INCB24360 (Epacadostat), a Highly Potent and Selective Indoleamine-2,3-dioxygenase 1 (IDO1) Inhibitor for Immuno-oncology.

A data-centric medicinal chemistry approach led to the invention of a potent and selective IDO1 inhibitor 4f, INCB24360 (epacadostat). The molecular structure of INCB24360 contains several previously unknown or underutilized functional groups in drug substances, including a hydroxyamidine, furazan, bromide, and sulfamide. These moieties taken together in a single structure afford a compound that falls outside of "drug-like" space. Nevertheless, the in vitro ADME data is consistent with the good cell permeability and oral bioavailability observed in all species (rat, dog, monkey) tested. The extensive intramolecular hydrogen bonding observed in the small molecule crystal structure of 4f is believed to significantly contribute to the observed permeability and PK. Epacadostat in combination with anti-PD1 mAb pembrolizumab is currently being studied in a phase 3 clinical trial in patients with unresectable or metastatic melanoma.

JAK-STAT pathway activation in malignant and nonmalignant cells contributes to MPN pathogenesis and therapeutic response.

UNLABELLED: The identification of JAK2/MPL mutations in patients with myeloproliferative neoplasms (MPN) has led to the clinical development of JAK kinase inhibitors, including ruxolitinib. Ruxolitinib reduces splenomegaly and systemic symptoms in myelofibrosis and improves overall survival; however, the mechanism by which JAK inhibitors achieve efficacy has not been delineated. Patients with MPN present with increased levels of circulating proinflammatory cytokines, which are mitigated by JAK inhibitor therapy. We sought to elucidate mechanisms by which JAK inhibitors attenuate cytokine-mediated pathophysiology. Single-cell profiling demonstrated that hematopoietic cells from myelofibrosis models and patient samples aberrantly secrete inflammatory cytokines. Pan-hematopoietic Stat3 deletion reduced disease severity and attenuated cytokine secretion, with similar efficacy as observed with ruxolitinib therapy. In contrast, Stat3 deletion restricted to MPN cells did not reduce disease severity or cytokine production. Consistent with these observations, we found that malignant and nonmalignant cells aberrantly secrete cytokines and JAK inhibition reduces cytokine production from both populations. SIGNIFICANCE: Our results demonstrate that JAK-STAT3-mediated cytokine production from malignant and nonmalignant cells contributes to MPN pathogenesis and that JAK inhibition in both populations is required for therapeutic efficacy. These findings provide novel insight into the mechanisms by which JAK kinase inhibition achieves therapeutic efficacy in MPNs.

Targeting JAK1/2 and mTOR in murine xenograft models of Ph-like acute lymphoblastic leukemia.

CRLF2 rearrangements, JAK1/2 point mutations, and JAK2 fusion genes have been identified in Philadelphia chromosome (Ph)-like acute lymphoblastic leukemia (ALL), a recently described subtype of pediatric high-risk B-precursor ALL (B-ALL) which exhibits a gene expression profile similar to Ph-positive ALL and has a poor prognosis. Hyperactive JAK/STAT and PI3K/mammalian target of rapamycin (mTOR) signaling is common in this high-risk subset. We, therefore, investigated the efficacy of the JAK inhibitor ruxolitinib and the mTOR inhibitor rapamycin in xenograft models of 8 pediatric B-ALL cases with and without CRLF2 and JAK genomic lesions. Ruxolitinib treatment yielded significantly lower peripheral blast counts compared with vehicle (P < .05) in 6 of 8 human leukemia xenografts and lower splenic blast counts (P < .05) in 8 of 8 samples. Enhanced responses to ruxolitinib were observed in samples harboring JAK-activating lesions and higher levels of STAT5 phosphorylation. Rapamycin controlled leukemia burden in all 8 B-ALL samples. Survival analysis of 2 representative B-ALL xenografts demonstrated prolonged survival with rapamycin treatment compared with vehicle (P < .01). These data demonstrate preclinical in vivo efficacy of ruxolitinib and rapamycin in this high-risk B-ALL subtype, for which novel treatments are urgently needed, and highlight the therapeutic potential of targeted kinase inhibition in Ph-like ALL.

The interplay between inhibition of JAK2 and HSP90.

A recent article by Weigert et al. published in The Journal of Experimental Medicine described the in vitro generation of synthetic mutations in Janus kinase 2 (JAK 2) that decreased the potency of JAK2 (or JAK1/JAK2) inhibitors in artificial systems. The authors found that heat shock protein 90 (HSP90) inhibitors circumvented the potency shift and suggested that HSP90 inhibition may abrogate JAK inhibitor resistance in these experimental systems. However, the clinical relevance of these laboratory-generated JAK2 mutations, which have not been identified to-date in patients treated with JAK inhibitors, and the therapeutic potential of HSP90 inhibitors in diseases involving aberrant JAK-STAT signaling remain to be determined.

A novel kinase inhibitor, INCB28060, blocks c-MET-dependent signaling, neoplastic activities, and cross-talk with EGFR and HER-3.

PURPOSE: The c-MET receptor tyrosine kinase plays important roles in the formation, progression, and dissemination of human cancer and presents an attractive therapeutic target. This study describes the preclinical characterization of INCB28060, a novel inhibitor of c-MET kinase. EXPERIMENTAL DESIGN: Studies were conducted using a series of in vitro and in vivo biochemical and biological experiments. RESULTS: INCB28060 exhibits picomolar enzymatic potency and is highly specific for c-MET with more than 10,000-fold selectivity over a large panel of human kinases. This inhibitor potently blocks c-MET phosphorylation and activation of its key downstream effectors in c-MET-dependent tumor cell lines. As a result, INCB28060 potently inhibits c-MET-dependent tumor cell proliferation and migration and effectively induces apoptosis in vitro. Oral dosing of INCB28060 results in time- and dose-dependent inhibition of c-MET phosphorylation and tumor growth in c-MET-driven mouse tumor models, and the inhibitor is well tolerated at doses that achieve complete tumor inhibition. In a further exploration of potential interactions between c-MET and other signaling pathways, we found that activated c-MET positively regulates the activity of epidermal growth factor receptors (EGFR) and HER-3, as well as expression of their ligands. These effects are reversed with INCB28060 treatment. Finally, we confirmed that circulating hepatocyte growth factor levels are significantly elevated in patients with various cancers. CONCLUSIONS: Activated c-MET has pleiotropic effects on multiple cancer-promoting signaling pathways and may play a critical role in driving tumor cell growth and survival. INCB28060 is a potent and selective c-MET kinase inhibitor that may have therapeutic potential in cancer treatment.

Preclinical evaluation of local JAK1 and JAK2 inhibition in cutaneous inflammation.

JAKs are required for signaling initiated by several cytokines (e.g., IL-4, IL-12, IL-23, thymic stromal lymphopoietin (TSLP), and IFNγ) implicated in the pathogenesis of inflammatory skin diseases such as psoriasis and atopic dermatitis (AD). Direct antagonism of cytokines, such as IL-12 and IL-23 using ustekinumab, has proven effective in randomized studies in psoriasis patients. We hypothesized that local inhibition of cytokine signaling using topical administration of INCB018424, a small molecule inhibitor of JAK1 and JAK2, would provide benefit similar to systemic cytokine neutralization. In cellular assays, INCB018424 inhibits cytokine-induced JAK/signal transducers and activators of transcription (STAT) signaling and the resultant production of inflammatory proteins (e.g., IL-17, monocyte chemotactic protein-1, and IL-22) in lymphocytes and monocytes, with half-maximal inhibitory concentration values <100 nM. In vivo, topical application of INCB018424 resulted in suppression of STAT3 phosphorylation, edema, lymphocyte infiltration, and keratinocyte proliferation in a murine contact hypersensitivity model and inhibited tissue inflammation induced by either intradermal IL-23 or TSLP. Topical INCB018424 was also well tolerated in a 28-day safety study in Gottingen minipigs. These results suggest that localized JAK1/JAK2 inhibition may be therapeutic in a range of inflammatory skin disorders such as psoriasis and AD. Clinical evaluation of topical INCB018424 is ongoing.

Safety and efficacy of INCB018424, a JAK1 and JAK2 inhibitor, in myelofibrosis.

BACKGROUND: Myelofibrosis is a Philadelphia chromosome­negative myeloproliferative neoplasm associated with cytopenias, splenomegaly, poor quality of life, and shortened survival. About half of patients with myelofibrosis carry a gain-of-function mutation in the Janus kinase 2 gene (JAK2 V617F) that contributes to the pathophysiology of the disease. INCB018424 is a potent and selective Janus kinase 1 (JAK1) and JAK2 inhibitor. METHODS: We conducted a phase 1−2 trial of INCB018424 in patients with JAK2 V617F−positive or JAK2 V617F−negative primary myelofibrosis, post­essential thrombocythemia myelofibrosis, or post­polycythemia vera myelofibrosis. RESULTS: A total of 153 patients received INCB018424 for a median duration of more than 14.7 months. The initial dose-escalation phase established 25 mg twice daily or 100 mg once daily as maximum tolerated doses, on the basis of reversible thrombocytopenia. A dose-dependent suppression of phosphorylated signal transducer and activator of transcription 3 (STAT3), a marker of JAK signaling, was demonstrated in patients with wild-type JAK2 and in patients with the JAK2 V617F mutation. We studied additional doses and established that a 15-mg twice-daily starting dose, followed by individualized dose titration, was the most effective and safest dosing regimen. At this dose, 17 of 33 patients (52%) had a rapid objective response (≥50% reduction of splenomegaly) lasting for 12 months or more, and this therapy was associated with grade 3 or grade 4 adverse events (mainly myelosuppression) in less than 10% of patients. Patients with debilitating symptoms, including weight loss, fatigue, night sweats, and pruritus, had rapid improvement. Clinical benefits were associated with a marked diminution of levels of circulating inflammatory cytokines that are commonly elevated in myelofibrosis. CONCLUSIONS: INCB018424 was associated with marked and durable clinical benefits in patients with myelofibrosis for whom no approved therapies existed. (Funded by Incyte; ClinicalTrials.gov number, NCT00509899.)

Selective inhibition of JAK1 and JAK2 is efficacious in rodent models of arthritis: preclinical characterization of INCB028050.

Inhibiting signal transduction induced by inflammatory cytokines offers a new approach for the treatment of autoimmune diseases such as rheumatoid arthritis. Kinase inhibitors have shown promising oral disease-modifying antirheumatic drug potential with efficacy similar to anti-TNF biologics. Direct and indirect inhibition of the JAKs, with small molecule inhibitors like CP-690,550 and INCB018424 or neutralizing Abs, such as the anti-IL6 receptor Ab tocilizumab, have demonstrated rapid and sustained improvement in clinical measures of disease, consistent with their respective preclinical experiments. Therefore, it is of interest to identify optimized JAK inhibitors with unique profiles to maximize therapeutic opportunities. INCB028050 is a selective orally bioavailable JAK1/JAK2 inhibitor with nanomolar potency against JAK1 (5.9 nM) and JAK2 (5.7 nM). INCB028050 inhibits intracellular signaling of multiple proinflammatory cytokines including IL-6 and IL-23 at concentrations <50 nM. Significant efficacy, as assessed by improvements in clinical, histologic and radiographic signs of disease, was achieved in the rat adjuvant arthritis model with doses of INCB028050 providing partial and/or periodic inhibition of JAK1/JAK2 and no inhibition of JAK3. Diminution of inflammatory Th1 and Th17 associated cytokine mRNA levels was observed in the draining lymph nodes of treated rats. INCB028050 was also effective in multiple murine models of arthritis, with no evidence of suppression of humoral immunity or adverse hematologic effects. These data suggest that fractional inhibition of JAK1 and JAK2 is sufficient for significant activity in autoimmune disease models. Clinical evaluation of INCB028050 in RA is ongoing.

Selective inhibition of IDO1 effectively regulates mediators of antitumor immunity.

Indoleamine 2,3-dioxygenase-1 (IDO1; IDO) mediates oxidative cleavage of tryptophan, an amino acid essential for cell proliferation and survival. IDO1 inhibition is proposed to have therapeutic potential in immunodeficiency-associated abnormalities, including cancer. Here, we describe INCB024360, a novel IDO1 inhibitor, and investigate its roles in regulating various immune cells and therapeutic potential as an anticancer agent. In cellular assays, INCB024360 selectively inhibits human IDO1 with IC(50) values of approximately 10nM, demonstrating little activity against other related enzymes such as IDO2 or tryptophan 2,3-dioxygenase (TDO). In coculture systems of human allogeneic lymphocytes with dendritic cells (DCs) or tumor cells, INCB024360 inhibition of IDO1 promotes T and natural killer (NK)-cell growth, increases IFN-gamma production, and reduces conversion to regulatory T (T(reg))-like cells. IDO1 induction triggers DC apoptosis, whereas INCB024360 reverses this and increases the number of CD86(high) DCs, potentially representing a novel mechanism by which IDO1 inhibition activates T cells. Furthermore, IDO1 regulation differs in DCs versus tumor cells. Consistent with its effects in vitro, administration of INCB024360 to tumor-bearing mice significantly inhibits tumor growth in a lymphocyte-dependent manner. Analysis of plasma kynurenine/tryptophan levels in patients with cancer affirms that the IDO pathway is activated in multiple tumor types. Collectively, the data suggest that selective inhibition of IDO1 may represent an attractive cancer therapeutic strategy via up-regulation of cellular immunity.

Preclinical characterization of the selective JAK1/2 inhibitor INCB018424: therapeutic implications for the treatment of myeloproliferative neoplasms.

Constitutive JAK2 activation in hematopoietic cells by the JAK2V617F mutation recapitulates myeloproliferative neoplasm (MPN) phenotypes in mice, establishing JAK2 inhibition as a potential therapeutic strategy. Although most polycythemia vera patients carry the JAK2V617F mutation, half of those with essential thrombocythemia or primary myelofibrosis do not, suggesting alternative mechanisms for constitutive JAK-STAT signaling in MPNs. Most patients with primary myelofibrosis have elevated levels of JAK-dependent proinflammatory cytokines (eg, interleukin-6) consistent with our observation of JAK1 hyperactivation. Accordingly, we evaluated the effectiveness of selective JAK1/2 inhibition in experimental models relevant to MPNs and report on the effects of INCB018424, the first potent, selective, oral JAK1/JAK2 inhibitor to enter the clinic. INCB018424 inhibited interleukin-6 signaling (50% inhibitory concentration [IC(50)] = 281nM), and proliferation of JAK2V617F(+) Ba/F3 cells (IC(50) = 127nM). In primary cultures, INCB018424 preferentially suppressed erythroid progenitor colony formation from JAK2V617F(+) polycythemia vera patients (IC(50) = 67nM) versus healthy donors (IC(50) > 400nM). In a mouse model of JAK2V617F(+) MPN, oral INCB018424 markedly reduced splenomegaly and circulating levels of inflammatory cytokines, and preferentially eliminated neoplastic cells, resulting in significantly prolonged survival without myelosuppressive or immunosuppressive effects. Preliminary clinical results support these preclinical data and establish INCB018424 as a promising oral agent for the treatment of MPNs.

Hydroxyamidine inhibitors of indoleamine-2,3-dioxygenase potently suppress systemic tryptophan catabolism and the growth of IDO-expressing tumors.

Malignant tumors arise, in part, because the immune system does not adequately recognize and destroy them. Expression of indoleamine-2,3-dioxygenase (IDO; IDO1), a rate-limiting enzyme in the catabolism of tryptophan into kynurenine, contributes to this immune evasion. Here we describe the effects of systemic IDO inhibition using orally active hydroxyamidine small molecule inhibitors. A single dose of INCB023843 or INCB024360 results in efficient and durable suppression of Ido1 activity in the plasma of treated mice and dogs, the former to levels seen in Ido1-deficient mice. Hydroxyamidines potently suppress tryptophan metabolism in vitro in CT26 colon carcinoma and PAN02 pancreatic carcinoma cells and in vivo in tumors and their draining lymph nodes. Repeated administration of these IDO1 inhibitors impedes tumor growth in a dose- and lymphocyte-dependent fashion and is well tolerated in efficacy and preclinical toxicology studies. Substantiating the fundamental role of tumor cell-derived IDO expression, hydroxyamidines control the growth of IDO-expressing tumors in Ido1-deficient mice. These activities can be attributed, at least partially, to the increased immunoreactivity of lymphocytes found in tumors and their draining lymph nodes and to the reduction in tumor-associated regulatory T cells. INCB024360, a potent IDO1 inhibitor with desirable pharmaceutical properties, is poised to start clinical trials in cancer patients.

Efficacy of the JAK2 inhibitor INCB16562 in a murine model of MPLW515L-induced thrombocytosis and myelofibrosis.

The discovery of JAK2 and MPL mutations in patients with myeloproliferative neoplasms (MPNs) provided important insight into the genetic basis of these disorders and led to the development of JAK2 kinase inhibitors for MPN therapy. Although recent studies have shown that JAK2 kinase inhibitors demonstrate efficacy in a JAK2V617F murine bone marrow transplantation model, the effects of JAK2 inhibitors on MPLW515L-mediated myeloproliferation have not been investigated. In this report, we describe the in vitro and in vivo effects of INCB16562, a small-molecule JAK2 inhibitor. INCB16562 inhibited proliferation and signaling in cell lines transformed by JAK2 and MPL mutations. Compared with vehicle treatment, INCB16562 treatment improved survival, normalized white blood cell counts and platelet counts, and markedly reduced extramedullary hematopoeisis and bone marrow fibrosis. We observed inhibition of STAT3 and STAT5 phosphorylation in vivo consistent with potent inhibition of JAK-STAT signaling. These data suggest JAK2 inhibitor therapy may be of value in the treatment of JAK2V617F-negative MPNs. However, we did not observe a decrease in the size of the malignant clone in the bone marrow of treated mice at the end of therapy, which suggests that JAK2 inhibitor therapy, by itself, was not curative in this MPN model.

INCB16562, a JAK1/2 selective inhibitor, is efficacious against multiple myeloma cells and reverses the protective effects of cytokine and stromal cell support.

Cytokines in the bone marrow of multiple myeloma patients activate Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways in tumor cells and promote tumor growth, survival, and drug resistance. INCB16562 was developed as a novel, selective, and orally bioavailable small-molecule inhibitor of JAK1 and JAK2 markedly selective over JAK3. The specific cellular activity of the inhibitor was demonstrated by its potent and dose-dependent inhibition of cytokine-dependent JAK/STAT signaling and cell proliferation in the absence of effects on Bcr-Abl-expressing cells. Treatment of myeloma cells with INCB16562 potently inhibited interleukin-6 (IL-6)-induced phosphorylation of STAT3. Moreover, the proliferation and survival of myeloma cells dependent on IL-6 for growth, as well as the IL-6-induced growth of primary bone marrow-derived plasma cells from a multiple myeloma patient, were inhibited by INCB16562. Induction of caspase activation and apoptosis was observed and attributed, at least in part, to the suppression of Mcl-1 expression. Importantly, INCB16562 abrogated the protective effects of recombinant cytokines or bone marrow stromal cells and sensitized myeloma cells to cell death by exposure to dexamethasone, melphalan, or bortezomib. Oral administration of INCB16562 antagonized the growth of myeloma xenografts in mice and enhanced the antitumor activity of relevant agents in combination studies. Taken together, these data suggest that INCB16562 is a potent JAK1/2 inhibitor and that mitigation of JAK/STAT signaling by targeting JAK1 and JAK2 will be beneficial in the treatment of myeloma patients, particularly in combination with other agents.

Combined inhibition of Janus kinase 1/2 for the treatment of JAK2V617F-driven neoplasms: selective effects on mutant cells and improvements in measures of disease severity.

PURPOSE: Deregulation of the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway is a hallmark for the Philadelphia chromosome-negative myeloproliferative diseases polycythemia vera, essential thrombocythemia, and primary myelofibrosis. We tested the efficacy of a selective JAK1/2 inhibitor in cellular and in vivo models of JAK2-driven malignancy. EXPERIMENTAL DESIGN: A novel inhibitor of JAK1/2 was characterized using kinase assays. Cellular effects of this compound were measured in cell lines bearing the JAK2V617F or JAK1V658F mutation, and its antiproliferative activity against primary polycythemiavera patient cells was determined using clonogenic assays. Antineoplastic activity in vivo was determined using a JAK2V617F-driven xenograft model, and effects of the compound on survival, organomegaly, body weight, and disease-associated inflammatory markers were measured. RESULTS: INCB16562 potently inhibited proliferation of cell lines and primary cells from PV patients carrying the JAK2V617F or JAK1V658F mutation by blocking JAK-STAT signaling and inducing apoptosis. In vivo, INCB16562 reduced malignant cell burden, reversed splenomegaly and normalized splenic architecture, improved body weight gains, and extended survival in a model of JAK2V617F-driven hematologic malignancy. Moreover, these mice suffered from markedly elevated levels of inflammatory cytokines, similar to advanced myeloproliferative disease patients, which was reversed upon treatment. CONCLUSIONS: These data showed that administration of the dual JAK1/2 inhibitor INCB16562 reduces malignant cell burden, normalizes spleen size and architecture, suppresses inflammatory cytokines, improves weight gain, and extends survival in a rodent model of JAK2V617F-driven hematologic malignancy. Thus, selective inhibitors of JAK1 and JAK2 represent a novel therapy for the patients with myeloproliferative diseases and other neoplasms associated with JAK dysregulation.

Discovery of potent competitive inhibitors of indoleamine 2,3-dioxygenase with in vivo pharmacodynamic activity and efficacy in a mouse melanoma model.

A hydroxyamidine chemotype has been discovered as a key pharmacophore in novel inhibitors of indoleamine 2,3-dioxygenase (IDO). Optimization led to the identification of 5l, which is a potent (HeLa IC(50) = 19 nM) competitive inhibitor of IDO. Testing of 5l in mice demonstrated pharmacodynamic inhibition of IDO, as measured by decreased kynurenine levels (>50%) in plasma and dose dependent efficacy in mice bearing GM-CSF-secreting B16 melanoma tumors.

Compelling P1 substituent affect on metalloprotease binding profile enables the design of a novel cyclohexyl core scaffold with excellent MMP selectivity and HER-2 sheddase inhibition.

A serendipitous discovery that the metalloprotease binding profile of a novel class of 2-carboxamide-3-hydroxamic acid piperidines could be significantly attenuated by the modification of the unexplored P1 substituent enabled the design and synthesis of a novel 2-carboxamide-1-hydroxamic acid cyclohexyl scaffold core that exhibited excellent HER-2 potency and unprecedented MMP-selectivity that we believe would not have been possible via conventional P1' perturbations.

Janus kinase inhibitor INCB20 has antiproliferative and apoptotic effects on human myeloma cells in vitro and in vivo.

Protein tyrosine kinases of the Janus kinase (JAK) family are associated with many cytokine receptors, which, on ligand binding, regulate important cellular functions such as proliferation, survival, and differentiation. In multiple myeloma, JAKs may be persistently activated due to a constant stimulation by interleukin (IL)-6, which is produced in the bone marrow environment. INCB20 is a synthetic molecule that potently inhibits all members of the JAK family with a 100- to 1,000-fold selectivity for JAKs over >70 other kinases. Treatment of multiple myeloma cell lines and patient tumor cells with INCB20 resulted in a significant and dose-dependent inhibition of spontaneous as well as IL-6-induced cell growth. Importantly, multiple myeloma cell growth was inhibited in the presence of bone marrow stromal cells. The IL-6 dependent cell line INA-6 was particularly sensitive to the drug (IC50<1 micromol/L). Growth suppression of INA-6 correlated with an increase in the percentage of apoptotic cells and inhibition of signal transducer and activator of transcription 3 phosphorylation. INCB20 also abrogated the protective effect of IL-6 against dexamethasone by blocking phosphorylation of SHP-2 and AKT. In contrast, AKT phosphorylation induced by insulin-like growth factor-I remained unchanged, showing selectivity of the compound. In a s.c. severe combined immunodeficient mouse model with INA-6, INCB20 significantly delayed INA-6 tumor growth. Our studies show that disruption of JAKs and downstream signaling pathways may both inhibit multiple myeloma cell growth and survival and overcome cytokine-mediated drug resistance, thereby providing the preclinical rationale for the use of JAK inhibitors as a novel therapeutic approach in multiple myeloma.

Design and identification of selective HER-2 sheddase inhibitors via P1' manipulation and unconventional P2' perturbations to induce a molecular metamorphosis.

In an effort to obtain a MMP selective and potent inhibitor of HER-2 sheddase (ADAM-10), the P1' group of a novel class of (6S,7S)-7-[(hydroxyamino)carbonyl]-6-carboxamide-5-azaspiro[2.5]octane-5-carboxylates was attenuated and the structure-activity relationships (SAR) will be discussed. In addition, it was discovered that unconventional perturbation of the P2' moiety could confer MMP selectivity, which was hypothesized to be a manifestation of the P2' group effecting global conformational changes.

Conversion of an MMP-potent scaffold to an MMP-selective HER-2 sheddase inhibitor via scaffold hybridization and subtle P1' permutations.

A series of beta-sulfonamide piperidine hydroxamates were prepared and shown to be potent inhibitors of the human epidermal growth factor receptor-2 (HER-2) sheddase with excellent selectivity against MMP-1, -2, -3, and -9. This was achieved by exploiting subtle differences within the otherwise highly conserved S(1)(') binding pocket of the active sites within the metalloprotease family. In addition, it was discovered that the introduction of polarity to the P(1) and P(1)(') groups reduced the projected human clearance.

Selective inhibition of ADAM metalloproteases as a novel approach for modulating ErbB pathways in cancer.

PURPOSE: ErbB receptor signaling pathways are important regulators of cell fate, and their dysregulation, through (epi)genetic alterations, plays an etiologic role in multiple cancers. ErbB ligands are synthesized as membrane-bound precursors that are cleaved by members of the ADAM family of zinc-dependent metalloproteases. This processing, termed ectodomain shedding, is essential for the functional activation of ErbB ligands. Recent studies suggest that elevated levels of ErbB ligands may circumvent the effectiveness of ErbB-targeted therapeutics. Here, we describe the discovery and preclinical development of potent, selective inhibitors of ErbB ligand shedding. EXPERIMENTAL DESIGN: A series of biochemical and cell-based assays were established to identify selective inhibitors of ErbB ligand shedding. The therapeutic potential of these compounds was assessed in multiple in vivo models of cancer and matrix metalloprotease-related toxicity. RESULTS: INCB3619 was identified as a representative selective, potent, orally bioavailable small-molecule inhibitor of a subset of ADAM proteases that block shedding of ErbB ligands. Administration of INCB3619 to tumor-bearing mice reduced ErbB ligand shedding in vivo and inhibited ErbB pathway signaling (e.g., phosphorylation of Akt), tumor cell proliferation, and survival. Further, INCB3619 synergized with clinically relevant cancer therapeutics and showed no overt or compounding toxicities, including fibroplasia, the dose-limiting toxicity associated with broad-spectrum matrix metalloprotease inhibitors. CONCLUSIONS: Inhibition of ErbB ligand shedding offers a potentially novel and well-tolerated therapeutic strategy for the treatment of human cancers and is currently being evaluated in the clinic.