Discovery of WRN inhibitor HRO761 with synthetic lethality in MSI cancers.

The Werner syndrome RecQ helicase WRN was identified as a synthetic lethal target in cancer cells with microsatellite instability (MSI) by several genetic screens1-6. Despite advances in treatment with immune checkpoint inhibitors7-10, there is an unmet need in the treatment of MSI cancers11-14. Here we report the structural, biochemical, cellular and pharmacological characterization of the clinical-stage WRN helicase inhibitor HRO761, which was identified through an innovative hit-finding and lead-optimization strategy. HRO761 is a potent, selective, allosteric WRN inhibitor that binds at the interface of the D1 and D2 helicase domains, locking WRN in an inactive conformation. Pharmacological inhibition by HRO761 recapitulated the phenotype observed by WRN genetic suppression, leading to DNA damage and inhibition of tumour cell growth selectively in MSI cells in a p53-independent manner. Moreover, HRO761 led to WRN degradation in MSI cells but not in microsatellite-stable cells. Oral treatment with HRO761 resulted in dose-dependent in vivo DNA damage induction and tumour growth inhibition in MSI cell- and patient-derived xenograft models. These findings represent preclinical pharmacological validation of WRN as a therapeutic target in MSI cancers. A clinical trial with HRO761 (NCT05838768) is ongoing to assess the safety, tolerability and preliminary anti-tumour activity in patients with MSI colorectal cancer and other MSI solid tumours.

The allosteric inhibitor ABL001 enables dual targeting of BCR-ABL1.

Chronic myeloid leukaemia (CML) is driven by the activity of the BCR-ABL1 fusion oncoprotein. ABL1 kinase inhibitors have improved the clinical outcomes for patients with CML, with over 80% of patients treated with imatinib surviving for more than 10 years. Second-generation ABL1 kinase inhibitors induce more potent molecular responses in both previously untreated and imatinib-resistant patients with CML. Studies in patients with chronic-phase CML have shown that around 50% of patients who achieve and maintain undetectable BCR-ABL1 transcript levels for at least 2 years remain disease-free after the withdrawal of treatment. Here we characterize ABL001 (asciminib), a potent and selective allosteric ABL1 inhibitor that is undergoing clinical development testing in patients with CML and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukaemia. In contrast to catalytic-site ABL1 kinase inhibitors, ABL001 binds to the myristoyl pocket of ABL1 and induces the formation of an inactive kinase conformation. ABL001 and second-generation catalytic inhibitors have similar cellular potencies but distinct patterns of resistance mutations, with genetic barcoding studies revealing pre-existing clonal populations with no shared resistance between ABL001 and the catalytic inhibitor nilotinib. Consistent with this profile, acquired resistance was observed with single-agent therapy in mice; however, the combination of ABL001 and nilotinib led to complete disease control and eradicated CML xenograft tumours without recurrence after the cessation of treatment.

A kinase inhibitor which specifically targets the ABL myristate pocket (STAMP), but unlike asciminib crosses the blood-brain barrier.

The ubiquitously expressed ABL1 and ABL2 protein kinases play many important roles in cell function. Although they have been implicated in neuron development, maintenance and signaling, there are no good tool compounds to evaluate the effects of ABL kinase inhibition in the brain. Asciminib is a recently approved drug that specifically and potently inhibits the tyrosine kinase activity of ABL1, ABL2 and that of the chimeric BCR-ABL1 oncoprotein which causes chronic myeloid leukemia. Herein we show that asciminib does not penetrate the intact blood-brain barrier (BBB) following administration to rats, which curtails its utility for assessing the in vivo effects of ABL kinase inhibition in the brain. However, we describe another specific ABL kinase inhibitor, possessing physicochemical characteristics suitable for BBB penetration, and which after administration (either i.v., i.p. or p.o.) to mice achieves substantial, pharmacologically relevant brain concentrations. This bipyridine compound (4) therefore has potential for elucidating the role of ABL kinases in the brain in non-clinical studies.

Disposition of asciminib, a potent BCR-ABL1 tyrosine kinase inhibitor, in healthy male subjects.

Asciminib is a potent, specific BCR-ABL1 inhibitor being developed for the treatment of patients with chronic myelogenous leukemia (CML) and Philadelphia chromosome positive acute lymphoblastic leukemia (Ph + ALL).Here, we present the results of human oral absorption, distribution, metabolism, excretion (ADME) and in vitro studies that together provide an overall understanding of the metabolism, distribution and clearance of asciminib in humans.Asciminib was rapidly absorbed with a maximum plasma concentration at two hours post-dose. Total radioactivity and asciminib showed similar terminal half-lives in plasma.Oral asciminib absorption ranged between a minimum of 33%, and a maximum of 57% based on the metabolite profiles of late time-point feces collections.Asciminib was eliminated mainly through feces via unchanged asciminib excretion and metabolism.Direct glucuronidation and oxidation were major metabolic pathways in human that were catalyzed predominantly by UDP-glucuronosyltransferase (UGT)2B7 and cytochrome P450 (CYP)3A4, respectively.The relative contribution of the glucuronidation pathway to the total clearance of asciminib via metabolism is estimated to range ∼28-58%, whereas the relative contribution of the oxidative pathway is estimated to range ∼37-64%, based upon the maximum oral absorption in humans.

Targeting conserved water molecules: design of 4-aryl-5-cyanopyrrolo[2,3-d]pyrimidine Hsp90 inhibitors using fragment-based screening and structure-based optimization.

Inhibitors of the Hsp90 molecular chaperone are showing promise as anti-cancer agents. Here we describe a series of 4-aryl-5-cyanopyrrolo[2,3-d]pyrimidine ATP competitive Hsp90 inhibitors that were identified following structure-driven optimization of purine hits revealed by NMR based screening of a proprietary fragment library. Ligand-Hsp90 X-ray structures combined with molecular modeling led to the rational displacement of a conserved water molecule leading to enhanced affinity for Hsp90 as measured by fluorescence polarization, isothermal titration calorimetry and surface plasmon resonance assays. This displacement was achieved with a nitrile group, presenting an example of efficient gain in binding affinity with minimal increase in molecular weight. Some compounds in this chemical series inhibit the proliferation of human cancer cell lines in vitro and cause depletion of oncogenic Hsp90 client proteins and concomitant elevation of the co-chaperone Hsp70. In addition, one compound was demonstrated to be orally bioavailable in the mouse. This work demonstrates the power of structure-based design for the rapid evolution of potent Hsp90 inhibitors and the importance of considering conserved water molecules in drug design.

Preclinical Evaluation of [11C]YC-72-AB85 for In Vivo Visualization of Heat Shock Protein 90 in Brain and Cancer with Positron Emission Tomography.

Aberrant Hsp90 has been implied in cancer and neurodegenerative disorders. The development of a suitable Hsp90 Positron emission tomography (PET) probe can provide in vivo quantification of the expression levels of Hsp90 as a biomarker for diagnosis and follow-up of cancer and central nervous system (CNS) disease progression. In this respect, [11C]YC-72-AB85 was evaluated as an Hsp90 PET probe in B16.F10 melanoma bearing mice and its brain uptake was determined in rats and nonhuman primate. In vitro binding of [11C]YC-72-AB85 to tissue slices of mouse B16.F10 melanoma, PC3 prostate carcinoma, and rodent brain was evaluated using autoradiography. Biodistribution of [11C]YC-72-AB85 was evaluated in healthy and B16.F10 melanoma mice. In vivo brain uptake was assessed by µPET studies in rats and a rhesus monkey. In vitro binding was deemed Hsp90-specific by blocking studies with heterologous Hsp90 inhibitors onalespib and SNX-0723. Saturable Hsp90 binding was observed in brain, tumor, blood, and blood-rich organs in mice. In combined pretreatment and displacement studies, reversible and Hsp90-specific binding of [11C]YC-72-AB85 was observed in rat brain. Dynamic µPET brain scans in baseline and blocking conditions in a rhesus monkey indicated Hsp90-specific binding. [11C]YC-72-AB85 is a promising PET tracer for in vivo visualization of Hsp90 in tumor and brain. Clear differences of Hsp90 binding to blood and blood-rich organs were observed in tumor vs control mice. Further, we clearly demonstrate, for the first time, binding to a saturable Hsp90 pool in brain of rats and a rhesus monkey.

Anti-myeloma activity of the novel 2-aminothienopyrimidine Hsp90 inhibitor NVP-BEP800.

The 90 kD heat shock protein (Hsp90) molecular chaperone sustains multiple components of oncogenic pathways and has recently emerged as a therapeutic target that is now being clinically tested in a number of malignancies. In order to address formulation issues and to deal with possible resistance mechanisms against small molecule Hsp90 inhibitors, a range of compounds based on different molecular scaffolds are now being developed. The present study preclinically tested the effects of the novel 2-aminothienopyrimidine class Hsp90 inhibitor NVP-BEP800, which is suitable for oral formulations, on multiple myeloma cells from established cell lines and on a larger cohort (n = 40) of primary myeloma samples. The drug effectively and specifically killed the majority of primary myeloma cells in coculture with bone marrow stromal cells and reliably entailed molecular consequences of Hsp90 blockade - such as survival pathway breakdown and client protein depletion - in multiple myeloma cells from cell lines as well as from patients. Collectively, the properties of this novel drug support clinical testing in multiple myeloma.

Discovery of a new class of catalytic topoisomerase II inhibitors targeting the ATP-binding site by structure based design. Part I.

Topoisomerase II is a validated target in oncology. Among the different ways of blocking the function of this enzyme, inhibiting its ATPase activity has been relatively less investigated. In an effort to identify topoisomerase II inhibitors of a novel type, exerting their action by this mechanism, we have designed a purine inhibitor scaffold targeting the ATP-binding site of the enzyme. Searching the Novartis compound collection for molecules containing this purine motif has allowed the identification of two micromolar hits providing access to a new class of catalytic topoisomerase II inhibitors.

Capmatinib (INC280) Is Active Against Models of Non-Small Cell Lung Cancer and Other Cancer Types with Defined Mechanisms of MET Activation.

PURPOSE: The selective MET inhibitor capmatinib is being investigated in multiple clinical trials, both as a single agent and in combination. Here, we describe the preclinical data of capmatinib, which supported the clinical biomarker strategy for rational patient selection. EXPERIMENTAL DESIGN: The selectivity and cellular activity of capmatinib were assessed in large cellular screening panels. Antitumor efficacy was quantified in a large set of cell line- or patient-derived xenograft models, testing single-agent or combination treatment depending on the genomic profile of the respective models. RESULTS: Capmatinib was found to be highly selective for MET over other kinases. It was active against cancer models that are characterized by MET amplification, marked MET overexpression, MET exon 14 skipping mutations, or MET activation via expression of the ligand hepatocyte growth factor (HGF). In cancer models where MET is the dominant oncogenic driver, anticancer activity could be further enhanced by combination treatments, for example, by the addition of apoptosis-inducing BH3 mimetics. The combinations of capmatinib and other kinase inhibitors resulted in enhanced anticancer activity against models where MET activation co-occurred with other oncogenic drivers, for example EGFR activating mutations. CONCLUSIONS: Activity of capmatinib in preclinical models is associated with a small number of plausible genomic features. The low fraction of cancer models that respond to capmatinib as a single agent suggests that the implementation of patient selection strategies based on these biomarkers is critical for clinical development. Capmatinib is also a rational combination partner for other kinase inhibitors to combat MET-driven resistance.

Structural States of Hdm2 and HdmX: X-ray Elucidation of Adaptations and Binding Interactions for Different Chemical Compound Classes.

Hdm2 (human MDM2, human double minute 2 homologue) counteracts p53 function by direct binding to p53 and by ubiquitin-dependent p53 protein degradation. Activation of p53 by inhibitors of the p53-Hdm2 interaction is being pursued as a therapeutic strategy in p53 wild-type cancers. In addition, HdmX (human MDMX, human MDM4) was also identified as an important therapeutic target to efficiently reactivate p53, and it is likely that dual inhibition of Hdm2 and HdmX is beneficial. Herein we report four new X-ray structures for Hdm2 and five new X-ray structures for HdmX complexes, involving different classes of synthetic compounds (including the worldwide highest resolutions for Hdm2 and HdmX, at 1.13 and 1.20 Å, respectively). We also reveal the key additive 18-crown-ether, which we discovered to enable HdmX crystallization and show its stabilization of various Lys residues. In addition, we report the previously unpublished details of X-ray structure determinations for eight further Hdm2 complexes, including the clinical trial compounds NVP-CGM097 and NVP-HDM201. An analysis of all compound binding modes reveals new and deepened insight into the possible adaptations and structural states of Hdm2 (e.g., flip of F55, flip of Y67, reorientation of H96) and HdmX (e.g., flip of H55, dimer induction), enabling key binding interactions for different compound classes. To facilitate comparisons, we used the same numbering for Hdm2 (as in Q00987) and HdmX (as in O15151, but minus 1). Taken together, these structural insights should prove useful for the design and optimization of further selective and/or dual Hdm2/HdmX inhibitors.

NVP-AUY922: a small molecule HSP90 inhibitor with potent antitumor activity in preclinical breast cancer models.

INTRODUCTION: Heat shock protein 90 (HSP90) is a key component of a multichaperone complex involved in the post-translational folding of a large number of client proteins, many of which play essential roles in tumorigenesis. HSP90 has emerged in recent years as a promising new target for anticancer therapies. METHODS: The concentrations of the HSP90 inhibitor NVP-AUY922 required to reduce cell numbers by 50% (GI50 values) were established in a panel of breast cancer cell lines and patient-derived human breast tumors. To investigate the properties of the compound in vivo, the pharmacokinetic profile, antitumor effect, and dose regimen were established in a BT-474 breast cancer xenograft model. The effect on HSP90-p23 complexes, client protein degradation, and heat shock response was investigated in cell culture and breast cancer xenografts by immunohistochemistry, Western blot analysis, and immunoprecipitation. RESULTS: We show that the novel small molecule HSP90 inhibitor NVP-AUY922 potently inhibits the proliferation of human breast cancer cell lines with GI50 values in the range of 3 to 126 nM. NVP-AUY922 induced proliferative inhibition concurrent with HSP70 upregulation and client protein depletion--hallmarks of HSP90 inhibition. Intravenous acute administration of NVP-AUY922 to athymic mice (30 mg/kg) bearing subcutaneous BT-474 breast tumors resulted in drug levels in excess of 1,000 times the cellular GI50 value for about 2 days. Significant growth inhibition and good tolerability were observed when the compound was administered once per week. Therapeutic effects were concordant with changes in pharmacodynamic markers, including HSP90-p23 dissociation, decreases in ERBB2 and P-AKT, and increased HSP70 protein levels. CONCLUSION: NVP-AUY922 is a potent small molecule HSP90 inhibitor showing significant activity against breast cancer cells in cellular and in vivo settings. On the basis of its mechanism of action, preclinical activity profile, tolerability, and pharmaceutical properties, the compound recently has entered clinical phase I breast cancer trials.

Entry into a new class of protein kinase inhibitors by pseudo ring design.

A pyrimidin-4-yl-urea motif forming a pseudo ring by intramolecular hydrogen bonding has been designed to mimic the pyrido[2,3-d]pyrimidin-7-one core structure of a well-established class of protein kinase inhibitors. Potent inhibition of a number of protein kinases was obtained with the first prototype compound synthesized to probe the design concept.

Discovery of Asciminib (ABL001), an Allosteric Inhibitor of the Tyrosine Kinase Activity of BCR-ABL1.

Chronic myelogenous leukemia (CML) arises from the constitutive activity of the BCR-ABL1 oncoprotein. Tyrosine kinase inhibitors (TKIs) that target the ATP-binding site have transformed CML into a chronic manageable disease. However, some patients develop drug resistance due to ATP-site mutations impeding drug binding. We describe the discovery of asciminib (ABL001), the first allosteric BCR-ABL1 inhibitor to reach the clinic. Asciminib binds to the myristate pocket of BCR-ABL1 and maintains activity against TKI-resistant ATP-site mutations. Although resistance can emerge due to myristate-site mutations, these are sensitive to ATP-competitive inhibitors so that combinations of asciminib with ATP-competitive TKIs suppress the emergence of resistance. Fragment-based screening using NMR and X-ray yielded ligands for the myristate pocket. An NMR-based conformational assay guided the transformation of these inactive ligands into ABL1 inhibitors. Further structure-based optimization for potency, physicochemical, pharmacokinetic, and drug-like properties, culminated in asciminib, which is currently undergoing clinical studies in CML patients.

Targeting Hsp90 for the treatment of cancer.

Heat shock protein (Hsp)90 is a molecular chaperone that is responsible for the correct folding of a large number of proteins, which allows these proteins to achieve their functional conformation. Client proteins of Hsp90 include many overexpressed or mutated oncogenes that are known to be critical for the transformed phenotype observed in tumors. The compounds 17-AAG (Kosan Biosciences Inc/National Cancer Institute) and 17-DMAG (Kosan Biosciences Inc/National Cancer Institute) are Hsp90 inhibitors that are derived from the prototypical ansamycin natural product Hsp90 inhibitor geldanamycin. These compounds have demonstrated preclinical efficacy in mouse xenograft models, and are now undergoing phase II and I clinical trials, respectively. Preclinical efficacy studies of these compounds are collated and discussed in this review. More recent disclosures of small-molecule Hsp90 inhibitors include purine and resorcinol analogs, and the first small-molecule Hsp90 compounds showing oral efficacy have been described. Inhibition of Hsp90 not only results in the degradation of client proteins, but also results in the induction of another chaperone, Hsp70. Hsp70 is known to be anti-apoptotic, and therefore the induction of Hsp70 may ultimately limit the efficacy of Hsp90 inhibitors under certain circumstances. Histone deacetylase inhibitors have recently been demonstrated to exert some of their effect through modulation of Hsp90 chaperoning activity, and some mechanistic aspects of this control are also discussed herein.

Biochemical and three-dimensional-structural study of the specific inhibition of protein kinase CK2 by [5-oxo-5,6-dihydroindolo-(1,2-a)quinazolin-7-yl]acetic acid (IQA).

IQA [[5-oxo-5,6-dihydro-indolo(1,2-a)quinazolin-7-yl]acetic acid] is a novel ATP/GTP site-directed inhibitor of CK2 ('casein kinase 2'), a pleiotropic and constitutively active protein kinase whose activity is abnormally high in transformed cells. The K (i) value of IQA (0.17 microM) is lower than those of other CK2 inhibitors reported so far. Tested at 10 microM concentration in the presence of 100 microM ATP, IQA almost suppresses CK2 activity in vitro, whereas it is ineffective or weakly effective on a panel of 44 protein kinases and on phosphoinositide 3-kinase. In comparison, other CK2 inhibitors, notably apigenin and quercetin, are more promiscuous. The in vivo efficacy of IQA has been assessed by using the fact that treatment of Jurkat cells with IQA inhibits endogenous CK2 in a dose-dependent manner. IQA has been co-crystallized with maize CK2alpha, which is >70% identical with its human homologue, and the structure of the complex has been determined at 1.68 A (1 A=0.1 nm) resolution. The inhibitor lies in the same plane occupied by the purine moiety of ATP with its more hydrophobic side facing the hinge region. Major contributions to the interaction are provided by hydrophobic forces and non-polar interactions involving the aromatic portion of the inhibitor and the hydrophobic residues surrounding the ATP-binding pocket, with special reference to the side chains of V53 (Val53), I66, M163 and I174. Consequently, mutants of human CK2alpha in which either V66 (the homologue of maize CK2alpha I66) or I174 is replaced by alanine are considerably less sensitive to IQA inhibition when compared with wild-type. These results provide new tools for deciphering the enigmatic role of CK2 in living cells and may pave the way for the development of drugs depending on CK2 activity.

Discovery of a potent and selective protein kinase CK2 inhibitor by high-throughput docking.

To assess the potential of protein kinase CK2 as a target for developing new antitumor agents, we have undertaken a search for inhibitors of this enzyme. As part of this effort, we report here the discovery of the potent and selective CK2 inhibitor (5-oxo-5,6-dihydroindolo[1,2-a]quinazolin-7-yl)acetic acid. We identified this inhibitor of a novel structural type by high-throughput docking of our corporate compound collection in the ATP binding site of a homology model of human CK2, using an appropriate protocol. The synthesis of the inhibitor as well as that of related analogues whose CK2 inhibitory activities give support to the binding mode proposed by the docking program is described. The results obtained suggest that virtual screening of a 3D database by molecular docking is a useful approach for lead finding provided that adapted postdocking filtering and reranking procedures are applied to the primary hit list.

Structure-based design and synthesis of 2-benzylidene-benzofuran-3-ones as flavopiridol mimics.

Novel 2-benzylidene-benzofuran-3-ones were designed and synthesized to mimic flavopiridol, a well-established inhibitor of cyclin-dependent kinases (CDKs) which is currently undergoing clinical evaluation. The underlying design concepts as well as the synthesis and structure-activity relationships (CDKs 1, 2, and 4 enzyme assays) of these mimics are described. Inhibitors of CDKs 1 and 2 that are more potent and selective than flavopiridol were obtained.

Development and implementation of a highly miniaturized confocal 2D-FIDA-based high-throughput screening assay to search for active site modulators of the human heat shock protein 90beta.

The beta isoform of the heat shock protein 90 (Hsp90beta) is a cellular chaperone required for the maturation of key proteins involved in growth response to extracellular factors as well as oncogenic transformation of various cell types. Compounds that inhibit the function of Hsp90beta are thus believed to have potential as novel anticancer drugs. To date, 2 fungal metabolites are known to inhibit Hsp90beta. However, insolubility and liver toxicity restrict the clinical use of these molecules. The limitation to identify novel and safe Hsp90beta inhibitors is that presently no suitable high-throughput screening assay is available. Here, the authors present the development of a homogenous assay based on 2-dimensional fluorescence intensity distribution analysis of tetramethyl-rhodamine (TAMRA)-labeled radicicol bound to Hsp90beta. Furthermore, the assay has been shown to be compatible with the confocal nanoscreening platform Mark II from Evotec-Technologies and can therefore be used for miniaturized high-throughput screening. The applied detection technology provides critical information about the nature of biomolecular interaction at the thermodynamic equilibrium, such as affinity constants and stoichiometric parameters of the binding. The assay is used to identify small molecular weight compounds displacing TAMRA-radicicol. Such compounds are believed to be important molecules in the discovery of novel anticancer drugs.

A drug resistance screen using a selective MET inhibitor reveals a spectrum of mutations that partially overlap with activating mutations found in cancer patients.

The emergence of drug resistance is a primary concern in any cancer treatment, including with targeted kinase inhibitors as exemplified by the appearance of Bcr-Abl point mutations in chronic myeloid leukemia (CML) patients treated with imatinib. In vitro approaches to identify resistance mutations in Bcr-Abl have yielded mutation spectra that faithfully recapitulated clinical observations. To predict resistance mutations in the receptor tyrosine kinase MET that could emerge during inhibitor treatment in patients, we conducted a resistance screen in BaF3 TPR-MET cells using the novel selective MET inhibitor NVP-BVU972. The observed spectrum of mutations in resistant cells was dominated by substitutions of tyrosine 1230 but also included other missense mutations and partially overlapped with activating MET mutations that were previously described in cancer patients. Cocrystallization of the MET kinase domain in complex with NVP-BVU972 revealed a key role for Y1230 in binding of NVP-BVU972, as previously reported for multiple other selective MET inhibitors. A second resistance screen in the same format with the MET inhibitor AMG 458 yielded a distinct spectrum of mutations rich in F1200 alterations, which is consistent with a different predicted binding mode. Our findings suggest that amino acid substitutions in the MET kinase domain of cancer patients need to be carefully monitored before and during treatment with MET inhibitors, as resistance may preexist or emerge. Compounds binding in the same manner as NVP-BVU972 might be particularly susceptible to the development of resistance through mutations in Y1230, a condition that may be addressed by MET inhibitors with alternative binding modes.

Preclinical antitumor activity of the orally available heat shock protein 90 inhibitor NVP-BEP800.

Heat shock protein 90 (Hsp90) is a ubiquitously expressed molecular chaperone with ATPase activity involved in the conformational maturation and stability of key signaling molecules involved in cell proliferation, survival, and transformation. Through its ability to modulate multiple pathways involved in oncogenesis, Hsp90 has generated considerable interest as a therapeutic target. NVP-BEP800 is a novel, fully synthetic, orally bioavailable inhibitor that binds to the NH(2)-terminal ATP-binding pocket of Hsp90. NVP-BEP800 showed activity against a panel of human tumor cell lines and primary human xenografts in vitro at nanomolar concentrations. In A375 melanoma and BT-474 breast cancer cell lines, NVP-BEP800 induced client protein degradation (including ErbB2, B-Raf(V600E), Raf-1, and Akt) and Hsp70 induction. Oral administration of NVP-BEP800 was well tolerated and induced robust antitumor responses in tumor xenograft models, including regression in the BT-474 breast cancer model. In these tumor models, NVP-BEP800 modulated Hsp90 client proteins and downstream signaling pathways at doses causing antitumor activity. NVP-BEP800 showed in vivo activity in a variety of dosing regimens covering daily to weekly schedules, potentially providing a high degree of flexibility in dose and schedule within the clinical setting. Overall, given the mechanism of action, preclinical activity profile, tolerability, and pharmaceutical properties, NVP-BEP800 is an exciting new oral Hsp90 inhibitor warranting further development. Mol Cancer Ther; 9(4); 906-19. (c)2010 AACR.