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Adobe PDF - MCT-07-0149--Suppl_Data.pdf from 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 (Kd) 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 IC50 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 GI50 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 GI50 for 24 h, resulting in 30% growth inhibition. The results indicate the therapeutic potential of the resorcinylic pyrazole/isoxazole amide analogues as HSP90 inhibitors. [Mol Cancer Ther 2007;6(4):1198–211]

ddPCR analysis of drug-resistance mutations shows they are pre-existing in cancer cell lines and quickly introduced into a population of HCT116 cells.

We describe the biological properties of NVP-AUY922, a novel resorcinylic isoxazole amide heat shock protein 90 (HSP90) inhibitor. NVP-AUY922 potently inhibits HSP90 (Kd = 1.7 nmol/L) and proliferation of human tumor cells with GI50 values of approximately 2 to 40 nmol/L, inducing G1-G2 arrest and apoptosis. Activity is independent of NQO1/DT-diaphorase, maintained in drug-resistant cells and under hypoxic conditions. The molecular signature of HSP90 inhibition, comprising induced HSP72 and depleted client proteins, was readily demonstrable. NVP-AUY922 was glucuronidated less than previously described isoxazoles, yielding higher drug levels in human cancer cells and xenografts. Daily dosing of NVP-AUY922 (50 mg/kg i.p. or i.v.) to athymic mice generated peak tumor levels at least 100-fold above cellular GI50. This produced statistically significant growth inhibition and/or regressions in human tumor xenografts with diverse oncogenic profiles: BT474 breast tumor treated/control, 21%; A2780 ovarian, 11%; U87MG glioblastoma, 7%; PC3 prostate, 37%; and WM266.4 melanoma, 31%. Therapeutic effects were concordant with changes in pharmacodynamic markers, including induction of HSP72 and depletion of ERBB2, CRAF, cyclin-dependent kinase 4, phospho-AKT/total AKT, and hypoxia-inducible factor-1α, determined by Western blot, electrochemiluminescent immunoassay, or immunohistochemistry. NVP-AUY922 also significantly inhibited tumor cell chemotaxis/invasion in vitro, WM266.4 melanoma lung metastases, and lymphatic metastases from orthotopically implanted PC3LN3 prostate carcinoma. NVP-AUY922 inhibited proliferation, chemomigration, and tubular differentiation of human endothelial cells and antiangiogenic activity was reflected in reduced microvessel density in tumor xenografts. Collectively, the data show that NVP-AUY922 is a potent, novel inhibitor of HSP90, acting via several processes (cytostasis, apoptosis, invasion, and angiogenesis) to inhibit tumor growth and metastasis. NVP-AUY922 has entered phase I clinical trials. [Cancer Res 2008;68(8):2850–60]

Supplementary Materials, Table 1 and Figures 1-3 from In vitro Biological Characterization of a Novel, Synthetic Diaryl Pyrazole Resorcinol Class of Heat Shock Protein 90 Inhibitors

Tables of biochemical and viability assays of resistant cell clones, fractional abundance of MPS1 mutants in different human cancer cell lines and refinement statistics of crystal structures of different MPS1 domains.

Expression of the p.M600T, p.Y568C and p.C604W MPS1 mutant constructs in DLD1 Flp-In TRex cells recues the spindle assembly checkpoint defect following AZ3146 treatment.

Supplementary Figure 6 from NVP-AUY922: A Novel Heat Shock Protein 90 Inhibitor Active against Xenograft Tumor Growth, Angiogenesis, and Metastasis

Supplementary Figure 3A-B from NVP-AUY922: A Novel Heat Shock Protein 90 Inhibitor Active against Xenograft Tumor Growth, Angiogenesis, and Metastasis

CCT251455 kills cancer cells by inhibiting the kinetochore recruitment of SAC protein.

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 HSP90β with comparable potency to 17-AAG and with similar ATP-competitive kinetics. X-ray crystallographic structures of the NH2-terminal domain of yeast Hsp90 complexed with CCT018159 or its analogues showed binding properties similar to radicicol. The mean cellular GI50 value of CCT018159 across a panel of human cancer cell lines, including melanoma, was 5.3 μmol/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 G1 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. [Cancer Res 2007;67(5):2206–16]

Supplementary Figure 5A-B from NVP-AUY922: A Novel Heat Shock Protein 90 Inhibitor Active against Xenograft Tumor Growth, Angiogenesis, and Metastasis

Supplementary Figure 4C-D from NVP-AUY922: A Novel Heat Shock Protein 90 Inhibitor Active against Xenograft Tumor Growth, Angiogenesis, and Metastasis

Description of the supplementary experimental procedures figure legends and references

Supplementary Figure 5C-D from NVP-AUY922: A Novel Heat Shock Protein 90 Inhibitor Active against Xenograft Tumor Growth, Angiogenesis, and Metastasis

Crystal structures of AZD3146 and ONCOII bound to MPS1-KD.

Expression of the p.S611G, p.I531M and Dbl MPS1 mutant constructs in DLD1 Flp-In TRex cells recues the spindle assembly checkpoint defect following AZ3146 treatment.

Supplementary Figure 2 from NVP-AUY922: A Novel Heat Shock Protein 90 Inhibitor Active against Xenograft Tumor Growth, Angiogenesis, and Metastasis

CCT251455-resistant HCT116 clones.

Supplementary Figures 1A-B from NVP-AUY922: A Novel Heat Shock Protein 90 Inhibitor Active against Xenograft Tumor Growth, Angiogenesis, and Metastasis

Supplementary Figure 1C from NVP-AUY922: A Novel Heat Shock Protein 90 Inhibitor Active against Xenograft Tumor Growth, Angiogenesis, and Metastasis

Supplementary Figure 4A-B from NVP-AUY922: A Novel Heat Shock Protein 90 Inhibitor Active against Xenograft Tumor Growth, Angiogenesis, and Metastasis

This article discusses a scoping review of position papers aimed at enhancing the quality of life of people with intellectual disabilities. It describes the approach taken to conducting the scoping...

There is growing international interest in service user involvement in social work education, but some service user groups are more likely to be included than others. This article explores the possibility of involving male service users who use intimate partner violence in Australian social work education. The article describes focus groups conducted separately with social work students and men who use intimate partner violence, which explored participants’ understandings of lived experience, service user involvement in social work education and the place of men who use intimate partner violence in the social work academy. The findings suggest it may be possible to involve previously excluded service user groups in social work education.

This paper examines methodological issues related to an innovative online qualitative research project that explored ‘safe spaces’ with mental health service users. The project used photovoice and focus groups conducted via Zoom videoconferencing software. Eleven participants shared photographs, discussing their meaning and significance in relation to ‘safe spaces’. The photographs were then synthesised into an artist's impression of a safe space and transcripts of the online photovoice discussion analysed thematically and triangulated with the artist's impression. The paper provides a reflexive discussion of the research process and explores methodological and ethical implications of conducting sensitive qualitative research in online spaces.

The personalisation of social care services in England predated the development of the Australian National Disability Insurance Scheme (NDIS). This commentary explores Rob Greig’s (2019) discussion...

This article considers the inclusion of mental health service users’ experiences and perspectives in professional education classrooms. After brief introductions to the authors’ backgrounds, the ar...

Renewed interest in covalent inhibitors of enzymes implicated in disease states has afforded several agents targeted at protein kinases of relevance to cancers., Renewed interest in covalent inhibitors of enzymes implicated in disease states has afforded several agents targeted at protein kinases of relevance to cancers. We now report the design, synthesis and biological evaluation of 6-ethynylpurines that act as covalent inhibitors of Nek2 by capturing a cysteine residue (Cys22) close to the catalytic domain of this protein kinase. Examination of the crystal structure of the non-covalent inhibitor 3-((6-cyclohexylmethoxy-7H-purin-2-yl)amino)benzamide in complex with Nek2 indicated that replacing the alkoxy with an ethynyl group places the terminus of the alkyne close to Cys22 and in a position compatible with the stereoelectronic requirements of a Michael addition. A series of 6-ethynylpurines was prepared and a structure activity relationship (SAR) established for inhibition of Nek2. 6-Ethynyl-N-phenyl-7H-purin-2-amine [IC50 0.15 μM (Nek2)] and 4-((6-ethynyl-7H-purin-2-yl)amino)benzenesulfonamide (IC50 0.14 μM) were selected for determination of the mode of inhibition of Nek2, which was shown to be time-dependent, not reversed by addition of ATP and negated by site directed mutagenesis of Cys22 to alanine. Replacement of the ethynyl group by ethyl or cyano abrogated activity. Variation of substituents on the N-phenyl moiety for 6-ethynylpurines gave further SAR data for Nek2 inhibition. The data showed little correlation of activity with the nature of the substituent, indicating that after sufficient initial competitive binding to Nek2 subsequent covalent modification of Cys22 occurs in all cases. A typical activity profile was that for 2-(3-((6-ethynyl-9H-purin-2-yl)amino)phenyl)acetamide [IC50 0.06 μM (Nek2); GI50 (SKBR3) 2.2 μM] which exhibited >5–10-fold selectivity for Nek2 over other kinases; it also showed > 50% growth inhibition at 10 μM concentration against selected breast and leukaemia cell lines. X-ray crystallographic analysis confirmed that binding of the compound to the Nek2 ATP-binding site resulted in covalent modification of Cys22. Further studies confirmed that 2-(3-((6-ethynyl-9H-purin-2-yl)amino)phenyl)acetamide has the attributes of a drug-like compound with good aqueous solubility, no inhibition of hERG at 25 μM and a good stability profile in human liver microsomes. It is concluded that 6-ethynylpurines are promising agents for cancer treatment by virtue of their selective inhibition of Nek2.

The education rankings of Australian students continue to slide further down the international scale, yet the dominant discourse in relation to both the cause and solution to this perceived decline remains focused on funding, curriculum and teacher quality whilst failing largely to take account of the broader social contexts in which education takes place. Drawing on findings from recent research in primary schools in regional Australia, this paper raises the question of Australian school students’ human right to an education and explores the role that school social workers can play in upholding this right. Discussion draws on international research, including studies that examine the outcomes of employing suitably qualified School Social Workers who can foster links and networks between student, family, school and community. It is argued that these networks create social capital that can serve to enable students to benefit from both the education to which they are entitled, and the connections that may empower them, as they move into adult life.

This article examines service user involvement in social work education. It discusses the challenges and ethical considerations of involving populations who may previously have been excluded from user involvement initiatives, raising questions about the benefits and challenges of their involvement. The article then provides discussion of an approach to service user involvement in social work education with one of these populations, men who use violence in their intimate relationships, and concludes by considering the implications of their involvement for the social work academy.

Purpose This paper aims to explore the place of disabled guests in the new world of hotel and holiday accommodation shaped by the sharing economy. Design/methodology/approach The paper uses Levitas’s (2013) Utopia as Method as a methodological tool to develop the hypothetical future scenarios, which are used to explore the place of disabled guests in peer-to-peer holiday accommodation. Findings Analysis of the hypothetical scenarios suggests that without state intervention, the place of disabled guests in both traditional hotels and peer-to-peer holiday accommodation is far from secure. Research limitations/implications This is a new area and the authors’ discussion is therefore tentative in its intent. Practical implications Planners and policymakers should consult with, and take account of, the needs of disabled people and other socially excluded groups when regulating shared economy enterprises. It may be helpful to put in place broader legislation for social inclusion rather than regulate peer-to-peer platforms. Any recourse to markets as a means of resolving access issues needs also to acknowledge the limited power of socially excluded groups within both traditional and sharing economy markets. Social implications The hypothetical scenarios discussed within this paper offer planners, policymakers and tourism stakeholders opportunities to think through the access and inclusion needs of disabled guests in the shared economy sector. Originality/value The paper extends discussion of hospitality and disability access to include shared economy approaches and the place of disabled guests in the new world of holiday accommodation shaped by the sharing economy.

Bruce Bonyhady's (2016) paper offers a clear and informative outline of the Australian National Disability Insurance Scheme (NDIS). As an economist with expertise in funds management and insurance,...

Darragh et al.'s research article explores the work of “Tutti Arts”, a multi-arts program for people with intellectual disabilities in Adelaide, South Australia. Tutti provides opportunities for yo...

This chapter begins with a note on terminology before outlining the origins and development of disability studies and, in particular, the social model of disability in the UK. It explores disability studies and critical disability studies, identifying key aspects of both approaches. The British social model makes a clear distinction between impairment and disability – it severs the causal link between impairment and disability. The central precept of the British social model of disability is the distinction between impairment and disability. In social model discussion, the term 'disabled people' is therefore used because this recognises that people with impairments are disabled by society's responses to impairment that is by barriers to their full inclusion in the mainstream of society. Historically, disability research was underpinned by individual model understandings of disability – research focused on individual deficit and was undertaken on, rather than with disabled people.

Stakeholder collaboration is increasingly being lauded as important in the development of accessible tourism. The purpose of this study is to explore how stakeholders collaborate in the development of accessible tourism. Drawing on research conducted in Western Australia, the study utilises qualitative approaches in its exploration. The evidence from the study strongly indicates that there is minimal collaboration between stakeholders in the development of accessible tourism. The findings suggest that when there are multiple and diverse stakeholders at play, an organic, circulatory and developmental approach to stakeholder collaboration should be adopted to innovatively move towards inclusive tourism – an ideal that aspires to equal access and inclusion for all. To this end, four emergent interrelated themes are considered: control and coordination, communication, clarity of roles and responsibilities and collaboration and integration. From these themes, a framework that can be applied to encourage collaboration is proposed.

Background: People (with and without learning disabilities) are living longer. Demographic ageing creates challenges and the leading policy response to these challenges is ‘active ageing’. ‘Active’ does not just refer to the ability to be physically and economically active, but also includes ongoing social and civic engagement in the communities of which older people are a part. Active ageing should apply to all citizens, including the experiences of older people with learning disabilities. Materials and Methods: This literature based paper explores the focus of active ageing discussions in relation to the general population drawing comparisons with the experiences of older people with learning disabilities. Results: It points out that older people with learning disabilities and their experiences are largely missing from broader policy discussions of active ageing. Conclusion: The paper concludes by arguing for inclusive research in active ageing which takes account of the concerns and interests of older people with learning disabilities.

Acquired resistance to therapy is perhaps the greatest challenge to effective clinical management of cancer. With several inhibitors of the mitotic checkpoint kinase MPS1 in preclinical development, we sought to investigate how resistance against these inhibitors may arise so that mitigation or bypass strategies could be addressed as early as possible. Toward this end, we modeled acquired resistance to the MPS1 inhibitors AZ3146, NMS-P715, and CCT251455, identifying five point mutations in the kinase domain of MPS1 that confer resistance against multiple inhibitors. Structural studies showed how the MPS1 mutants conferred resistance by causing steric hindrance to inhibitor binding. Notably, we show that these mutations occur in nontreated cancer cell lines and primary tumor specimens, and that they also preexist in normal lymphoblast and breast tissues. In a parallel piece of work, we also show that the EGFR p.T790M mutation, the most common mutation conferring resistance to the EGFR inhibitor gefitinib, also preexists in cancer cells and normal tissue. Our results therefore suggest that mutations conferring resistance to targeted therapy occur naturally in normal and malignant cells and these mutations do not arise as a result of the increased mutagenic plasticity of cancer cells. Cancer Res; 75(16); 3340–54. ©2015 AACR.

Disability is a widespread phenomenon, indeed a potentially universal one as life expectancies rise. Within the academic world, it has relevance for all disciplines yet is often dismissed as a niche market or someone else’s domain. This collection explores how academic avoidance of disability studies and disability theory is indicative of social prejudice and highlights, conversely, how the academy can and does engage with disability studies. This innovative book brings together work in the humanities and the social sciences, and draws on the riches of cultural diversity to challenge institutional and disciplinary avoidance. Divided into three parts, the first looks at how educational institutions and systems implicitly uphold double standards, which can result in negative experiences for staff and students who are disabled. The second part explores how disability studies informs and improves a number of academic disciplines, from social work to performance arts. The final part shows how more diverse cultural engagement offers a way forward for the academy, demonstrating ways in which we can make more explicit the interdisciplinary significance of disability studies – and, by extension, disability theory, activism, experience, and culture. Disability, Avoidance and the Academy: Challenging Resistance will interest students and scholars of disability studies, education studies and cultural studies.