Brian Blagg, Ph.D. - US grants

COBRE; CRISP; Cancer Treatment; Center of Biomedical Research Excellence; Centers of Research Excellence; Chaperone; Client; Computer Retrieval of Information on Scientific Projects Database; EC 1.14.13.39; EDRF Synthase; Endothelium-Derived Growth Factor Synthase; Enzymes; FK506 binding protein 5; FK506 binding protein 59; FK506-binding protein 4; FKBP4; FKBP51; FKBP52; FKBP54; FKBP59; Funding; GRP94; Genes, p53; Grant; Guanylyl Cyclase-Activating Factor Synthase; HSP 56; HSP-90; HSP56; HSP90; Heat-Shock Proteins 90; Immunophilins; Individual; Institution; Investigators; L-Arginine,NADPH[{..}]oxygen oxidoreductase (nitric-oxide-forming); Malignant Neoplasm Therapy; Malignant Neoplasm Treatment; Molecular Chaperones; NADPH-Diaphorase; NIH; NO Synthase; Names; National Institutes of Health; National Institutes of Health (U.S.); Nitric Oxide Synthase; Nitric-Oxide Synthetase; P53; PPIase; Peptides; Peptidyl-Prolyl cis-trans-Isomerase; Peptidylproline cis-trans-isomerase; Peptidylprolyl Isomerase; Proline Isomerase; Proline Rotamase; Prolyl Isomerase; Protein-Tyrosine Kinases, src; Proteins; Receptor Protein; Regulatory Protein; Research; Research Personnel; Research Resources; Researchers; Resources; Role; Source; TP53; TP53 gene; TRP53; Telomerase; Tumor Protein p53 Gene; United States National Institutes of Health; anticancer therapy; cancer therapy; cyclophilin; gene product; genetic regulatory protein; heat shock-binding immunophilin; heat-shock protein 56; hsp90 Family; immunophilin; mutant; p59 heat shock-binding immunophilin; protein folding; raf Kinases; raf MAP Kinase Kinase Kinases; raf Proto-Oncogene Proteins; raf Serine-Theonine Protein Kinases; receptor; regulatory gene product; social role; src Kinases; src Tyrosine Kinases; src-Family Kinases; src-Family Tyrosine Kinases; steroid hormone receptor; tacrolimus binding protein 4; tacrolimus binding protein 5

DESCRIPTION (Provided by the applicant): Hsp90 is a molecular chaperone responsible for folding nascent polypeptides into their biologically active, three-dimensional conformations. Disruption of the Hsp90 protein folding process results in the simultaneous inhibition of multiple enzymes that are essential for malignant cell growth. In fact, proteins represented in all six hallmarks of cancer are dependent on Hsp90 for conformational activation, and several of these Hsp90 client proteins are individually sought after cancer chemotherapeutic targets. Consequently, Hsp90 inhibition offers a promising new target for the development of anticancer chemotherapeutic agents because multiple signaling pathways can be simultaneously inhibited by disruption of the Hsp90 protein folding machinery. This application aims to develop new high-throughput assays for the identification of new lead compounds that inhibit Hsp90. A cell lysate assay will be developed to screen for inhibitors that prevent the renaturation of a well-studied Hsp90 client protein that has excellent bio-luminescence properties. It is proposed that molecules capable of inhibiting this renaturation process will be subsequently analyzed for their method of Hsp90 inhibition by two independent assays aimed at identifying both N- and C-terminal ATP binding site inhibitors. Alternatively, each of these assays should be independently capable of identifying new inhibitors of Hsp90 in a complimentary manner. At the completion of this project, we expect to provide three independent assays for high-throughput detection and evaluation of inhibitors that disrupt the Hsp90 protein folding process. The therapeutic potential of these compounds could be immense, as subsequent modification of these lead compounds should lead to the development of novel high-affinity Hsp90 antagonists for the treatment of cancer.

[unreadable] DESCRIPTION (provided by applicant): The 90 kDa heat shock proteins are proving to be extraordinary cancer chemotherapeutic targets as evidenced by the fact that 26 clinical trials are currently in progress. Unfortunately, all of these trials are based upon the N-terminal inhibitor, geldanamycin, which has serious formulation difficulties and produces toxicity unrelated to Hsp90 inhibition by virtue of its redox-active and electrophilic quinone ring. Only one total synthesis of this molecule has been reported, and the overall yield was 0.017% after 43 steps, suggesting that the preparation of analogues would be difficult. More recently, Neckers and coworkers determined that Hsp90 contains a C-terminal ATP binding site that bound coumarin antibiotics competitively versus ATP. Like N-terminal inhibitors, inhibitors of the C-terminal binding domain also cause the degradation of Hsp90-dependent client proteins involved in tumor cell growth and proliferation. A major drawback of the coumarin antibiotics is that they bind weakly to Hsp90 (IC50 approximately 700 micromolar); however, they remain the most potent C-terminal inhibitors described in the literature. We have recently identified a compound that is 700-7000 x's more active than the coumarin antibiotic in collaboration with Len Neckers and Jeff Holzbeierlein. This molecule has been evaluated in several tumor cell lines and has potent activity against a wide range of Hsp90 client proteins. In an effort to increase the potency of our lead molecule, we have proposed to incorporate functionalities that exist in the nucleotide specific substrates onto our lead compound to afford additional interactions with the Hsp90 C-terminal binding site. In addition, we propose to evaluate our lead compound in murine xenograft models of prostate cancer. Finally, it is proposed that by compromising the Hsp90 protein folding machinery with low doses of our lead compound, or any improved analogue, it will provide an acceptable concentration of other clinically used anti-tumor agents to induce apoptosis without detrimental side effects. As a consequence of these studies, we believe we can provide a basis upon which new inhibitors of the Hsp90 protein folding process can be developed without the deleterious side effects of the geldanamycin derivatives that are currently plagued in clinical trials. [unreadable] [unreadable] [unreadable]

Hsp90 is a molecular chaperone responsible for folding nascent polypeptides into biologically active native structures. Many of the proteins dependent upon HspQO for conformational maturation are directly associated with malignant growth and proliferation. Inhibition of Hsp90 results in the destabilization of Hsp90-client protein complexes, resulting in ubiquitination and proteasomal degradation of the protein substrate. Consequently, inhibitors of Hsp90 represent a promising new approach toward the treatment of cancer because multiple chemotherapeutic targets can be simultaneously targeted. Two natural products with potent activity against Hsp90 have been co-crystallized with HspQO and the structures solved. Based on these co-crystal structures, new inhibitors of Hsp90 have been designed, docked to the N-terminal ATP binding site, and initial compounds synthesized. Preliminary testing of these compounds has resulted in the identification of several molecules with exceptional activity. In addition, new inhibitors of the C-terminal nucleotide binding site have been prepared and evaluated. One C-terminal inhibitor was shown to be >700 x more active than novobiocin. The objectives of this proposal are to prepare new Hsp90 inhibitors with increased affinity and solubility, as compared to the known natural product inhibitors. These molecules will be evaluated for their biological activity against HspQO by a coupled assay (N-terminal ATP binding site), a cellular HspQO client protein degradation assay, and by cytotoxicity studies. In collaboration with other researchers, the co-crystal structure of these molecules bound to Hsp90 will be solved and in vivo studies of these molecules will be performed in murine xenograft models of prostate and breast cancer.

The determination of intracellular accumulation and target selectivity/specificity is an essential and challenging aspect of modern probe development. Although controversial rules have been proposed to account for the bioavailability of orally administered drugs, techniques to determine target selectivity and intracellular compartmentalization of drugs within living cells have not been adequately developed. A lead compound identified from high-throughput screening is generally not suitable for in vivo biological use until a systematic evaluation of the small molecule in cells has taken place. This step is required because highthroughput biochemical and cellular assays are not representative of the multiple different physicochemical environments that exist within living organisms. For example, upon exposure to a small molecule, numerous organelles and compartments within the cell can sequester compounds and prevent association with the desired biological target. Correspondingly, the development of small molecule enzyme inhibitors and receptor modulators is often limited by an inability to target specific proteins. Molecular probes derived from biologically active small molecules have the potential to shed light on these issues. However, many efforts to develop optimal molecular probes suffer the following pitfalls: (1) the inability to accurately monitor where biologically active compounds accumulate within cells, (2) the inability to accurately know which biological targets are truly affected upon exposure of cells to small molecules, and (3) the inability to accurately determine the relative selectivity of compounds for multiple protein targets in their native environment. Previous attempts to address these issues have often used linked fluorophores and other labels that can intrinsically promote differential accumulation of the small molecule away from the site of biological relevance. Consequently, a general systematic protocol is needed to efficiently evaluate the intracellular accumulation and target selectivity of molecular probes. To circumvent these bottlenecks, we propose to develop two complementary approaches to identify proteins that bind small molecules. The first approach will utilize fluorescent probes that readily diffuse through cellular membranes, but do not localize in any compartment. These fluorescent probes will then be used in pulsechase experiments to react with protein-bound inhibitors and monitored via confocal microscopy to elucidate the extent of intracellular sequestration of the fluorescent small molecule. In addition, these probes will be utilized to determine target selectivity by modification of a tether to allow covalent attachment to bound proteins within the cell. Immunopurification and subsequent LCMS will be used to isolate the proteins linked to these fluorophores and determine their identity. A second approach for isolating targets will employ a novel yeast 3- hybrid screen to identify proteins encoded by cDNA libraries that activate transcription by binding to chemical inducers of dimerization (CIDs). To accomplish these objectives, we propose the following specific aims: 1. Develop methodology for visualizing and identifying intracellular protein targets using fluorescent molecular probes. We propose to develop optimal cell-permeable fluorophores, multifunctional tethers, approaches for elucidation of localization of probes, and methods for target identification (ID) using pulse-chase affinity labeling and applications of selective antibodies. 2. Develop yeast three-hybrid (Y3H) systems as tools for the identification of protein targets of small molecules. We propose to combine yeast genetic screening with fluorescence-activated cell sorting (FACS), develop methods to facilitate the penetration of small molecules into living yeast cells, and improve the utility of yeast genetic systems for screening of small molecules and target ID. This proposal is innovative because it aims to address some of the most difficult problems associated with delivering probe molecules identified through high-throughput screening to researchers engaged in real-world biomedical science. Two novel approaches are presented that have the potential to identify and ultimately eliminate undesired cellular trafficking issues and properties that could interfere with targeting of a particular probe. By associating this project with a Specialized Chemistry Center, we also intend to develop general tagging techniques that could be employed as needed in collaboration with other MLPCN groups.

? DESCRIPTION (provided by applicant): Hsp90 is a molecular chaperone that is responsible for the conformational maturation of more than 200 client protein substrates, many of which are directly associated with cell signaling, and thus, are often hijacked during malignant transformation. Consequently, through Hsp90 inhibition, multiple signaling pathways can be disrupted simultaneously. As a result, Hsp90 has emerged as a promising anti-cancer target, and there are currently 17 inhibitors undergoing clinical evaluation. Unfortunately, all of these molecule bind to the Hsp90 N-terminal binding site, and also induce the pro-survival heat shock response at the same concentration they inhibit the Hsp90 protein folding machinery. The net result is generally, cytostatic activity and the potential for chemotherapeutic resistance. Unlike N-terminal inhibitors, C-terminal inhibitors can segregate these activities, which have led to unforeseen opportunities for the development of useful anti-cancer agents. In fact, C-terminal inhibitors do not induce the heat shock response and consequently, induce apoptosis against many cancer cells with high differential selectivity. The first C-terminal inhibitor identified was novobiocin, which manifests an IC50 value of ~700 micromolar. During the past few years, we have modified this coumarin antibiotic and transformed it into a potential clinical lead compound that exhibits ~100 nM activity. In this proposal, we aim to further develop this class of compounds and to evaluate them in animal models of head and neck squamous cell carcinoma in an effort to provide additional evidence to support their clinical application against a varietyof cancers.

PROJECT SUMMARY Heat shock protein 90 (Hsp90) and Hsp70 are molecular chaperones that facilitate protein folding, maturation and clearance. It is well recognized that targeting Hsp90 and Hsp70 with small molecule drugs may improve proteostatic neurodegenerative diseases by enhancing the clearance of neurotoxic protein aggregates. However, we have identified that pharmacologically targeting molecular chaperones has therapeutic value in treating peripheral neuropathies whose etiology is independent of proteostasis. The traditional small molecule approach for targeting Hsp90 has relied on drugs that inhibit its N-terminal ATPase activity. Lamentably, the translational success of these drugs has been hindered by the complex chemical biology of targeting the Hsp90 N-terminal ATP binding site. In contrast, we have developed ?novologues? as small molecule inhibitors of the Hsp90 C-terminal domain that circumvent limitations that have confounded the clinical prospects of Hsp90 as a therapeutic target. Novologues are orally bioavailable and non-toxic molecules that promote a cytoprotective response that constitutes a paradigm shift for the treatment of diabetic peripheral neuropathy (DPN) and potentially, demyelinating neuropathies (DymN). Our published and unpublished data support that novologues improve physiologic measures of DPN and DymN in an Hsp70- dependent manner by improving mitochondrial bioenergetics in DPN and inhibiting the induction of c-jun, a negative regulator of myelination, that contributes to DymN. Thus, our central hypothesis is that modulating the activity of molecular chaperones with novologues will provide an innovative and novel approach for the treatment of both DPN and some forms of DymN. Our efforts over the four years of NIH funding have led to the licensing of our lead novologue, KU-596, to a pharmaceutical company and its advancement to Phase I studies for treating DPN. To build upon this success, we have identified attributes and detriments of KU- 596. Specific Aim 1 focuses on the rationale design and development of ?noviomimetics? as a new class of molecules that will improve on the chemical biology of novologues by increasing potency, requiring fewer synthetic steps and exhibiting better metabolic stability. Specific Aim 2 will use two cell-based screening assays to examine the ability to improve mitochondrial bioenergetics in diabetic sensory neurons or increase the degradation of the transcription factor, c-jun. Importantly, we provide evidence that these biochemical readouts are prognostic for in vivo efficacy to improve DPN and DymN, respectively. Compounds that advance through the cell-based assay will undergo ADME screening to aid selecting candidates for in vivo testing in a mouse model of DPN and two models that recapitulate a human demyelinating motor neuropathy. The outcome of this work will extend the success of our drug development program and identify noviomimetics as a new class of neuroprotective compounds for treating peripheral neuropathies.                

DESCRIPTION (provided by applicant): To date, there are very limited therapies for patients with metastatic castrate resistant prostate cancer. Due to the heterogeneity of prostate cancer no one specific oncogene, mutation, signaling pathways, or risk factor has been described in prostate cancer. Multiple pathways have been described to contribute to both the development as well as progression of prostate cancer. Hsp90 has been shown to be over-expressed in prostate cancer and serves as a hub for multiple signaling pathways that contribute to malignant growth and proliferation, making it an excellent target for the development of inhibitors. In contrast to N-terminal inhibitors that induce the pro-survival heat shock response, C-terminal inhibitors do not, and therefore provide a new paradigm in cancer research. Specifically, we aim to prepare conformationally biased analogues of new Hsp90 C-terminal inhibitors, evaluate them through a series of novel assays in vitro, and then administer select compounds to an orthotopic model of prostate cancer to determine their efficacy. Upon completion of this project, we aim to produce compounds amenable to further evaluation in clinical trials for prostate cancer.

DESCRIPTION (provided by applicant): Over 100,000 people in the US suffer from primary open-angle glaucoma (POAG) caused by mutations in the MYOC gene. This form of POAG results from optic nerve damage caused by the death of a protective cell network called the trabecular meshwork (TM). TM cell death occurs in these cases because mutant myocilin abnormally accumulates into toxic aggregates. This mechanism is reminiscent of neurodegenerative diseases, such as Alzheimer's, Huntington's and Parkinson's, where abnormal proteins accumulate in neurons and lead to cell death. In fact, TM cells are long-lived just like neurons. Moreover, mutations that cause earlier POAG onset also make myocilin aggregate more readily, similar to proteins associated with neurodegenerative diseases. Thus, both types of diseases can be considered proteostasis disorders, meaning that long-lived cells (neurons and TM) progressively lose their ability to prevent the toxic accumulation of mutant proteins with age. Thus, strategies aimed at restoring proteostasis in TM cells could be beneficial for glaucoma, just as they have proven for neurodegenerative disease. Through a series of studies, we determined that the Grp94 chaperone (an Hsp90 isoform) that resides in the endoplasmic reticulum, mistakenly preserves mutant myocilin in cells. Importantly, Grp94 only affects misfolded myocilin: Properly folded and functioning myocilin is unaffected by Grp94 manipulation. Grp94 recognizes only myocilin that is misfolded due to either mutations or impaired glycosylation: But Grp94 is unable to clear this misfolded myocilin, and instead, preserves it, causing its toxic accumulation. Thus, myocilin misfolding disrupts proteostasis by mistakenly engaging the Grp94 chaperone. We have shown that the clearance of toxic myocilin can be accelerated simply by inhibiting Grp94! Our team has developed the first isoform selective Grp94 inhibitor termed BnIm. Because the list of Grp94-dependent substrates is small, compared to other Hsp90 isoforms, the toxicity profile for this Grp94 inhibitor also appears low. Therefore, we propose to validate and improve upon this Grp94 inhibitor for the treatment of myocilin-associated POAG by establishing structure-activity relationships of Grp94 inhibitors to elucidate mechanisms of misfolded myocilin triage. We will also evaluate the biological efficacy of Grp94 inhibitors towards mutant myocilin in disease relevant systems and then work to develop Grp94 inhibitors with greater efficacy and biological activity towards misfolded myocilin. These studies will result in a new suite of Grp94 modulators and demonstrate that Grp94 is a novel clinical target to treat glaucoma caused by misfolded myocilin. In addition, mechanisms identified herein that clarify how Grp94 regulates myocilin triage could provide new insights for other proteostasis diseases.

The 90 kDa heat shock proteins (Hsp90) are responsible for the maturation of approximately 200 client protein substrates, most of which are associated with signaling cascades that regulate cellular growth and proliferation. Therefore, Hsp90 inhibition provides a novel approach toward the treatment of cancer as numerous signaling cascades can be derailed through inhibition of the Hsp90-dependent protein folding process. In this application, we propose to develop selective inhibitors of the Hsp90 Isoforms, Grp94, Hsp90?, and Hsp90?, using a structure-guided approach. In addition, we will perform side by side evaluation of these compounds against pan-inhibitors both in vitro and in vivo to validate our approach. Together, these methods will provide a new approach for selective inhibition of Hsp90 and will provide a new paradigm for anti-cancer drug development.

Abstract The development of new methods to treat cancer is greatly needed. In this application, we have identified a macrocyclic small molecule that exhibits high differential selectivity for cancer cells vs. normal cells and aim to develop more simplified analogs of this molecule in an effort to maximize efficiency as well as to decrease the overall number of steps, which will likely lead to clinically useful molecules for the treatment of cancer. In addition, we aim to interrogate the activity manifested by these compounds in both cellular and animal models of cancer, with the goal of providing preclinical data to support the use of such compounds for translational development.

The development of new methods to treat cancer is greatly needed. The Hsp90 protein folding machinery has been the target of significant interest for the development of anti-cancer agents. However, Hsp90 is responsible for the folding of ~300 protein folding substrates, which may lead to undesired on-target toxicity and may be responsible for many of the Hsp90-targeted drugs that have failed in clinical trials. Through a structure-based approach, an isoform-selective inhibitor of Hsp90 has been identified and shown to exhibit good selectivity and affinity against particular cancers. Consequently, the goal of this application is to optimize this new inhibitory scaffold for great affinity/efficacy, to evaluate the role of a specific Hsp90 isoform in cancer, and to validate this isoform as a target for the treatment of bladder cancer.