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Insulin-like growth factor-I receptor (IGF-IR) and its ligands, IGF-I and IGF-II, are up-regulated in a variety of human cancers. In tumors, such as colorectal, non–small cell lung, ovarian, and pediatric cancers, which may drive their own growth and survival through autocrine IGF-II expression, the role of IGF-IR is especially critical. Here, we present a novel small-molecule IGF-IR kinase inhibitor, cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo[1,5-a]pyrazin-8-ylamine (PQIP), which displayed a cellular IC50 of 19 nmol/L for inhibition of ligand-dependent autophosphorylation of human IGF-IR with 14-fold cellular selectivity relative to the human insulin receptor. PQIP showed minimal activity against a panel of 32 other protein kinases. It also abolished the ligand-induced activation of downstream phosphorylated AKT and phosphorylated extracellular signal-regulated kinase 1/2 in both IGF-IR transfectant cells and a GEO human colorectal cancer cell line. Analysis of GEO cells revealed a significant level of both phosphorylated IGF-IR and IGF-II expression. Furthermore, inactivation of IGF-II in conditioned GEO culture medium by a neutralizing antibody diminished IGF-IR activation, indicating the presence of a functional IGF-II/IGF-IR autocrine loop in GEO cells. Once daily oral dosing of PQIP induced robust antitumor efficacy in GEO xenografts. The antitumor efficacy correlated with the degree and duration of inhibition of tumor IGF-IR phosphorylation in vivo by this compound. Moreover, when mice were treated for 3 days with a dose of PQIP that maximally inhibited tumor growth, only minor changes in blood glucose were observed. Thus, PQIP represents a potent and selective IGF-IR kinase inhibitor that is especially efficacious in an IGF-II–driven human tumor model. [Mol Cancer Ther 2007;6(8):2158–67]

Supplementary Material from A novel, potent, and selective insulin-like growth factor-I receptor kinase inhibitor blocks insulin-like growth factor-I receptor signaling in vitro and inhibits insulin-like growth factor-I receptor–dependent tumor growth in vivo

Supplementary Figures 1-6 from Feedback Mechanisms Promote Cooperativity for Small Molecule Inhibitors of Epidermal and Insulin-Like Growth Factor Receptors

Supplementary Table 1 from Feedback Mechanisms Promote Cooperativity for Small Molecule Inhibitors of Epidermal and Insulin-Like Growth Factor Receptors

Supplementary Methods from Feedback Mechanisms Promote Cooperativity for Small Molecule Inhibitors of Epidermal and Insulin-Like Growth Factor Receptors

Supplementary Figure Legends from Feedback Mechanisms Promote Cooperativity for Small Molecule Inhibitors of Epidermal and Insulin-Like Growth Factor Receptors

This Letter describes the medicinal chemistry effort towards a series of novel imidazo[1,5-a]pyrazine derived inhibitors of ACK1. Virtual screening led to the discovery of the initial hit, and subsequent exploration of structure-activity relationships and optimization of drug metabolism and pharmacokinetic properties led to the identification of potent, selective and orally bioavailable ACK1 inhibitors.

Epidermal growth factor receptor (EGFR) and insulin-like growth factor-I receptor (IGF-IR) can cooperate to regulate tumor growth and survival, and synergistic growth inhibition has been reported for combined blockade of EGFR and IGF-IR. However, in preclinical models, only a subset of tumors exhibit high sensitivity to this combination, highlighting the potential need for patient selection to optimize clinical efficacy. Herein, we have characterized the molecular basis for cooperative growth inhibition upon dual EGFR and IGF-IR blockade and provide biomarkers that seem to differentiate response. We find for epithelial, but not for mesenchymal-like, tumor cells that Akt is controlled cooperatively by EGFR and IGF-IR. This correlates with synergistic apoptosis and growth inhibition in vitro and growth regression in vivo upon combined blockade of both receptors. We identified two molecular aspects contributing to synergy: (a) inhibition of EGFR or IGF-IR individually promotes activation of the reciprocal receptor; (b) inhibition of EGFR-directed mitogen-activated protein kinase (MAPK) shifts regulation of Akt from EGFR toward IGF-IR. Targeting the MAPK pathway through downstream MAPK/extracellular signal-regulated kinase kinase (MEK) antagonism similarly promoted IGF-driven pAkt and synergism with IGF-IR inhibition. Mechanistically, we find that inhibition of the MAPK pathway circumvents a negative feedback loop imposed on the IGF-IR– insulin receptor substrate 1 (IRS-1) signaling complex, a molecular scenario that parallels the negative feedback loop between mTOR-p70S6K and IRS-1 that mediates rapamycin-directed IGF-IR signaling. Collectively, these data show that resistance to inhibition of MEK, mTOR, and EGFR is associated with enhanced IGF-IR–directed Akt signaling, where all affect feedback loops converging at the level of IRS-1. [Cancer Res 2008;68(20):8322–32]

The insulin-like growth factor-1 receptor (IGF1R) is a receptor tyrosine kinase (RTK) that has a critical role in mitogenic signalling during embryogenesis and an antiapoptotic role in the survival and progression of many human tumours. Here, we present the crystal structure of the tyrosine kinase domain of IGF1R (IGF1RK), in its unphosphorylated state, in complex with a novel compound, cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo[1,5-a]pyrazin-8-ylamine (PQIP), which we show is a potent inhibitor of both the unphosphorylated (basal) and phosphorylated (activated) states of the kinase. PQIP interacts with residues in the ATP-binding pocket and in the activation loop, which confers specificity for IGF1RK and the highly related insulin receptor (IR) kinase. In this crystal structure, the IGF1RK active site is occupied by Tyr1135 from the activation loop of an symmetry (two-fold)-related molecule. This dimeric arrangement affords, for the first time, a visualization of the initial trans-phosphorylation event in the activation loop of an RTK, and provides a molecular rationale for a naturally occurring mutation in the activation loop of the IR that causes type II diabetes mellitus.

Phospholipase C-γ1 (PLC-γ1) activation has been reported to enhance cell survival during the cellular response to oxidative stress. We studied the role of protein kinase C (PKC) pathways in mediating PLC-γ1 survival signalling in oxidative stress by using mouse embryonic fibroblasts genetically deficient in PLC-γ1 (Plcg1−/−) and its wild type (Plcg1+/+). PLC-γ1 was activated by H2O2 treatment in a dose- and time-dependent manner. Activation of PKC was also markedly increased in both cell lines treated with H2O2 (1–5mM), but with low doses (50–200μM), PKC activation was considerably decreased in Plcg1−/− cells. After treatment with H2O2, PKC-dependent phosphorylation of Bcl-2 and cell viability of Plcg1−/− cells decreased dramatically and caspase-3-like activity increased significantly compared with that of the wild-type cells. Furthermore, pretreatment of Plcg1+/+ cells with PKC-specific inhibitor decreased levels of PKC-dependent Bcl-2 phosphorylation, enhanced caspase-3 activity and their sensitivity to H2O2. On the contrary, treatment of Plcg1−/− cells with PKC-specific activator increased the Bcl-2 phosphorylation, decreased caspase-3 activity and improved their survival. These results suggest that PLC-γ1 mediates survival signalling in oxidative-stress response by PKC-dependent phosphorylation of Bcl-2 and inhibition of caspase-3.

The consequences of heat-induced phospholipase C-gamma1 (PLC-gamma1) phosphorylation are not known. We investigated the role of PLC-gamma1 activation and its downstream targets during the cellular response to heat stress using mouse embryonic fibroblasts genetically deficient in PLC-gamma1 (Plcg1 null MEF) and its wild type (wt MEF) as models. Treatment of wt MEF with heat resulted in temperature- and heating duration-dependent tyrosine phosphorylation of PLC-gamma1. HSP70 synthesis and the activation of extracellular signal-regulated kinases 1/2 (ERK1/2) and c-Jun N-terminal protein kinase (JNK) increased equally following heat treatment in both cell lines. However, heat-induced protein kinase C (PKC) activation was dramatically reduced in Plcg1 null MEF compared with wt MEF. Importantly, the mitochondrial localization of PKCalpha, PKC-dependent phosphorylation of Bcl-2, and cell viability in Plcg1 null MEF following heat treatment, were significantly decreased compared with the wild type. Furthermore, pretreatment with bryostatin-1, a PKC activator, enhanced Bcl-2 phosphorylation and cellular resistance to heat-induced apoptosis in Plcg1 null MEF. Taken together, these results suggest that PLC-gamma1 activation enhances cell survival through the PKC-dependent phosphorylation of Bcl-2 during the cellular response to heat stress.

The nonreceptor tyrosine kinase FAK ("focal adhesion kinase") is a key mediator of integrin signaling events controlling cellular responses to the extracellular matrix, including spreading, migration, proliferation, and survival. Integrin-ligand interactions stimulate FAK tyrosine phosphorylation and activation of FAK signaling functions. Here evidence is presented that the FAK autophosphorylation site Tyr-397 mediates a direct interaction with the C-terminal Src homology 2 domain of phospholipase C (PLC)-gamma1 and that this is required for both adhesion-dependent association of the two molecules and increased inositol phosphate production in mouse embryo fibroblasts. Overexpression of FAK and PLC-gamma1 in COS-7 cells increases PLC-gamma1 enzymatic activity and tyrosine phosphorylation, also dependent on FAK Tyr-397. However, FAK appears incapable of directly phosphorylating PLC-gamma1. These observations suggest a role for FAK in recruiting PLC-gamma1 to the plasma membrane at sites of cell-matrix adhesion and there promoting its enzymatic activity, possibly by releasing the repression caused by intramolecular interactions of the PLC-gamma1 Src homology domains and/or by positioning it for phosphorylation by associated Src-family kinases. These findings expand the known signaling functions of FAK and provide mechanistic insight into integrin-stimulation of PLC-gamma1.

The activation of many tyrosine kinases leads to the phosphorylation and activation of phospholipase C-γ1 (PLC-γ1). To examine the biological function of this protein, homologous recombination has been used to selectively disrupt the Plcg1 gene in mice. Homozygous disruption of Plcg1 results in embryonic lethality at approximately embryonic day (E) 9.0. Histological analysis indicates that Plcg1 (−/−) embryos appear normal at E 8.5 but fail to continue normal development and growth beyond E 8.5–E9.0. These results clearly demonstrate that PLC-γ1 with, by inference, its capacity to mobilize second messenger molecules is an essential signal transducing molecule whose absence is not compensated by other signaling pathways or other genes encoding PLC isozymes.

Background: The IGF-1 receptor (IGF-1R) has been implicated in the promotion of tumorigenesis, metastasis and resistance to cancer therapies. Therefore, this receptor has become a major focus for the development of anticancer agents. Results: Our lead optimization efforts that blended structure-based design and empirical medicinal chemistry led to the discovery of OSI-906, a novel small-molecule dual IGF-1R/insulin receptor (IR) kinase inhibitor. OSI-906 potently and selectively inhibits autophosphorylation of both human IGF-1R and IR, displays in vitro antiproliferative effects in a variety of tumor cell lines and shows robust in vivo anti-tumor efficacy in an IGF-1R-driven xenograft model when administered orally once daily. Conclusion: OSI-906 is a novel, potent, selective and orally bioavailable dual IGF-1R/IR kinase inhibitor with favorable preclinical drug-like properties, which has demonstrated in vivo efficacy in tumor models and is currently in clinical testing.

Preclinical and emerging clinical evidence suggests that inhibiting insulin-like growth factor 1 receptor (IGF-1R) signaling may offer a promising therapeutic strategy for the treatment of several types of cancer. This Letter describes the medicinal chemistry effort towards a series of 8-amino-imidazo[1,5-a]pyrazine derived inhibitors of IGF-1R which features a substituted quinoline moiety at the C1 position and a cyclohexyl linking moiety at the C3 position. Lead optimization efforts which included the optimization of structure-activity relationships and drug metabolism and pharmacokinetic properties led to the identification of compound 9m, a potent, selective and orally bioavailable inhibitor of IGF-1R with in vivo efficacy in an IGF-driven mouse xenograft model.

Insulin-like growth factor (IGF) signaling plays a critical role in the growth and differentiation of many tissues, particularly in prenatal growth and puberty. The IGF axis is also implicated in various pathophysiological conditions, and is believed to play a crucial role in tumorigenesis (Pollak et al. 2004).

Insulin-like growth factor-I receptor (IGF-IR) and its ligands, IGF-I and IGF-II, are up-regulated in a variety of human cancers. In tumors, such as colorectal, non–small cell lung, ovarian, and pediatric cancers, which may drive their own growth and survival through autocrine IGF-II expression, the role of IGF-IR is especially critical. Here, we present a novel small-molecule IGF-IR kinase inhibitor, cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo[1,5-a]pyrazin-8-ylamine (PQIP), which displayed a cellular IC50 of 19 nmol/L for inhibition of ligand-dependent autophosphorylation of human IGF-IR with 14-fold cellular selectivity relative to the human insulin receptor. PQIP showed minimal activity against a panel of 32 other protein kinases. It also abolished the ligand-induced activation of downstream phosphorylated AKT and phosphorylated extracellular signal-regulated kinase 1/2 in both IGF-IR transfectant cells and a GEO human colorectal cancer cell line. Analysis of GEO cells revealed a significant level of both phosphorylated IGF-IR and IGF-II expression. Furthermore, inactivation of IGF-II in conditioned GEO culture medium by a neutralizing antibody diminished IGF-IR activation, indicating the presence of a functional IGF-II/IGF-IR autocrine loop in GEO cells. Once daily oral dosing of PQIP induced robust antitumor efficacy in GEO xenografts. The antitumor efficacy correlated with the degree and duration of inhibition of tumor IGF-IR phosphorylation in vivo by this compound. Moreover, when mice were treated for 3 days with a dose of PQIP that maximally inhibited tumor growth, only minor changes in blood glucose were observed. Thus, PQIP represents a potent and selective IGF-IR kinase inhibitor that is especially efficacious in an IGF-II–driven human tumor model. [Mol Cancer Ther 2007;6(8):2158–67]

A series of novel, potent quinolinyl-derived imidazo[1,5-a]pyrazine IGF-IR (IGF-1R) inhibitors--most notably, cis-3-(3-azetidin-1-ylmethylcyclobutyl)-1-(2-phenylquinolin-7-yl)imidazo[1,5-a]pyrazin-8-ylamine (AQIP)--is described. Synthetic details, structure-activity relationships, and in vitro biological activity are reported for the series. Key in vitro and in vivo biological results for AQIP are reported, including: inhibition of ligand-stimulated autophosphorylation of IGF-IR and downstream pathways in 3T3/huIGFIR cells; inhibition of proliferation and induction of DNA fragmentation in human tumor cell lines; a pharmacokinetic profile suitable for once-per-day oral dosing; antitumor activity in a 3T3/huIGFIR xenograft model; and effects on insulin and glucose levels.

A series of novel 8-amino-1,3-disubstituted-imidazo[1,5-a]pyrazines was designed and synthesized as IGF-IR inhibitors.

OBJECTIVE: To investigate the expression of phospholipase C-gamma1(PLC-gamma1) in human fetal tissues. METHODS: Serial sections 5mgr;m in thickness were prepared from paraformadehyde-fixed and paraffin-embedded normal human fetal tissues including the cartilage, the cartilage membrane and the muscle tissues. The distribution of PLC-gamma1 expression was examined immunohistochemically in the sections. RESULTS: Immunoreactive PLC-gamma1 was readily detected in the cells of the cartilage, the cartilage membrane and the muscl tissues, which was largely confined within the cytoplasm. CONCLUSION: PLC-gamma1 is essential for the early development and cell proliferation of human embryos.

The consequences of heat-induced phospholipase C-gamma1 (PLC-gamma1) phosphorylation are not known. We investigated the role of PLC-gamma1 activation and its downstream targets during the cellular response to heat stress using mouse embryonic fibroblasts genetically deficient in PLC-gamma1 (Plcg1 null MEF) and its wild type (wt MEF) as models. Treatment of wt MEF with heat resulted in temperature- and heating duration-dependent tyrosine phosphorylation of PLC-gamma1. HSP70 synthesis and the activation of extracellular signal-regulated kinases 1/2 (ERK1/2) and c-Jun N-terminal protein kinase (JNK) increased equally following heat treatment in both cell lines. However, heat-induced protein kinase C (PKC) activation was dramatically reduced in Plcg1 null MEF compared with wt MEF. Importantly, the mitochondrial localization of PKCalpha, PKC-dependent phosphorylation of Bcl-2, and cell viability in Plcg1 null MEF following heat treatment, were significantly decreased compared with the wild type. Furthermore, pretreatment with bryostatin-1, a PKC activator, enhanced Bcl-2 phosphorylation and cellular resistance to heat-induced apoptosis in Plcg1 null MEF. Taken together, these results suggest that PLC-gamma1 activation enhances cell survival through the PKC-dependent phosphorylation of Bcl-2 during the cellular response to heat stress.

To explore the functional role of phospholipase C-gamma1 (PLC-gamma1) in the induction of immediate early genes (IEGs), we have examined the influence of Plcg1 gene disruption on the expression of 14 IEG mRNAs induced by platelet-derived growth factor (PDGF). Plcg1-null embryos were used to produce immortalized fibroblasts genetically deficient in PLC-gamma1 (Null cells), and retroviral infection of those cells was used to derive PLC-gamma1 re-expressing cells (Null+ cells). In terms of PDGF activation of PDGF receptor tyrosine phosphorylation as well as the mitogen-activated protein kinases Erk1 and Erk2, Null and Null+ cells responded equivalently. However, the PDGF-dependent expression of all IEG mRNAs was diminished in cells lacking PLC-gamma1. The expression of FIC, COX-2, KC, JE, and c-fos mRNAs were most strongly compromised, as the stimulation of these genes was reduced by more than 90% in cells lacking PLC-gamma1. The combination of PMA and ionomycin, downstream analogs of PLC activation, did provoke expression of mRNAs for these IEGs in the Null cells. We conclude that PLC-gamma1 is necessary for the maximal expression of many PDGF-induced IEGs and is essential for significant induction of at least five IEGs.

The role of intracellular Ca2+ pools in the regulation of growth factor signal transduction pathways and mitogenesis is not well understood. We have examined the roles of basal and transiently mobilized Ca2+ in the regulation of MAP kinases by EGF. To assess the influence of Ca2+ transients we utilized Plcg1−/− and Plcg1+/+ mouse embryonic fibroblasts, while BAPTA/AM was employed to chelate total intracellular Ca2+ in the same cell lines. The MAP kinases erk-1, erk-2 and erk-5 exhibited similar patterns of activation in wild-type and Plcg1−/− cells treated with EGF. However, pretreatment with BAPTA/AM significantly increased and prolonged erk-1 and erk-2 activation in both cell types. In contrast, BAPTA/AM prevented the EGF activation of erk-5 in wild-type and Plcg1−/− cells. These data indicate that basal Ca2+, but not growth factor provoked Ca2+ transients, has a significant influence on the activation of these MAP kinases. AG1478, a specific EGF receptor kinase inhibitor, abolished the prolonged erk-1 and erk-2 activation produced by EGF in cells pretreated with BAPTA/AM. This indicates that the prolonged activation of erk-1 and erk-2 produced in the presence of BAPTA/AM requires continuous signaling from the EGF receptor kinase.

A ubiquitous signaling event in hormonal responses is the phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4, 5-bisphosphate to produce the metabolite second messenger molecules inositol 1,4,5-trisphosphate and diacylglycerol. The former provokes a transient increase in intracellular free Ca(2+), while the latter serves as a direct activator of protein kinase C. In tyrosine kinase-dependent signaling pathways this reaction is mediated by the PLC-gamma isozymes. These are direct substrates of many tyrosine kinases in a wide variety of cell types. The mechanism of PLC-gamma activation involves its association with and phosphorylation by receptor and non-receptor tyrosine kinases, as well as interaction with specialized adaptor molecules and, perhaps, other second messenger molecules. However, the biochemistry of PLC-gamma is at a more advanced state than a clear understanding of exactly how this signaling element functions in the generation of a mitogenic response.

Two approaches have been utilized to investigate the role of individual SH2 domains in growth factor activation of phospholipase C-gamma1 (PLC-gamma1). Surface plasmon resonance analysis indicates that the individual N-SH2 and C-SH2 domains are able to specifically recognize a phosphotyrosine-containing peptide corresponding to Tyr 1021 of the platelet-derived growth factor (PDGF) beta receptor. To assess SH2 function in the context of the full-length PLC-gamma1 molecule as well as within the intact cell, PLC-gamma1 SH2 domain mutants, disabled by site-directed mutagenesis of the N-SH2 and/or C-SH2 domain(s), were expressed in Plcg1(-/-) fibroblasts. Under equilibrium incubation conditions (4 degrees C, 40 min), the N-SH2 domain, but not the C-SH2 domain, was sufficient to mediate significant PLC-gamma1 association with the activated PDGF receptor and PLC-gamma1 tyrosine phosphorylation. When both SH2 domains in PLC-gamma1 were disabled, the double mutant did not associate with activated PDGF receptors and was not tyrosine phosphorylated. However, no single SH2 mutant was able to mediate growth factor activation of Ca2+ mobilization or inositol 1,4,5-trisphosphate (IP3) formation. Subsequent kinetic experiments demonstrated that each single SH2 domain mutant was significantly impaired in its capacity to mediate rapid association with activated PDGF receptors and become tyrosine phosphorylated. Hence, when assayed under physiological conditions necessary to achieve a rapid biological response (Ca2+ mobilization and IP3 formation), both SH2 domains of PLC-gamma1 are essential to growth factor responsiveness.

Platelet-derived growth factor (PDGF) activates phospholipase D (PLD) in mouse embryo fibroblasts (MEFs). In order to investigate a role for phospholipase C-gamma1 (PLC-gamma1), we used targeted disruption of the Plcg1 gene in the mouse to develop Plcg1(+/+) and Plcg1(-/-) cell lines. Plcg1(+/+) MEFs treated with PDGF showed a time- and dose-dependent increase in the production of total inositol phosphates that was substantially reduced in Plcg1(-/-) cells. Plcg1(+/+) cells also showed a PDGF-induced increase in PLD activity that had a similar dose dependence to the PLC response but was down-regulated after 15 min. Phospholipase D activity, however, was markedly reduced in Plcg1(-/-) cells. The PDGF-induced inositol phosphate formation and the PLD activity that remained in the Plcg1(-/-) cells could be attributed to the presence of phospholipase C-gamma2 (PLC-gamma2) in the Plcg1(-/-) cells. The PLC-gamma2 expressed in the Plcg1(-/-) cells was phosphorylated on tyrosine in response to PDGF treatment, and a small but significant fraction of the Plcg1(-/-) cells showed Ca2+ mobilization in response to PDGF, suggesting that the PLC-gamma2 expressed in the Plcg1(-/-) cells was activated in response to PDGF. The inhibition of PDGF-induced phospholipid hydrolysis in Plcg1(-/-) cells was not due to differences in the level of PDGF receptor or in the ability of PDGF to cause autophosphorylation of the receptor. Upon treatment of the Plcg1(-/-) cells with oleoylacetylglycerol and the Ca2+ ionophore ionomycin to mimic the effect of PLC-gamma1, PLD activity was restored. The targeted disruption of Plcg1 did not result in universal changes in the cell signaling pathways of Plcg1(-/-) cells, because the phosphorylation of mitogen-activated protein kinase was similar in Plcg1(+/+) and Plcg1(-/-) cells. Because increased plasma membrane ruffles occurred in both Plcg1(+/+) and Plcg1(-/-) cells following PDGF treatment, it is possible neither PLC nor PLD are necessary for this growth factor response. In summary, these data indicate that PLC-gamma is required for growth factor-induced activation of PLD in MEFs.

Correct development of the limb is dependent on coordination between three distinct signaling centers. Recently, fibroblast growth factor-4 has been identified as a crucial determinant of AER function, which directs limb bud outgrowth, and Sonic hedgehog has been identified as a signaling molecule that mediates ZPA function, which specifies anterior-posterior patterning in the developing limb bud. In addition, Shh and FGF-4 reciprocally reinforce each other’s expression via a positive feedback loop, providing a molecular basis for the coordination of limb bud outgrowth and anterior-posterior patterning. The mechanisms by which these signaling centers come to occupy their normal positions in the posterior limb bud during development are not understood. Here we identify and characterize Alx-4, a gene that encodes a paired-type homeodomain protein. Alx-4 is expressed in several populations of mesenchymal cells, including mesenchymal cells in the anterior limb bud, and mice homozygous for targeted disruption of the Alx-4 gene have multiple abnormalities, including preaxial polydactyly. The polydactyly is associated with the formation of an ectopic anterior ZPA, as indicated by anterior expression of Sonic hedgehog, HoxD13 and fibroblast growth factor-4. The expression of other candidate regulators of anterior-posterior positional information in the limb bud, including HoxB8 and Gli3, is not altered in Alx-4 mutant embryos. By chromosomal mapping experiments, Alx-4 is tightly linked to Strong’s luxoid, a polydactylous mouse mutant. The results identify Alx-4 as a determinant of anterior-posterior positional identity in the limb and a component of a regulatory program that restricts ZPA formation to the posterior limb bud mesenchyme.

Activated Cdc42-associated kinase (ACK1) is a non-receptor tyrosine kinase originally identified by virtue of its binding to GTP-bound small GTPase Cdc42. Considerable attention has been paid to ACK1’s involvement in cancer in recent years. For example, gene amplification and over-expression of ACK1 were found in multiple cancers including lung, ovarian and prostate cancers and were associated with poor prognosis and metastatic phenotypes. Activated ACK1 has been shown to phosphorylate and activate androgen receptor function and to promote the progression of prostate cancer. More recently, activated ACK1 was found to phosphorylate and promote the activation of Akt, a protein kinase that plays a central role in growth, proliferation and cell survival in various cancers. Taken together, these literature data suggest that ACK1 is a potential target for cancer treatment. Several series of ACK1 inhibitors have been previously disclosed in literature. Unfortunately, compounds from these series suffer from poor oral pharmacokinetic (PK) properties which have prevented them from being utilized further for in vivo studies. Therefore, there is a clear need for potent, selective and orally bioavailable small molecule ACK1 inhibitors to further probe its role in cancer, both in the in vitro and in vivo setting. This report describes the medicinal chemistry effort towards a series of novel imidazo[1,5-a]pyrazine derived inhibitors of ACK1. Virtual screening led to the discovery of the initial hit, and subsequent exploration of structure-activity relationships and optimization of drug metabolism and pharmacokinetic properties led to the identification of potent, selective and orally bioavailable ACK1 inhibitors. Citation Format: Meizhong Jin, Jing Wang, Andrew Kleinberg, Mridula Kadalbajoo, Kam W. Siu, Andrew Cooke, Mark Bittner, Yan Yao, April Thelemann, Qunsheng Ji, Shripad Bhagwat, Kristen M. Mulvihill, Josef A. Rechka, Jonathan A. Pachter, Andrew P. Crew, David Epstein, Mark J. Mulvihill. Discovery of imidazo[1,5-a]pyrazine derived potent, selective and orally bioavailable ACK1 inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2463. doi:10.1158/1538-7445.AM2013-2463