and the histone arginine methyltransferase PRMT5 was decreased by 17AAG. PRMT5 was shown to be a novel HSP90-binding partner and potential client protein. Cellular protein acetylation was reduced by 17AAG, which was shown to have an antagonistic interaction on cell proliferation with the histone deacetylase inhibitor trichostatin A. This mRNA and protein expression analysis has provided new insights into the complex molecular pharmacology of 17AAG and suggested new genes and proteins that may be involved in response to the drug or be potential biomarkers of drug action.

In vitro biological characterization of a novel, synthetic diaryl pyrazole resorcinol class of heat shock protein 90 inhibitors.

The molecular chaperone heat shock protein 90 (HSP90) has emerged as an exciting molecular target. Derivatives of the natural product geldanamycin, such as 17-allylamino-17-demethoxy-geldanamycin (17-AAG), were the first HSP90 ATPase inhibitors to enter clinical trial. Synthetic small-molecule HSP90 inhibitors have potential advantages. Here, we describe the biological properties of the lead compound of a new class of 3,4-diaryl pyrazole resorcinol HSP90 inhibitor (CCT018159), which we identified by high-throughput screening. CCT018159 inhibited human HSP90beta with comparable potency to 17-AAG and with similar ATP-competitive kinetics. X-ray crystallographic structures of the NH(2)-terminal domain of yeast Hsp90 complexed with CCT018159 or its analogues showed binding properties similar to radicicol. The mean cellular GI(50) value of CCT018159 across a panel of human cancer cell lines, including melanoma, was 5.3 mumol/L. Unlike 17-AAG, the in vitro antitumor activity of the pyrazole resorcinol analogues is independent of NQO1/DT-diaphorase and P-glycoprotein expression. The molecular signature of HSP90 inhibition, comprising increased expression of HSP72 protein and depletion of ERBB2, CDK4, C-RAF, and mutant B-RAF, was shown by Western blotting and quantified by time-resolved fluorescent-Cellisa in human cancer cell lines treated with CCT018159. CCT018159 caused cell cytostasis associated with a G(1) arrest and induced apoptosis. CCT018159 also inhibited key endothelial and tumor cell functions implicated in invasion and angiogenesis. Overall, we have shown that diaryl pyrazole resorcinols exhibited similar cellular properties to 17-AAG with potential advantages (e.g., aqueous solubility, independence from NQO1 and P-glycoprotein). These compounds form the basis for further structure-based optimization to identify more potent inhibitors suitable for clinical development.

Inhibition of the heat shock protein 90 molecular chaperone in vitro and in vivo by novel, synthetic, potent resorcinylic pyrazole/isoxazole amide analogues.

Although the heat shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) shows clinical promise, potential limitations encourage development of alternative chemotypes. We discovered the 3,4-diarylpyrazole resorcinol CCT018159 by high-throughput screening and used structure-based design to generate more potent pyrazole amide analogues, exemplified by VER-49009. Here, we describe the detailed biological properties of VER-49009 and the corresponding isoxazole VER-50589. X-ray crystallography showed a virtually identical HSP90 binding mode. However, the dissociation constant (K(d)) of VER-50589 was 4.5 +/- 2.2 nmol/L compared with 78.0 +/- 10.4 nmol/L for VER-49009, attributable to higher enthalpy for VER-50589 binding. A competitive binding assay gave a lower IC(50) of 21 +/- 4 nmol/L for VER-50589 compared with 47 +/- 9 nmol/L for VER-49009. Cellular uptake of VER-50589 was 4-fold greater than for VER-49009. Mean cellular antiproliferative GI(50) values for VER-50589 and VER-49009 for a human cancer cell line panel were 78 +/- 15 and 685 +/- 119 nmol/L, respectively, showing a 9-fold potency gain for the isoxazole. Unlike 17-AAG, but as with CCT018159, cellular potency of these analogues was independent of NAD(P)H:quinone oxidoreductase 1/DT-diaphorase and P-glycoprotein expression. Consistent with HSP90 inhibition, VER-50589 and VER-49009 caused induction of HSP72 and HSP27 alongside depletion of client proteins, including C-RAF, B-RAF, and survivin, and the protein arginine methyltransferase PRMT5. Both caused cell cycle arrest and apoptosis. Extent and duration of pharmacodynamic changes in an orthotopic human ovarian carcinoma model confirmed the superiority of VER-50589 over VER-49009. VER-50589 accumulated in HCT116 human colon cancer xenografts at levels above the cellular GI(50) for 24 h, resulting in 30% growth inhibition. The results indicate the therapeutic potential of the resorcinylic pyrazole/isoxazole amide analogues as HSP90 inhibitors.

Inhibitors of the heat shock response: biology and pharmacology.

A number of human diseases can be linked to aberrations in protein folding which cause an imbalance in protein homeostasis. Molecular chaperones, including heat shock proteins, act to assist protein folding, stability and activity in the cell. Attention has begun to focus on modulating the expression and/or activity of this group of proteins for the treatment of a wide variety of human diseases. This review will describe the progress made to date in developing pharmacological modulators of the heat shock response, including both agents which affect the entire heat shock response and those that specifically target the HSP70 and HSP90 chaperone families.

Discovery of a Chemical Probe Bisamide (CCT251236): An Orally Bioavailable Efficacious Pirin Ligand from a Heat Shock Transcription Factor 1 (HSF1) Phenotypic Screen.

Phenotypic screens, which focus on measuring and quantifying discrete cellular changes rather than affinity for individual recombinant proteins, have recently attracted renewed interest as an efficient strategy for drug discovery. In this article, we describe the discovery of a new chemical probe, bisamide (CCT251236), identified using an unbiased phenotypic screen to detect inhibitors of the HSF1 stress pathway. The chemical probe is orally bioavailable and displays efficacy in a human ovarian carcinoma xenograft model. By developing cell-based SAR and using chemical proteomics, we identified pirin as a high affinity molecular target, which was confirmed by SPR and crystallography.

Targeting of multiple signalling pathways by heat shock protein 90 molecular chaperone inhibitors.

The last decade has seen the molecular chaperone heat shock protein 90 (HSP90) emerge as an exciting target for cancer therapy. This is because HSP90 is involved in maintaining the conformation, stability, activity and cellular localisation of several key oncogenic client proteins. These include, amongst others, ERBB2, C-RAF, CDK4, AKT/PKB, steroid hormone receptors, mutant p53, HIF-1alpha , survivin and telomerase hTERT. Therefore, modulation of this single drug target offers the prospect of simultaneously inhibiting all the multiple signalling pathways and biological processes that have been implicated in the development of the malignant phenotype. The chaperone function of HSP90 requires the formation of a multichaperone complex, which is dependent on the hydrolysis of ATP and ADP/ATP exchange. Most current inhibitors of HSP90 act as nucleotide mimetics, which block the intrinsic ATPase activity of this molecular chaperone. The first-in-class inhibitor to enter and complete phase I clinical trials was the geldanamycin analogue, 17-allylamino-17-demethoxygeldanamycin. The results of these trials have demonstrated that HSP90 is a valid drug target. Evidence of clinical activity has been seen in patients with melanoma, breast and prostate cancer. This article provides a personal perspective of the present efforts to increase our understanding of the molecular and cellular consequences of HSP90 inhibition, with examples from work in our own laboratory. We also review the discovery and development of novel small-molecule inhibitors and discuss alternative approaches to inhibit HSP90 activity, both of which offer exciting prospects for the future.

Second-Generation HSP90 Inhibitor Onalespib Blocks mRNA Splicing of Androgen Receptor Variant 7 in Prostate Cancer Cells.

Resistance to available hormone therapies in prostate cancer has been associated with alternative splicing of androgen receptor (AR) and specifically, the expression of truncated and constitutively active AR variant 7 (AR-V7). The transcriptional activity of steroid receptors, including AR, is dependent on interactions with the HSP90 chaperone machinery, but it is unclear whether HSP90 modulates the activity or expression of AR variants. Here, we investigated the effects of HSP90 inhibition on AR-V7 in prostate cancer cell lines endogenously expressing this variant. We demonstrate that AR-V7 and full-length AR (AR-FL) were depleted upon inhibition of HSP90. However, the mechanisms underlying AR-V7 depletion differed from those for AR-FL. Whereas HSP90 inhibition destabilized AR-FL and induced its proteasomal degradation, AR-V7 protein exhibited higher stability than AR-FL and did not require HSP90 chaperone activity. Instead, HSP90 inhibition resulted in the reduction of AR-V7 mRNA levels but did not affect total AR transcript levels, indicating that HSP90 inhibition disrupted AR-V7 splicing. Bioinformatic analyses of transcriptome-wide RNA sequencing data confirmed that the second-generation HSP90 inhibitor onalespib altered the splicing of at least 557 genes in prostate cancer cells, including AR. These findings indicate that the effects of HSP90 inhibition on mRNA splicing may prove beneficial in prostate cancers expressing AR-V7, supporting further clinical investigation of HSP90 inhibitors in malignancies no longer responsive to androgen deprivation. Cancer Res; 76(9); 2731-42. ©2016 AACR.

A real-time brain-machine interface combining motor target and trajectory intent using an optimal feedback control design.

Real-time brain-machine interfaces (BMI) have focused on either estimating the continuous movement trajectory or target intent. However, natural movement often incorporates both. Additionally, BMIs can be modeled as a feedback control system in which the subject modulates the neural activity to move the prosthetic device towards a desired target while receiving real-time sensory feedback of the state of the movement. We develop a novel real-time BMI using an optimal feedback control design that jointly estimates the movement target and trajectory of monkeys in two stages. First, the target is decoded from neural spiking activity before movement initiation. Second, the trajectory is decoded by combining the decoded target with the peri-movement spiking activity using an optimal feedback control design. This design exploits a recursive Bayesian decoder that uses an optimal feedback control model of the sensorimotor system to take into account the intended target location and the sensory feedback in its trajectory estimation from spiking activity. The real-time BMI processes the spiking activity directly using point process modeling. We implement the BMI in experiments consisting of an instructed-delay center-out task in which monkeys are presented with a target location on the screen during a delay period and then have to move a cursor to it without touching the incorrect targets. We show that the two-stage BMI performs more accurately than either stage alone. Correct target prediction can compensate for inaccurate trajectory estimation and vice versa. The optimal feedback control design also results in trajectories that are smoother and have lower estimation error. The two-stage decoder also performs better than linear regression approaches in offline cross-validation analyses. Our results demonstrate the advantage of a BMI design that jointly estimates the target and trajectory of movement and more closely mimics the sensorimotor control system.

Mode of cell death induced by the HSP90 inhibitor 17-AAG (tanespimycin) is dependent on the expression of pro-apoptotic BAX.

Inhibitors of the molecular chaperone heat shock protein 90 (HSP90) are of considerable current interest as targeted cancer therapeutic agents because of the ability to destabilize multiple oncogenic client proteins. Despite their resulting pleiotropic effects on multiple oncogenic pathways and hallmark traits of cancer, resistance to HSP90 inhibitors is possible and their ability to induce apoptosis is less than might be expected. Using an isogenic model for BAX knockout in HCT116 human colon carcinoma cells, we demonstrate the induction of BAX-dependent apoptosis at pharmacologically relevant concentrations of the HSP90 inhibitor 17-AAG both in vitro and in tumor xenografts in vivo. Removal of BAX expression by homologous recombination reduces apoptosis in vitro and in vivo but allows a lower level of cell death via a predominantly necrotic mechanism. Despite reducing apoptosis, the loss of BAX does not alter the overall sensitivity to 17-AAG in vitro or in vivo. The results indicate that 17-AAG acts predominantly to cause a cytostatic antiproliferative effect rather than cell death and further suggest that BAX status may not alter the overall clinical response to HSP90 inhibitors. Other agents may be required in combination to enhance tumor-selective killing by these promising drugs. In addition, there are implications for the use of apoptotic endpoints in the assessment of the activity of molecularly targeted agents.

Neural population partitioning and a concurrent brain-machine interface for sequential motor function.

Although brain-machine interfaces (BMIs) have focused largely on performing single-targeted movements, many natural tasks involve planning a complete sequence of such movements before execution. For these tasks, a BMI that can concurrently decode the full planned sequence before its execution may also consider the higher-level goal of the task to reformulate and perform it more effectively. Using population-wide modeling, we discovered two distinct subpopulations of neurons in the rhesus monkey premotor cortex that allow two planned targets of a sequential movement to be simultaneously held in working memory without degradation. Such marked stability occurred because each subpopulation encoded either only currently held or only newly added target information irrespective of the exact sequence. On the basis of these findings, we developed a BMI that concurrently decodes a full motor sequence in advance of movement and can then accurately execute it as desired.

Targeting HSP70: the second potentially druggable heat shock protein and molecular chaperone?

The HSF1-mediated stress response pathway is steadily gaining momentum as a critical source of targets for cancer therapy. Key mediators of this pathway include molecular chaperones such as heat shock protein (HSP) 90. There has been considerable progress in targeting HSP90 and the preclinical efficacy and signs of early clinical activity of HSP90 inhibitors have provided proof-of-concept for targeting this group of proteins. The HSP70 family of molecular chaperones are also key mediators of the HSF-1-stress response pathway and have multiple additional roles in protein folding, trafficking and degradation, as well as regulating apoptosis. Genetic and biochemical studies have supported the discovery of HSP70 inhibitors which have the potential for use as single agents or in combination to enhance the effects of classical chemotherapeutic or molecularly targeted agents including HSP90 inhibitors. Here we provide a perspective on the progress made so far in designing agents which target the HSP70 family.

Death by chaperone: HSP90, HSP70 or both?

HSP70 family members are highly conserved proteins that function as molecular chaperones. Their principle role is to aid protein folding and promote the correct cellular localizations of their respective substrates. The function of HSP70 isoforms can be exhibited independently or with the HSP90 chaperone system in which HSP70 is important for substrate recruitment. In addition to their chaperone role, HSP70 isoforms promote cell survival by inhibiting apoptosis at multiple points within both the intrinsic and extrinsic cell death pathways. Consistent with this cytoprotective function, increased expression of HSP70 isoforms is commonly associated with the malignant phenotype. We recently reported that dual silencing of the major constitutive (HSC70) and inducible (HSP72) isoforms of HSP70 in cancer cells could phenocopy the effects of a pharmacologic HSP90 inhibitor to induce proteasome-dependent degradation of HSP90 client proteins CRAF, CDK4 and ERBB2. This was accompanied by a G(1) cell cycle arrest and extensive apoptosis which was not seen in non-tumorigenic human cell lines. Here we discuss the possible implications of our research for the development of HSP70 family modulators which offer not only the possibility of inhibiting HSP70 activity but also the simultaneous inhibition of HSP90, resulting in extensive tumor-specific apoptosis.

Dual targeting of HSC70 and HSP72 inhibits HSP90 function and induces tumor-specific apoptosis.

Heat-shock protein 70 (HSP70) isoforms contribute to tumorigenesis through their well-documented antiapoptotic activity and via their role as cochaperones for the HSP90 molecular chaperone. HSP70 expression is induced following treatment with HSP90 inhibitors, which may attenuate the cell death effects of this class of inhibitor. Here we show that silencing either heat-shock cognate 70 (HSC70) or HSP72 expression in human cancer cell lines has no effect on HSP90 activity or cell proliferation. However, simultaneously reducing the expression of both of these isoforms induces proteasome-dependent degradation of HSP90 client proteins, G1 cell-cycle arrest, and extensive tumor-specific apoptosis. Importantly, simultaneous silencing of HSP70 isoforms in nontumorigenic cell lines does not result in comparable growth arrest or induction of apoptosis, indicating a potential therapeutic window.