Target occupancy biomarker assay development using a conformation-selective antibody against small-molecule-bound TNF.

Background: An antibody specific to small-molecule inhibitor-bound TNF has enabled the development of target occupancy biomarker assays to support the development of novel treatments for autoimmune disorders. Materials & methods: ELISAs were developed for inhibitor-bound and total TNF to determine the percentage of TNF occupancy in samples from stimulated blood. Inhibitor-saturated samples allowed measurement of total and inhibitor-bound TNF in a single electrochemiluminescence immunoassay. Results: TNF occupancy was proportional to inhibitor concentration in plasma samples. An electrochemiluminescence method for inhibitor-bound TNF was validated for use as a potential clinical occupancy biomarker assay. Conclusion: Development of these assays has allowed measurement of a target occupancy biomarker, which has supported progression of the first small-molecule inhibitors of TNF.

A conformation-selective monoclonal antibody against a small molecule-stabilised signalling-deficient form of TNF.

We have recently described the development of a series of small-molecule inhibitors of human tumour necrosis factor (TNF) that stabilise an open, asymmetric, signalling-deficient form of the soluble TNF trimer. Here, we describe the generation, characterisation, and utility of a monoclonal antibody that selectively binds with high affinity to the asymmetric TNF trimer-small molecule complex. The antibody helps to define the molecular dynamics of the apo TNF trimer, reveals the mode of action and specificity of the small molecule inhibitors, acts as a chaperone in solving the human TNF-TNFR1 complex crystal structure, and facilitates the measurement of small molecule target occupancy in complex biological samples. We believe this work defines a role for monoclonal antibodies as tools to facilitate the discovery and development of small-molecule inhibitors of protein-protein interactions.

WNT3A-loaded exosomes enable cartilage repair.

Cartilage defects repair poorly. Recent genetic studies suggest that WNT3a may contribute to cartilage regeneration, however the dense, avascular cartilage extracellular matrix limits its penetration and signalling to chondrocytes. Extracellular vesicles actively penetrate intact cartilage. This study investigates the effect of delivering WNT3a into large cartilage defects in vivo using exosomes as a delivery vehicle. Exosomes were purified by ultracentrifugation from conditioned medium of either L-cells overexpressing WNT3a or control un-transduced L-cells, and characterized by electron microscopy, nanoparticle tracking analysis and marker profiling. WNT3a loaded on exosomes was quantified by western blotting and functionally characterized in vitro using the SUPER8TOPFlash reporter assay and other established readouts including proliferation and proteoglycan content. In vivo pathway activation was assessed using TCF/Lef:H2B-GFP reporter mice. Wnt3a loaded exosomes were injected into the knees of mice, in which large osteochondral defects were surgically generated. The degree of repair was histologically scored after 8 weeks. WNT3a was successfully loaded on exosomes and resulted in activation of WNT signalling in vitro. In vivo, recombinant WNT3a failed to activate WNT signalling in cartilage, whereas a single administration of WNT3a loaded exosomes activated canonical WNT signalling for at least one week, and eight weeks later, improved the repair of osteochondral defects. WNT3a assembled on exosomes, is efficiently delivered into cartilage and contributes to the healing of osteochondral defects.

Discovery of a novel series of quinoxalines as inhibitors of c-Met kinase.

A series of quinoxaline inhibitors of c-Met kinase is described. The postulated binding mode was confirmed by an X-ray crystal structure and optimisation of the series was performed on the basis of this structure. Future directions for development of the series are discussed together with the identification of a novel quinoline scaffold.

Discovery of 4-azaindoles as novel inhibitors of c-Met kinase.

A series of 4-azaindole inhibitors of c-Met kinase is described. The postulated binding mode was confirmed by an X-ray crystal structure and series optimisation was performed on the basis of this structure. Future directions for series development are discussed.

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.

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.

TNFR1 membrane reorganization promotes distinct modes of TNFα signaling.

Signaling by the ubiquitously expressed tumor necrosis factor receptor 1 (TNFR1) after ligand binding plays an essential role in determining whether cells exhibit survival or death. TNFR1 forms distinct signaling complexes that initiate gene expression programs downstream of the transcriptional regulators NFκB and AP-1 and promote different functional outcomes, such as inflammation, apoptosis, and necroptosis. Here, we investigated the ways in which TNFR1 was organized at the plasma membrane at the nanoscale level to elicit different signaling outcomes. We confirmed that TNFR1 forms preassembled clusters at the plasma membrane of adherent cells in the absence of ligand. After trimeric TNFα binding, TNFR1 clusters underwent a conformational change, which promoted lateral mobility, their association with the kinase MEKK1, and activation of the JNK/p38/NFκB pathway. These phenotypes required a minimum of two TNFR1-TNFα contact sites; fewer binding sites resulted in activation of NFκB but not JNK and p38. These data suggest that distinct modes of TNFR1 signaling depend on nanoscale changes in receptor organization.

Small molecules that inhibit TNF signalling by stabilising an asymmetric form of the trimer.

Tumour necrosis factor (TNF) is a cytokine belonging to a family of trimeric proteins; it has been shown to be a key mediator in autoimmune diseases such as rheumatoid arthritis and Crohn's disease. While TNF is the target of several successful biologic drugs, attempts to design small molecule therapies directed to this cytokine have not led to approved products. Here we report the discovery of potent small molecule inhibitors of TNF that stabilise an asymmetrical form of the soluble TNF trimer, compromising signalling and inhibiting the functions of TNF in vitro and in vivo. This discovery paves the way for a class of small molecule drugs capable of modulating TNF function by stabilising a naturally sampled, receptor-incompetent conformation of TNF. Furthermore, this approach may prove to be a more general mechanism for inhibiting protein-protein interactions.

Phase I pharmacokinetic and pharmacodynamic study of 17-allylamino, 17-demethoxygeldanamycin in patients with advanced malignancies.

PURPOSE: To study the toxicity and pharmacokinetic-pharmacodynamic profile of 17-allylamino, 17- demethoxygeldanamycin (17-AAG) and to recommend a dose for phase II trials. PATIENTS AND METHODS: This was a phase I study examining a once-weekly dosing schedule of 17-AAG. Thirty patients with advanced malignancies were treated. RESULTS: The highest dose level reached was 450 mg/m(2)/week. The dose-limiting toxicities (DLTs) encountered were grade 3 diarrhea in three patients (one at 320 mg/m(2)/week and two at 450 mg/m(2)/week) and grade 3 to 4 hepatotoxicity (AST/ALT) in one patient at 450 mg/m(2)/week. Two of nine DLTs were at the highest dose level. Two patients with metastatic melanoma had stable disease and were treated for 15 and 41 months, respectively. The dose versus area under the curve-relationship for 17-AAG was linear (r(2) = .71) over the dose range 10 to 450 mg/m(2)/week, with peak plasma concentrations of 8,998 mug/L (standard deviation, 2,881) at the highest dose level. After the demonstration of pharmacodynamic changes in peripheral blood leukocytes, pre- and 24 hours post-treatment, tumor biopsies were performed and demonstrated target inhibition (c-RAF-1 inhibition in four of six patients, CDK4 depletion in eight of nine patients and HSP70 induction in eight of nine patients) at the dose levels 320 and 450 mg/m(2)/week. It was not possible to reproducibly demonstrate these changes in biopsies taken 5 days after treatment. CONCLUSION: It has been possible to demonstrate that 17-AAG exhibits a tolerable toxicity profile with therapeutic plasma concentrations and target inhibition for 24 hours after treatment and some indications of clinical activity at the dose level 450 mg/m(2)/week. We recommend this dose for phase II clinical trials.

Engagement of CD22 on B cells with the monoclonal antibody epratuzumab stimulates the phosphorylation of upstream inhibitory signals of the B cell receptor.

The binding of antigen to the B cell receptor (BCR) results in a cascade of signalling events that ultimately drive B cell activation. Uncontrolled B cell activation is regulated by negative feedback loops that involve inhibitory co-receptors such as CD22 and CD32B that exert their functions following phosphorylation of immunoreceptor tyrosine-based inhibition motifs (ITIMs). The CD22-targeted antibody epratuzumab has previously been shown to inhibit BCR-driven signalling events, but its effects on ITIM phosphorylation of CD22 and CD32B have not been properly evaluated. The present study therefore employed both immunoprecipitation and flow cytometry approaches to elucidate the effects of epratuzumab on direct phosphorylation of key tyrosine (Tyr) residues on both these proteins, using both transformed B cell lines and primary human B cells. Epratuzumab induced the phosphorylation of Tyr(822) on CD22 and enhanced its co-localisation with SHP-1. Additionally, in spite of high basal phosphorylation of other key ITIMs on CD22, in primary human B cells epratuzumab also enhanced phosphorylation of Tyr(807), a residue involved in the recruitment of Grb2. Such initiation events could explain the effects of epratuzumab on downstream signalling in B cells. Finally, we were able to demonstrate that epratuzumab stimulated the phosphorylation of Tyr(292) on the low affinity inhibitory Fc receptor CD32B which would further attenuate BCR-induced signalling. Together, these data demonstrate that engagement of CD22 with epratuzumab leads to the direct phosphorylation of key upstream inhibitory receptors of BCR signalling and may help to explain how this antibody modulates B cell function.

Genes and proteins governing the cellular sensitivity to HSP90 inhibitors: a mechanistic perspective.

HSP90 inhibitors such as 17AAG have the major therapeutic advantage that they exert downstream inhibitory effects on multiple oncogenic client proteins. They therefore block several mission critical cancer-causing pathways and have the potential to modulate all of the hallmark biological features of malignancy. Consistent with this combinatorial anti-oncogenic profile, 17AAG exhibits broad-spectrum antitumour activity against cultured cancer cell lines and in vivo animal models. However, there are clear differences in sensitivity between various cancer cell lines and it is quite possible that some tumour types or individual patients will be more responsive in the clinic than others. We describe the methods used to investigate the genes and proteins involved in the mechanism of action of HSP90 inhibitors and discuss the significance of these for cellular sensitivity. Methods used involve the conventional cell and molecular biology techniques, together with the more recent application of high throughput global technologies such as gene expression microarrays and proteomics. Selected examples that seem to play a role in sensitivity to HSP90 inhibitors are highlighted and the potential relevance to the response of cancer patients is discussed. Important determinants of response include: 1) Dependence upon key HSP90 client proteins such as ERBB2, steroid hormone receptors and AKT/PKB; 2) Levels of HSP90 family members and co-chaperones, such as HSP70 and AHA1; and 3) expression of various cell cycle and apoptotic regulators. In the case of 17AAG, metabolic enzymes such as NQO1 and membrane efflux pumps are also important for sensitivity.

CDP7657, an anti-CD40L antibody lacking an Fc domain, inhibits CD40L-dependent immune responses without thrombotic complications: an in vivo study.

INTRODUCTION: CD40 ligand (CD40L) blockade has demonstrated efficacy in experimental autoimmune models. However, clinical trials of hu5c8, an anti-human CD40L IgG1 antibody, in systemic lupus erythematosus (SLE) were halted due to an increased incidence of thrombotic events. This study evaluated CDP7657, a high affinity PEGylated monovalent Fab' anti-CD40L antibody fragment, to assess whether an Fc-deficient molecule retains efficacy while avoiding the increased risk of thrombotic events observed with hu5c8. METHODS: The potency and cross-reactivity of CDP7657 was assessed in in vitro assays employing human and non-human primate leukocytes, and the capacity of different antibody formats to activate platelets in vitro was assessed using aggregometry and dense granule release assays. Given the important role CD40L plays in regulating humoral immunity, in vivo efficacy was assessed by investigating the capacity of Cynomolgus monkeys to generate immune responses to the tetanus toxoid antigen while the potential to induce thrombotic events in vivo was evaluated after repeat dosing of antibodies to Rhesus monkeys. A PEGylated anti-mouse CD40L was generated to assess efficacy in the New Zealand Black/White (NZB/W) mouse model of SLE. RESULTS: CDP7657 dose-dependently inhibited antigen-specific immune responses to tetanus toxoid in Cynomolgus monkeys, and in contrast to hu5c8, there was no evidence of pulmonary thrombovasculopathy in Rhesus monkeys. Aglycosyl hu5c8, which lacks Fc receptor binding function, also failed to induce thrombotic events in Rhesus monkeys. In vitro experiments confirmed that antibody constructs lacking an Fc, including CDP7657, did not induce human or monkey platelet activation. A PEGylated monovalent Fab' anti-mouse CD40L antibody also inhibited disease activity in the NZB/W mouse model of SLE after administration using a therapeutic dosing regimen where mice received antibodies only after they had displayed severe proteinuria. CONCLUSIONS: These findings demonstrate for the first time that anti-CD40L antibodies lacking a functional Fc region do not induce thrombotic events in Rhesus monkeys and fail to activate platelets in vitro but, nevertheless retain pharmacological activity and support the investigation of CDP7657 as a potential therapy for systemic lupus erythematosus and other autoimmune diseases.

HSP90 as a new therapeutic target for cancer therapy: the story unfolds.

Current anticancer drug development strategies involve identifying novel molecular targets which are crucial for tumourigenesis. The molecular chaperone heat shock protein (HSP) 90 is of interest as an anticancer drug target because of its importance in maintaining the conformation, stability and function of key oncogenic client proteins involved in signal transduction pathways leading to proliferation, cell cycle progression and apoptosis, as well as other features of the malignant phenotype such as invasion, angiogenesis and metastasis. The natural product HSP90 inhibitors geldanamycin and radicicol exert their antitumour effect by inhibiting the intrinsic ATPase activity of HSP90, resulting in degradation of HSP90 client proteins via the ubiquitin proteosome pathway. Anticancer selectivity may derive from the simultaneous combinatorial effects of HSP90 inhibitors on multiple cancer targets and pathways. 17-allylamino, 17-demethoxygeldanamycin (17AAG), a geldanamycin derivative, showed good activity and cancer selectivity in preclinical models and has now progressed to Phase I clinical trial in cancer patients with encouraging initial results. Phase II trials including combination studies with cytotoxic agents are now being planned and these should allow the therapeutic activity of 17AAG to be determined. Second generation HSP90 inhibitors may be designed to overcome some of the drawbacks of 17AAG, including limited oral bioavailability and solubility. They could also be engineered to target specific functions of HSP90, which may not only provide greater molecular selectivity and clinical benefit but may also increase understanding of the complex functions of this molecular chaperone. HSP90 inhibitors provide proof of concept for drugs directed at HSP90 and protein folding and this principle may be applicable to other medical conditions involving protein aggregation and stability.

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.

The identification, synthesis, protein crystal structure and in vitro biochemical evaluation of a new 3,4-diarylpyrazole class of Hsp90 inhibitors.

High-throughput screening identified the 3,4-diarylpyrazole CCT018159 as a novel and potent (7.1 microM) inhibitor of Hsp90 ATPase activity. Here, we describe the synthesis of CCT018159 and a number of close analogues together with data on their biochemical properties. Some initial structure-activity relationships are discussed, as well as the crystal structure of CCT018159 bound to Hsp90.

Activation of the ATPase activity of hsp90 by the stress-regulated cochaperone aha1.

Client protein activation by Hsp90 involves a plethora of cochaperones whose roles are poorly defined. A ubiquitous family of stress-regulated proteins have been identified (Aha1, activator of Hsp90 ATPase) that bind directly to Hsp90 and are required for the in vivo Hsp90-dependent activation of clients such as v-Src, implicating them as cochaperones of the Hsp90 system. In vitro, Aha1 and its shorter homolog, Hch1, stimulate the inherent ATPase activity of yeast and human Hsp90. The identification of these Hsp90 cochaperone activators adds to the complex roles of cochaperones in regulating the ATPase-coupled conformational changes of the Hsp90 chaperone cycle.