Search

Androgens, through their actions on the androgen receptor (AR), are required for the development of the prostate and contribute to the pathologic growth dysregulation observed in prostate cancers. Consequently, androgen ablation has become an essential component of the pharmacotherapy of prostate cancer. In this study, we explored the utility of targeting processes downstream of AR as an alternate approach for therapy. Specifically, we show that the serum and glucocorticoid-regulated kinase 1 (SGK1) gene is an androgen-regulated target gene in cellular models of prostate cancer. Furthermore, functional serum- and glucocorticoid-regulated kinase 1 (SGK1) protein, as determined by the phosphorylation of its target Nedd4-2, was also increased with androgen treatment. Importantly, we determined that RNA interference–mediated knockdown of SGK1 expression attenuates the androgen-mediated growth of the prostate cancer cell line LNCaP. Given these findings, we explored the utility of SGK1 as a therapeutic target in prostate cancer by developing and evaluating a small-molecule inhibitor of this enzyme. From these studies emerged GSK650394, a competitive inhibitor that quantitatively blocks the effect of androgens on LNCaP cell growth. Thus, in addition to androgen ablation, inhibition of pathways downstream of AR is likely to have therapeutic utility in prostate cancer. [Cancer Res 2008;68(18):7475–83]

Supplementary Data from Development of a Small-Molecule Serum- and Glucocorticoid-Regulated Kinase-1 Antagonist and Its Evaluation as a Prostate Cancer Therapeutic

The RAS/MAPK pathway is one of the most commonly dysregulated signaling pathways found in human cancers. KRAS, HRAS and NRAS genes, which play a critical role in transducing the signal from receptor tyrosine kinases to the downstream target proteins such as BRAF and MEK1/2, are frequently mutated in most solid tumors. The extracellular signal-regulated kinases, ERK1 and ERK2 (ERK1/2), which are downstream of MEK and RAF kinases, serve as a key node in this crucial signaling pathway in tumor cells. Targeting ERK1/2 offers a promising therapeutic strategy for a broad range of cancers, particularly the ones driven by RAS mutations, and in overcoming resistance to MEK/BRAF inhibitors. In this report, we present discovery and characterization of a novel orally bioavailable compound, ASN007, that showed potent inhibition of ERK1/2 kinases in a biochemical assay with IC50 = 2 nM for both targets. Interestingly, ASN007 displayed a much slower dissociation rate (longer target residence time) as compared to other ERK inhibitors. ASN007 showed potent antiproliferative activity that was selective for RAS/MAPK pathway dependent cancer cell lines, and did not affect the growth of cell lines harboring PI3K pathway or receptor tyrosine kinase mutations. Of note, ASN007 showed much more potent and broader activity against a panel of RAS mutant cell lines as compared to other ERK1/2 inhibitors. In a panel of 96 KRAS mutant cell lines, ASN007 showed strong activity across all KRAS subtype mutations, including G12C, G12D, G12V and G13D. In tumor xenograft models harboring KRAS and NRAS mutations, a daily oral dosing regimen of ASN007 provided strong tumor growth inhibition and was well tolerated. ASN007 also showed strong single-agent antitumor activity in colorectal PDX models with various KRAS subtype mutations including G12C, G12D, and G12S. A clinical phase 1 trial of ASN007 in patients with BRAF and KRAS, HRAS and NRAS mutant melanoma, colorectal, non-small lung and pancreatic cancers is currently ongoing (NCT03415126). Citation Format: Sanjeeva P. Reddy, Scott K. Thompson, Helen Usansky, Roger A. Smith, Louis Denis, Sandeeep Gupta. ASN007, an oral ERK1/2 inhibitor, shows strong antitumor activity across a panel of KRAS subtype mutant cancer models [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B05.

The RAS/RAF/MEK pathway is frequently dysregulated in a broad range of cancers through mutations in several targets such as KRAS, NRAS, HRAS, BRAF, NF1 and receptor tyrosine kinases. Although MEK and BRAF inhibitors improve the outcome for patients with BRAF mutant melanoma and lung cancers, the duration of response is limited due to reactivation of the pathway. The extracellular-signal-regulated kinases, ERK1 and ERK2 (ERK1/2), which are downstream of MEK and BRAF kinases, serve as a key node in this crucial signaling pathway. Targeting ERK1/2 offers a promising therapeutic strategy for a broad range of cancers, particularly the ones driven by RAS mutations, and in overcoming resistance to MEK/BRAF inhibitors. Here, we present discovery and characterization of a novel orally bioavailable compound, ASN007, that showed inhibition of ERK1/2 kinases in a biochemical assay with IC50 = 2 nM for both targets. Interestingly, ASN007 displayed a much slower dissociation rate (longer target residence time) as compared to other ERK inhibitors. ASN007 inhibited the phosphorylation of ERK1/2 substrates such as RSK1, FRA1 and Elk1 in various cell lines with low nM IC50 values. ASN007 showed potent anti-proliferative activity that was selective for MAPK-pathway dependent cancer cell lines. Indeed, ASN007 showed much more robust activity against RAS mutant cell lines as compared to other ERK1/2 inhibitors. In a daily oral dosing regimen, ASN007 demonstrated strong tumor growth inhibition in BRAF, KRAS and NRAS mutant xenograft models in mice and was well tolerated at efficacious doses. Of note, ASN007 showed strong efficacy using various intermittent dosing regimens. It also showed a strong anti-tumor activity in colorectal PDX models with various KRAS mutations. Furthermore, ASN007 showed regression of tumor growth in a melanoma PDX model that was resistant to both MEK and BRAF inhibitors. In addition to the single agent activity, greater efficacy was observed when ASN007 was combined with immunotherapeutic agents such as an anti-PD-1 antibody or an IDO-1 inhibitor. ASN007 does not exhibit significant inhibition of CYP enzymes or the hERG channel. A Phase 1 trial with ASN007 in patients with BRAF and K-, H- and N-RAS mutant melanoma, colorectal, non-small lung and pancreatic cancers is being planned. Citation Format: Sanjeeva P. Reddy, Niranjan S. Rao, Roger A. Smith, Scott K. Thompson, Sarper Toker. Strong antitumor activity of ASN007, an oral ERK1/2 inhibitor, in PDX tumor models with MAP kinase pathway alterations including KRAS mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5783.

The extracellular-signal-regulated kinases, ERK1 and ERK2 (ERK1/2), are key players in the RAS/RAF/MEK signaling pathway. This pathway is frequently hyper-activated in a wide range of cancers through mutations in upstream targets such as BRAF, RAS, and receptor tyrosine kinases. Inhibition of ERK1/2 offers a promising therapeutic strategy for these cancers, particularly the ones driven by RAS mutations. In order to inhibit this critical node of the RAS/RAF/MEK pathway, we discovered and characterized ASN007, a novel compound that shows potent inhibition of ERK1/2 kinases in a biochemical assay with low single-digit nM IC50 values. ASN007 showed a slow dissociation rate (long target residence time) as compared to several other known ERK inhibitors. In mechanistic cell-based assays, ASN007 inhibited the phosphorylation of ERK1/2 substrates such as RSK1, FRA1, and Elk1 in various cell lines. ASN007 showed single-digit nanomolar antiproliferative activity that was selective for MAPK-pathway dependent cancer cell lines. Indeed, ASN007 showed much more potent activity against a broader panel of KRAS, NRAS, and HRAS mutant cell lines compared to other ERK1/2 inhibitors such as BVD-523 and GDC-0994. In a daily oral dosing regimen, ASN007 demonstrated strong inhibition of tumor growth in multiple BRAF and KRAS mutant xenograft models in mice and was well tolerated at efficacious doses. Of note, ASN007 also showed strong efficacy using various intermittent dosing regimens. ASN007 has a good pharmacokinetic profile in preclinical species and does not exhibit significant inhibition of CYP enzymes or hERG channel. IND-enabling studies are ongoing in preparation for a phase 1 clinical trial to evaluate oral ASN007 in patients with BRAF and K- , H- , and N-RAS mutant cancers Citation Format: Sanjeeva P. Reddy, Dhanalakshmi Sivanandhan, Purushottam Dewang, Niranjan Rao, Roger A. Smith, Scott K. Thompson. ASN007, a novel oral ERK inhibitor, shows robust antitumor activity in RAS mutant cancer models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B150.

We have designed and synthesized a novel series of pyrrolidinones as progesterone receptor partial agonists. Compounds from this series had improved AR selectivity, rat pharmacokinetic properties, and in vivo potency compared to the lead compound. In addition, these compounds had improved selectivity against hERG channel inhibition.

In an effort to determine which regions of IL-8 are involved in interactions with its receptors, eight peptides were designed to correspond to distinct exposed regions of the IL-8 monomer, using the proton NMR-derived structure of the dimer as a basis. The peptides were evaluated singularly, and as equimolar mixtures of two to six peptides, in an IL-8 receptor binding assay and found to have no binding interaction with either alpha or beta IL-8 receptor as single peptides or mixtures of two peptides. In contrast, one of these peptides having the sequence AVLPRSAKEL, which corresponds to the N-terminal 10 amino acid residues of the 77 amino acid form of IL-8, exhibited potent chemotactic activity in human neutrophils. These results indicate that there is no contiguous ligand that can be designed based on the NMR and X-ray determined structure of IL-8 and that there may be multiple receptors responsible for neutrophil activation and chemotaxis.

Selective progesterone receptor modulators (SPRMs) have been suggested as therapeutic agents for treatment of gynecological disorders. One such SPRM, asoprisnil, was recently in clinical trials for treatment of uterine fibroids and endometriosis. We present the crystal structures of progesterone receptor (PR) ligand binding domain complexed with asoprisnil and the corepressors nuclear receptor corepressor (NCoR) and SMRT. This is the first report of steroid nuclear receptor crystal structures with ligand and corepressors. These structures show PR in a different conformation than PR complexed with progesterone (P4). We profiled asoprisnil in PR-dependent assays to understand further the PR-mediated mechanism of action. We confirmed previous findings that asoprisnil demonstrated antagonism, but not agonism, in a PR-B transfection assay and the T47D breast cancer cell alkaline phosphatase activity assay. Asoprisnil, but not RU486, weakly recruited the coactivators SRC-1 and AIB1. However, asoprisnil strongly recruited the corepressor NCoR in a manner similar to RU486. Unlike RU486, NCoR binding to asoprisnil-bound PR could be displaced with equal affinity by NCoR or TIF2 peptides. We further showed that it weakly activated T47D cell gene expression of Sgk-1 and PPL and antagonized P4-induced expression of both genes. In rat leiomyoma ELT3 cells, asoprisnil demonstrated partial P4-like inhibition of cyclooxygenase (COX) enzymatic activity and COX-2 gene expression. In the rat uterotrophic assay, asoprisnil demonstrated no P4-like ability to oppose estrogen. Our data suggest that asoprisnil differentially recruits coactivators and corepressors compared to RU486 or P4, and this specific cofactor interaction profile is apparently insufficient to oppose estrogenic activity in rat uterus.

Mifepristone is a non-selective antagonist of 3-oxosteroid receptors with both abortifacient and anti-endometriotic activities. Non-steroidal mimetics of mifepristone and progesterone are important templates for modulation of the progesterone receptor (PR). For our PR program, we sought an unexplored, synthetically accessible non-steroidal mimetic of mifepristone, suitable for parallel synthesis of analogues. Docking of compounds into a PR homology model identified 4-substituted pyrazolines, which, when synthesized and tested, exhibited functional antagonism of PR.

The RAS-RAF-MEK and PI3K-AKT-mTOR pathways are two major signaling pathways involved in human cancer. Components of these two pathways are frequently mutated in a wide variety of solid tumors. Concurrent double mutations in the two pathways are also observed quite often in a broad range of tumor types. Additionally, inhibition of one of these pathways often leads to the upregulation of the other pathway and development of resistance. In preclinical models, combined inhibition of both pathways has been shown to impart greater efficacy as compared to inhibition of either pathway alone. ASN003 is a novel, highly selective, small-molecule inhibitor of both RAS-RAF and PI3K pathways, discovered using a rational design approach. ASN003 shows potent inhibitory activity against B-RAF and PI3K kinases (low nM IC50). Within the PI3K family, ASN003 has high selectivity for inhibition of PI3Kα and PI3Kδ over PI3Kβ. In a panel of 292 kinases, ASN003 showed high selectivity for inhibiting B-RAF and PI3 kinases, and associated mutant kinases. In cell-based mechanistic studies, ASN003 inhibited phosphorylation of ERK, AKT and S6, and showed strong antiproliferative activity (IC50 = 60-300 nM) in cell lines with B-RAF and PI3K pathway mutations as well as in vemurafenib-resistant cell lines. In pharmacodynamic studies in multiple tumor models, ASN003 showed strong inhibition of the phosphorylation of downstream targets of B-RAF and PI3K, confirming appropriate target engagement. In in vivo efficacy studies, ASN003 showed strong tumor growth inhibition or regression in multiple tumor xenograft models, including A375 (B-Raf V600E mutation), RKO (B-Raf V600E and PIK3CA mutations), and A2058 (B-Raf V600E mutation and PTEN loss). We now report that ASN003 also showed strong tumor growth inhibition (>80%) in a patient-derived xenograft (PDX) model established from a relapsed patient with progressive B-Raf mutant melanoma who showed initial response to vemurafenib. Sequencing analysis showed that the vemurafenib resistant tumor acquired a concurrent PIK3CA mutation. Dual targeting of the B-RAF and PI3K pathways with ASN003 has the potential to treat and/or prevent the acquired resistance to selective B-RAF inhibitors, and may also treat a broader patient population and provide greater efficacy and survival benefit than selective B-RAF inhibitors or selective PI3K pathway inhibitors alone. ASN003 is currently in Phase I clinical development in patients with advanced solid tumors, including tumors with B-Raf V600 mutation, PI3 kinase pathway alterations or PTEN loss. Citation Format: Scott K. Thompson, Roger A. Smith, Niranjan Rao, Michael J. Wick, Sanjeeva P. Reddy. ASN003, a highly selective inhibitor of B-Raf and PI3 kinases, shows strong antitumor activity in B-Raf inhibitor resistant patient-derived xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 158. doi:10.1158/1538-7445.AM2017-158

Spleen tyrosine kinase (SYK) and Janus kinase (JAK) play important roles in the pathogenesis of various types of lymphomas, solid tumors, and myeloproliferative and inflammation disorders. Inhibition of both SYK and JAK kinases has been shown to suppress tumor growth in various preclinical and/or clinical studies. ASN002 is a novel and potent dual inhibitor of SYK and JAK kinases with IC50 values of 4-46 nM in biochemical assays. In mechanistic cell-based studies, ASN002 strongly suppressed the SYK and JAK family kinase signaling pathways as measured in pLAT and pSTAT assays, respectively. When profiled in a panel of 178 cell lines, the compound showed strong anti-proliferative activity in many lymphoid/leukemia cell lines, including SU-DHL-6, SU-DHL-4, OCI-LY10, H929 and Pfeiffer. The proliferation of many solid tumor cell lines representing bladder, lung and colorectal cancer tumor types was also potently inhibited. Importantly, it also strongly inhibited the growth of ibrutinib and idelalisib resistant cell lines. ASN002 was highly efficacious in inhibiting the tumor growth in several tumor xenograft models which include Pfeiffer (diffuse large B-cell lymphoma, DLBCL), SU-DHL-6 (DLBCL), H929 (myeloma), HEL (erythroleukemia), and RT112 (bladder). ASN002 shows little to no inhibition of CYP450 isozymes suggesting low potential for drug-drug interactions. ASN002 is currently being evaluated in a Phase I/II clinical study in patients with lymphomas (DLBCL, mantle cell lymphoma and follicular lymphoma) and solid tumors. Citation Format: Sanjeeva P. Reddy, Niranjan Rao, David Zammit, Scott K. Thompson, Roger A. Smith, Louis Denis. ASN002: A novel dual SYK/JAK inhibitor with strong antitumor activity in both hematological and solid tumor xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4204. doi:10.1158/1538-7445.AM2017-4204

The steady-state kinetics of a full-length and truncated form of the type 2 human methionine aminopeptidase (hMetAP2) were analyzed by continuous monitoring of the amide bond cleavage of various peptide substrates and methionyl analogues of 7-amido-4-methylcoumarin (AMC) and p-nitroaniline (pNA), utilizing new fluorescence-based and absorbance-based assay substrates and a novel coupled-enzyme assay method. The most efficient substrates for hMetAP2 appeared to be peptides of three or more amino acids for which the values of k(cat)/K(m) were approximately 5 x 10(5) M(-1) min(-1). It was found that while the nature of the P1' residue of peptide substrates dictates the substrate specificity in the active site of hMetAP2, the P2' residue appears to play a key role in the kinetics of peptidolysis. The catalytic efficiency of dipeptide substrates was found to be at least 250-fold lower than those of the tripeptides. This substantially diminished catalytic efficiency of hMetAP2 observed with the alternative substrates MetAMC and MetpNA is almost entirely due to the reduction in the turnover rate (k(cat)), suggesting that cleavage of the amide bond is at least partially rate-limiting. The 107 N-terminal residues of hMetAP2 were not required for either the peptidolytic activity of the enzyme or its stability. Steady-state kinetic comparison and thermodynamic analyses of an N-terminally truncated form and full-length enzyme yielded essentially identical kinetic behavior and physical properties. Addition of exogenous Co(II) cation was found to significantly activate the full-length hMetAP2, while Zn(II) cation, on the other hand, was unable to activate hMetAP2 under any concentration that was tested.

The nature of the inhibition of thiol proteases by a new class of mechanism-based inhibitors, 1,5-diacylcarbohydrazides, is described. These potent, time-dependent, active-site spanning inhibitors include compounds that are selective for cathepsin K, a cysteine protease unique to osteoclasts. The 1,5-diacylcarbohydrazides are slow substrates for members of the papain superfamily with inhibition resulting from slow enzyme decarbamylation. Enzyme-catalyzed hydrolysis of 2,2'-N, N'-bis(benzyloxycarbonyl)-L- leucinylcarbohydrazide is accompanied by formation of a hydrazide-containing product and a carbamyl-enzyme intermediate that is sufficiently stable to be observed by mass spectrometry and NMR. Stopped-flow studies yield a saturation limited value of 43 s(-)(1) for the rate of cathepsin K acylation by 2,2'N, N'-bis(benzyloxycarbonyl)-L-leucinylcarbohydrazide. Inhibition potency varies among proteases tested as reflected by 2-3 orders of magnitude differences in K(i) and K(obs)/I, but all eventually form the same stable covalent intermediate. Reactivation rates are equivalent for all enzymes tested (1 x 10(-)(4) s(-)(1)), indicating hydrolysis of a common carbamyl-enzyme form. NMR spectroscopic studies with cathepsin K and 2,2'-N,N'-bis(benzyloxycarbonyl)-L-leucinylcarbohydrazide provide evidence of inhibitor cleavage to generate a covalent carbamyl-enzyme intermediate rather than a tetrahedral complex. The product Cbz-leu-hydrazide does not appear enzyme-bound after cleavage in the NMR spectra, suggesting that the stable inhibited form of the enzyme is the thioester complex. 1, 5-diacylcarbohydrazides represent a new class of unreactive cysteine protease inhibitors that share a common mechanism of action across members of the papain superfamily. Both S and S' subsite interactions are exploited in achieving high selectivity and potency.

Cathepsin K (EC 3.4.22.38) is a cysteine protease of the papain superfamily which is selectively expressed within the osteoclast. Several lines of evidence have pointed to the fact that this protease may play an important role in the degradation of the bone matrix. Potent and selective inhibitors of cathepsin K could be important therapeutic agents for the control of excessive bone resorption. Recently a series of peptide aldehydes have been shown to be potent inhibitors of cathepsin K. In an effort to design more selective and metabolically stable inhibitors of cathepsin K, a series of electronically attenuated alkoxymethylketones and thiomethylketones inhibitors have been synthesized. The X-ray co-crystal structure of one of these analogues in complex with cathepsin K shows the inhibitor binding in the primed side of the enzyme active site with a covalent interaction between the active site cysteine 25 and the carbonyl carbon of the inhibitor. ©

Nerve sheath tumors of the upper airway are extremely uncommon. Asthma, with its associated wheezing and dyspnea, is one of the most commonly encountered respiratory diseases in the pediatric population. Perhaps as a consequence, tumors and other disorders affecting the upper airway, which often present in a similar or identical manner to asthma, are commonly misdiagnosed. Since airway obstruction, bleeding, and ultimate respiratory failure are potential complications of upper airway disease, timely diagnosis is critical. We report two cases of peripheral nerve sheath tumor, one endotracheal, and one subglottic, with a twofold purpose: (1) as case reports of nonmalignant nerve sheath tumor presenting in extremely uncommon locations, and (2) to emphasize the importance of history, physical examination, and diagnostic testing in making a prompt and accurate diagnosis.

We have shown previously that cathepsin K, a recently identified member of the papain superfamily of cysteine proteases, is expressed selectively in osteoclasts and is the predominant cysteine protease in these cells. Based upon its abundant cell type-selective expression, potent endoprotease activity at low pH and cellular localization at the bone interface, cathepsin K has been proposed to play a specialized role in osteoclast-mediated bone resorption. In this study, we evaluated a series of peptide aldehydes and demonstrated that they are potent cathepsin K inhibitors. These compounds inhibited osteoclast-mediated bone resorption in fetal rat long bone (FRLB) organ cultures in vitro in a concentration-dependent manner. Selected compounds were also shown to inhibit bone resorption in a human osteoclast-mediated assay in vitro. Chz-Leu-Leu-Leu-H (in vitro enzyme inhibition Ki,app = 1.4 nM) inhibited parathyroid hormone (PTH)-stimulated resorption in the FRLB assay with an IC-50 of 20 nM and inhibited resorption by isolated human osteoclasts cultured on bovine cortical bone slices with an IC-50 of 100 nM. In the adjuvant-arthritic (AA) rat model, in situ hybridization studies demonstrated high levels of cathepsin K expression in osteoclasts at sites of extensive bone loss in the distal tibia. Cbz-Leu-Leu-Leu-H (30 mg/kg, intraperitoneally) significantly reduced this bone loss, as well as the associated hind paw edema. In the thyroparathyriodectomized rat model, Cbz-Leu-Leu-Leu-H inhibited the increase in blood ionized calcium induced by a 6 h infusion of PTH. These data indicate that inhibitors of cathepsin K are effective at reducing osteoclast-mediated bone resorption and may have therapeutic potential in diseases of excessive bone resorption such as rheumatoid arthritis or osteoporosis.

Human cathepsin K is a recently identified protein with high primary sequence homology to members of the papain cysteine protease superfamily including cathepsins S, L, and B and is selectively expressed in osteoclasts (Drake, F. H., Dodds, R., James, I., Connor, J., Debouck, C., Richardson, S., Lee, E., Rieman, D., Barthlow, R., Hastings, G., and Gowen, M.(1996) J. Biol. Chem. 271, 12511-12516). To characterize its catalytic properties, cathepsin K has been expressed in baculovirus-infected SF21 cells and the soluble recombinant protein isolated from growth media was purified. Purified protein includes an inhibitory pro-leader sequence common to this family of protease. Conditions for enzyme activation upon removal of the pro-sequence have been identified. Fluorogenic peptides have been identified as substrates for mature cathepsin K. In addition, two protein components of bone matrix, collagen and osteonectin, have been shown to be substrates of the activated protease. Cathepsin K is inhibited by E-64 and leupeptin, but not by pepstatin, EDTA, phenylmethylsulfonyl fluoride, or phenanthroline, consistent with its classification within the cysteine protease class. Leupeptin has been characterized as a slow binding inhibitor of cathepsin K (kobs/[I] = 273,000 M–1•s–1). Cathepsin K may represent the elusive protease implicated in degradation of protein matrix during bone resorption and represents a novel molecular target in treatment of disease states associated with excessive bone loss such as osteoporosis.

A novel series of hydroxyethylene-based peptidomimetics that contain 2-substituted nitrogen heterocycles as P1′-P2′ amide bond isosteres has been prepared and evaluated as inhibitors of HIV-1 protease and in vitro HIV-1 replication. Many of these compounds exhibit inhibition constants in the low to subnanomolar range. Structure-activity relationships are discussed.

A general and efficient synthetic route to the marasmane sequiterpenes (±)-isovelleral (2) and (±)-stearoylvelutinal (1b) is described. Total syntheses of two other naturally occurring sesquiterpenes, deconjugated anhydrolactarorufin A (5) and lactarorufin A (6), were achieved using an acid-catalyzed ring expansion. All four syntheses are highly stereoselective and do not require the use of any protecting groups. Finally, the protic acid-catalyzed degradation of velutinal (1a) was investigated in an effort to chemically induce the biologically important conversion of velutinal (1a) to isovelleral (2). The experimental results thus obtained indicate that an enzymatic mechanism for the key transformation of velutinal(1a) into isovelleral(2) is more plausible than one that is acid-catalyzed

The previously reported pyrrolidine class of progesterone receptor partial agonists demonstrated excellent potency but suffered from serious liabilities including hERG blockade and high volume of distribution in the rat. The basic pyrrolidine amine was intentionally converted to a sulfonamide, carbamate, or amide to address these liabilities. The evaluation of the degree of partial agonism for these non-basic pyrrolidine derivatives and demonstration of their efficacy in an in vivo model of endometriosis is disclosed herein.

The lead serum and glucocorticoid-related kinase 1 (SGK1) inhibitors 4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid (1) and {4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid (2) suffer from low DNAUC values in rat, due in part to formation and excretion of glucuronic acid conjugates. These PK/glucuronidation issues were addressed either by incorporating a substituent on the 3-phenyl ring ortho to the key carboxylate functionality of 1 or by substituting on the group in between the carboxylate and phenyl ring of 2. Three of these analogs have been identified as having good SGK1 inhibition potency and have DNAUC values suitable for in vivo testing.

Using the X-ray crystal structure of an amide-based progesterone receptor (PR) partial agonist bound to the PR ligand binding domain, a novel PR partial agonist class containing a pyrrolidine ring was designed. Members of this class of N-alkylpyrrolidines demonstrate potent and highly selective partial agonism of the progesterone receptor, and one of these analogs was shown to be efficacious upon oral dosing in the OVX rat model of estrogen opposition.

Two classes of amino acid-derived heterocyclic progesterone receptor ligands were developed to address the metabolic issues posed by the dimethyl amide functionality of the lead compound (1). The tetrazole-derived ligands behaved as potent partial agonists, while the 1,2,4-triazole ligands behaved as potent full agonists.

Androgens, through their actions on the androgen receptor (AR), are required for the development of the prostate and contribute to the pathologic growth dysregulation observed in prostate cancers. Consequently, androgen ablation has become an essential component of the pharmacotherapy of prostate cancer. In this study, we explored the utility of targeting processes downstream of AR as an alternate approach for therapy. Specifically, we show that the serum and glucocorticoid-regulated kinase 1 (SGK1) gene is an androgen-regulated target gene in cellular models of prostate cancer. Furthermore, functional serum- and glucocorticoid-regulated kinase 1 (SGK1) protein, as determined by the phosphorylation of its target Nedd4-2, was also increased with androgen treatment. Importantly, we determined that RNA interference–mediated knockdown of SGK1 expression attenuates the androgen-mediated growth of the prostate cancer cell line LNCaP. Given these findings, we explored the utility of SGK1 as a therapeutic target in prostate cancer by developing and evaluating a small-molecule inhibitor of this enzyme. From these studies emerged GSK650394, a competitive inhibitor that quantitatively blocks the effect of androgens on LNCaP cell growth. Thus, in addition to androgen ablation, inhibition of pathways downstream of AR is likely to have therapeutic utility in prostate cancer. [Cancer Res 2008;68(18):7475–83]

A novel series of 1H-indol-1-yl tertiary amine LXR agonists has been designed. Compounds from this series were potent agonists with good rat pharmacokinetic parameters. In addition, the crystal structure of an LXR agonist bound to LXRα will be disclosed.

High-throughput screening for inhibitors of the human metalloprotease, methionine aminopeptidase-2 (MetAP2), identified a potent class of 3-anilino-5-benzylthio-1,2,4-triazole compounds. Efficient array and interative synthesis of triazoles led to rapid SAR development around the aniline, benzylthio, and triazole moeities. Evaluation of these analogs in a human MetAP2 enzyme assay led to the identification of several inhibitors with potencies in the 50-100 picomolar range. The deleterious effects on inhibitor potency by methylation of the anilino-triazole nitrogens, as well as the X-ray crystal structure of triazole 102 bound in the active site of MetAP2, confirm the key interactions between the triazole nitrogens, the active site cobalt atoms, and the His-231 side-chain. The structure has also provided a rationale for interpreting SAR within the triazole series. Key aniline (2-isopropylphenyl) and sulfur substituents (furanylmethyl) identified in the SAR studies led to the identification of potent inhibitors (103 and 104) of endothelial cell proliferation. Triazoles 103 and 104 also exhibited dose-dependent activity in an aortic ring tissue model of angiogenesis highlighting the potential utility of MetAP2 inhibitors as anticancer agents.

Various genes in RAS-RAF and PI3K pathways are frequently mutated in a wide variety of solid tumors. Concurrent double mutations are also observed quite often in a broad range of tumor types. Combined inhibition of both pathways has been shown to impart greater efficacy in multiple tumor models in animals. In order to simultaneously block these pathways, we have discovered and characterized several molecules showing dual inhibition of the RAS-RAF and PI3K pathways. ASN003 has been identified as a lead compound with potent and highly selective inhibitory activity against B-RAF, PI3K and mTOR kinases (low nM IC50). Within the PI3K family, ASN003 has high selectivity for inhibition of PI3Kα and PI3Kδ over PI3Kβ. In cell-based mechanistic studies, ASN003 did not increase phospho-ERK levels in the KRAS mutant cell line HCT116 and thus may not cause paradoxical activation of RAS/MAPK pathway in tumors. Consistent with its dual inhibition profile, ASN003 showed strong antiproliferative activity in cell lines with B-RAF and PI3K pathway mutations as well as vemurafenib (B-RAF inhibitor)-resistant cell lines. ASN003 suppressed the phosphorylation of downstream targets of B-RAF, PI3K and mTOR in pharmacodynamic studies in multiple tumor models, indicating appropriate target engagement. In in vivo efficacy studies, ASN003 showed regression in a B-RAFV600E mutant A375 xenograft model and also caused significant tumor growth inhibition in RKO and A2058 tumor models, which harbor mutations in PIK3CA or PTEN genes, respectively, in addition to the B-RAF V600E mutation. Dual targeting of the B-RAF and PI3K pathways with ASN003 has the potential to treat and/or prevent the acquired resistance to selective B-RAF inhibitors, and may also treat a broader patient population and provide greater efficacy and survival benefit than selective B-RAF inhibitors or selective PI3K pathway inhibitors alone. Due to the lack of paradoxical activation, treatment with ASN003 may not cause skin toxicities seen with pure B-RAF inhibitors. ASN003 is currently in preclinical development and is expected to enter Phase I/II clinical trials by early 2016. Citation Format: Scott K. Thompson, Mahaboobi Jaleel, Vijay Kumar Nyavanandi, Murali Ramachandra, Hosahalli Subramanya, Aravind Basavaraju, Vaibhav Sihorkar, Roger A. Smith, Niranjan Rao, Sandeep Gupta, Sanjeeva P. Reddy. ASN003, a unique B-RAF inhibitor with additional selective activity against PI3K and mTOR kinases, shows strong antitumor activity in multiple xenograft models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B100.

Spleen tyrosine kinase (SYK) and Janus kinase (JAK) play important roles in the pathogenesis of various types of lymphomas, solid tumors, myeloproliferative and inflammation disorders. Inhibition of these targets has been shown to suppress tumor growth in various preclinical and/or clinical studies. Most B-cell malignancies are driven by the B-cell receptor (BCR) pathway, and SYK plays a critical role in this pathway by initiating and amplifying the signal from the receptor. JAK kinases (JAK1, JAK2, JAK3 and TYK2), upon stimulation by various growth factors and cytokines, phosphorylate signal transducers and activators of transcription family proteins (STAT1 -5) which activate various downstream target genes. ASN002 is a novel and potent dual inhibitor of SYK and JAK kinases with IC50 values of 5-46 nM in biochemical assays. In mechanistic cell-based studies involving IgE and cytokine stimulations, ASN002 strongly suppressed the SYK and JAK family kinase signaling pathways measured as pLAT and pSTAT levels, respectively. The compound showed anti-proliferative activity in a broad panel of human cancer cell lines including DHL6, DHL4, OCI-LY10, H929, Pfeiffer, HT-1376, and Lovo, suggesting activity in both solid and hematological tumor types. In a multiple myeloma (H929) xenograft model, ASN002 exhibited significant efficacy in inhibiting tumor growth (>95%). It also significantly delayed the onset of hind limb paralysis in the human erythroleukemia (HEL) mouse model. ASN002 has good oral bioavailability, metabolic stability, is not a Pgp substrate, and shows little to no inhibition of CYP450 isozymes. ASN002 showed a favorable safety profile in rat and dog toxicology studies. A Phase I/II clinical study is being planned for the evaluation of ASN002 in lymphomas (diffuse large B cell lymphoma, mantle cell lymphoma, follicular lymphoma) and solid tumors. Citation Format: Sanjeeva Reddy, Nitin K. Damle, Aranapakam M. Venkatesan, Scott K. Thompson, Niranajan Rao, Roger A. Smith, Sandeep Gupta. ASN002: A novel dual SYK/JAK inhibitor with strong antitumor activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 792. doi:10.1158/1538-7445.AM2015-792

The extension of a previously reported cathepsin K azepanone-based inhibitor template to the design and synthesis of potent and selective inhibitors of the homologous cysteine protease cathepsin L is detailed. Structure-activity studies examining the effect of inhibitor selectivity as a function of the P3 and P2 binding elements of the potent cathepsin K inhibitor 1 revealed that incorporation of either a P3 quinoline-8-carboxamide or a naphthylene-1-carboxamide led to increased selectivity for cathepsin L over cathepsin K. Substitution of the P2 leucine of 1 with either a phenylalanine or a beta-naphthylalanine also resulted in an increased selectivity for cathepsin L over cathepsin K. Molecular modeling studies with the inhibitors docked within the active sites of both cathepsins L and K have rationalized the observed selectivities. Optimization of cathepsin L binding by the combination of the P3 naphthylene-1-carboxamide with the P2 beta-naphthylalanine provided 15, which is a potent, selective, and competitive inhibitor of human cathepsin L with a K(i) = 0.43 nM.

Objective Although many studies have examined the effects of systemic corticosteroid therapy (SCT) on the clinical course of infectious mononucleosis (IM), few have evaluated the influence of these studies on treatment patterns and outcomes. The purpose of this study was to review current therapeutic strategies and outcomes in uncomplicated and complicated IM. Design Retrospective case series. Setting Tertiary care center. Patients We identified 206 immunocompetent patients with IM diagnosed during the previous 5 years. Patient information, including age, sex, history and physical findings at presentation, pertinent laboratory data, management practices, and treatment outcomes, were analyzed. Interventions Systemic corticosteroid therapy was used in 44.7% of patients. Evaluation of treatment indications for SCT revealed that 8.0% of the study population qualified by traditional criteria for the use of corticosteroids; 92.0% of patients received SCT for other indications. Factors associated with the observed increase in corticosteroid use included a history of repeat visits, inpatient admission, and otolaryngology consultation. Main Outcome Measures Diagnosis was made on the basis of a positive heterophil antibody test (monospot test) with appropriate clinical findings (97.5% of patients) or by the presence of lymphocytosis with appropriate clinical findings (2.4% of patients). Results Systemic corticosteroid therapy was not positively associated with fever, decreased oral intake, tonsillar hypertrophy, or duration of symptoms. No significant differences in incidence of disease complications, rates of hospital admission, or length of hospital stay were noted between the steroid and nonsteroid treatment groups. Conclusions Despite consistent and uniform acceptance in the medical literature that SCT in the setting of IM should be reserved for patients with impending airway obstruction, corticosteroids continue to be used on a much broader scale at this tertiary care institution. This observation suggests that clinicians see value in SCT for treatment of IM beyond the classically accepted reasons. Moreover, despite previous reports of possible adverse consequences of SCT in IM, our review failed to demonstrate any such trend.

Objective To examine the effects of estrogen suppression on age-related changes in distortion product otoacoustic emissions (DPOAEs) and contralateral suppression (CS) of DPOAEs in CBA mice. Study design and setting Young CBA mice received a slow-release shoulder implantation of either tamoxifen or placebo. Serial DPOAEs and CS of DPOAEs were obtained at 3-week intervals over a period of 9 weeks. Results Although DPOAEs were maintained over the study interval, CS of DPOAEs decreased significantly with age in the experimental group. No such declines were observed in either the control animals or the untreated male mice. Conclusions Estrogen suppression negatively affects the MOC efferent feedback system. Significance Our results support the hypothesis that estrogen plays an important role in the maintenance of auditory integrity. Additionally, our findings raise intriguing questions about auditory effects of hormonal shifts in humans resulting from menopause, hormone supplements such as oral contraceptives, and hormone replacement therapy as well as antiestrogens.

Inhibitors of human methionine aminopeptidase type 2 (hMetAP2) are of interest as potential treatments for cancer. A new class of small molecule reversible inhibitors of hMetAP2 was discovered and optimized, the 4-aryl-1,2,3-triazoles. Compound 24, a potent inhibitor of cobalt-activated hMetAP2, also inhibits human and mouse endothelial cell growth. Using a mouse matrigel model, this reversible hMetAP2 inhibitor was also shown to inhibit angiogenesis in vivo.

Substituted 3-(phenylamino)-1H-pyrrole-2,5-diones were identified from a high throughput screen as inducers of human ATP binding cassette transporter A1 expression. Mechanism of action studies led to the identification of GSK3987 as an LXR ligand. GSK3987 recruits the steroid receptor coactivator-1 to human LXRalpha and LXRbeta with EC(50)s of 40 nM, profiles as an LXR agonist in functional assays, and activates LXR though a mechanism that is similar to first generation LXR agonists.

Objectives/Hypothesis: The purpose of the study was to evaluate the effectiveness of external auditory canal foreign body removal attempts by health care practitioners and to explore outcomes of patients with unsuccessful initial removal attempts. Study Design: Retrospective case series. Methods: A case series of patients presenting with external auditory canal foreign body to the emergency department of the authors' institution (Strong Memorial Hospital, University of Rochester Medical Center) over a 3-year period was studied retrospectively. Medical records were reviewed, and information including age at presentation, type of foreign body, side of presentation, length of time in place, signs and symptoms at presentation, management practices, and outcomes was recorded. Results: One hundred sixty-two patients with a diagnosis of external auditory canal foreign body were identified. Emergency personnel successfully managed 67% of patients using direct visualization techniques, and 33% required otolaryngological consultation. Otolaryngologists used otomicroscopy with standard otological instruments as their mainstay of management. Analysis of successfully managed emergency department cases revealed that 82% of foreign bodies were irregularly shaped objects with soft, graspable parts. Conversely, 72% of foreign bodies in otolaryngology referrals were firm, rounded objects such as beads and beans. Analysis of patients referred to otolaryngologists revealed a requirement for removal under anesthesia of 19%. Patients with a history of one or more removal attempts before emergency department evaluation universally failed further direct visualization techniques. A disproportionate number of these patients eventually required operative intervention and/or had tympanic membrane perforation. Patients with a history of previous removal attempt(s) who were referred directly to otolaryngologists were more likely to be successfully managed without general anesthesia. Conclusion: Emergency room personnel successfully manage the majority of patients with foreign bodies of the external auditory canal. For patients with firm, rounded objects, direct otolaryngology consultation without further manipulation should be strongly considered. Patients who have had previous removal attempts should not undergo further manipulation in the emergency department but rather should be referred directly to an otolaryngologist.

The structure-based design and synthesis of lactam-constrained azapeptide inhibitors of human cathepsin K are described. Enhanced stability to proteolytic cleavage over acyclic analogues is discussed.

Using binding models which were based on the X-ray crystal structure of an amino acid-based active site-spanning inhibitor complexed with cathepsin K, Cbz-leucine mimics have been developed, leading ultimately to the design of a potent cathepsin K inhibitor free of amino acid components. These mimics, which consist of alpha-substituted biphenylacetyl groups in place of Cbz-leucine moieties, effectively mimic all aspects of the Cbz-leucine moieties which are important for inhibitor binding. The predicted directions of binding for the inhibitors were confirmed by mass spectral analysis of their complexes with cathepsin K, which gave results consistent with acylation of the enzyme and loss of the acylhydrazine portion of the inhibitor which binds on the S' side of the active site. The binding models were found to be very predictive of relative inhibitor potency as well as direction of inhibitor binding. These results strengthen the validity of a strategy involving iterative cycles of structure-based design and inhibitor synthesis and evaluation for the discovery of non-peptide inhibitors.

D.F. VEBER, R.W. MARQUIS, Y. RU, D.S. YAMASHITA, H.-J. OH, S.K. THOMPSON, S.M. HALBERT, T.J. CARR, J.G. GLEASON, T.A. TOMASZEK, JR. M.A. LEVY, M. BOSSARD, D.G. TEW, I.E. JAMES, J. BRIAND, S.A. CARR, D. ZYMBRYKI, L. LEE-RYKACZEWSKI, R.L. DESJARLAIS, M.S. McQUENEY, K.J. D’ALESSIO, B. ZHAO, C.A. JANSON, S.S. ABDEL-MEGUID and W.W. SMITH SmithKline Beecham Pharmaceuticals, 709 Swedeland Rd., King of Prussia, PA 19406

Papain has been used as a surrogate enzyme in a drug design effort to obtain potent and selective inhibitors of cathepsin K, a new member of the papain superfamily of cysteine proteases that is selectively and highly expressed in osteoclasts and is implicated in bone resorption. Here we report the crystal structures of two papain-inhibitor complexes and the rational design of novel cathepsin K inhibitors. Unlike previously known crystal structures of papain-inhibitor complexes, our papain structures show ligand binding extending deep within the S'-subsites. The two inhibitor complexes, carbobenzyloxyleucinyl-leucinyl-leucinal and carbobenzyloxy-L-leucinyl-L-leucinyl methoxymethyl ketone, were refined to 2.2- and 2.5-A resolution with R-factors of 0.190 and 0. 217, respectively. The S'-subsite interactions with the inhibitors are dominated by an aromatic-aromatic stacking and an oxygen-aromatic ring edge interaction. The knowledge of S'-subsite interactions led to a design strategy for an inhibitor spanning both subsites and yielded a novel, symmetric inhibitor selective for cathepsin K. Simultaneous exploitation of both S- and S'-sites provides a general strategy for the design of cysteine protease inhibitors having high specificity to their target enzymes.

Peptidomimetic cathepsin K inhibitors have been designed using binding models which were based on the X-ray crystal structure of an amino acid-based, active site-spanning inhibitor complexed with cathepsin K. These inhibitors, which contain a benzyloxybenzoyl group in place of a Cbz-leucine moiety, maintained good inhibitory potency relative to the amino acid-based inhibitor, and the binding models were found to be very predictive of relative inhibitor potency. The binding mode of one of the inhibitors was confirmed by X-ray crystallography, and the crystallographically determined structure is in close qualitative agreement with the initial binding model. These results strengthen the validity of a strategy involving iterative cycles of structure-based design, inhibitor synthesis and evaluation, and crystallographic structure determination for the discovery of peptidomimetic inhibitors.

We have investigated the inhibitor specificity for the proteases of the human immunodeficiency viruses, types 1 and 2. Using a series of related inhibitors, the P1' side chain was confirmed to play a significant role in determining both the absolute and relative affinity for the enzymes. To further define the residues in the enzymes responsible for the difference in affinity, chimeric proteins were constructed in which domains of the respective proteases were exchanged at the genetic level. The results of these studies demonstrated that inhibitor affinity is conferred by a combination of the active site residues (32, 47, and 82) along with a loop comprised of residues 31 and 33-37, which lies outside of the active site cavity. These results are discussed in terms of existing structural data.

The RAS-RAF-MAPK and PI3K pathways are two major signaling pathways involved in the initiation and progression of a broad range of tumors. These two pathways are frequently activated in the majority of solid tumors through mutations in various components of the pathways. Inhibition of one pathway often leads to the activation of the other pathway. Preclinical studies have shown that simultaneous inhibition of these two pathways leads to greater efficacy in a broader range of tumor types. For example, despite the presence of the B-RAFV600E mutation, colorectal cancer tumors do not respond to B-RAF selective inhibitors. In contrast, the combination of B-RAF or MEK inhibitors with PI3K pathway inhibitors shows significant tumor growth inhibition in colorectal cancer xenograft models in mice. Currently, there are several clinical trials ongoing using combinations of inhibitors targeting both pathways. We employed a rational drug design approach to discover and develop a single compound with dual inhibition of both RAS-RAF-MAPK and PI3K pathways. EN3352 was identified as a lead compound with potent inhibitory activity against B-RAF, PI3K and mTOR kinases, with low nanomolar IC50 values. Profiling of EN3352 in a panel of 292 kinases showed that it is highly selective for these three kinases. Within the PI3K family, EN3352 is selective for inhibition of PI3Kα versus PI3Kβ. EN3352 showed broader anti-proliferative activity in tumor cell lines compared to the B-Raf selective inhibitors, vemurafenib and dabrafenib. EN3352 has good oral bioavailability in preclinical species and showed inhibition of phosphorylation of the downstream targets of B-RAF, PI3K and mTOR in various tumor models in mice. EN3352 showed regression in a B-RAFV600E mutant A375 xenograft model and also showed significant tumor growth inhibition in a RKO model, which harbors mutations in both B-RAF and PIK3CA genes. Dual targeting of the B-RAF and PI3K pathways with EN3352 has the potential to treat and/or prevent the acquired resistance to selective B-RAF inhibitors, and may also treat a broader patient population and provide greater efficacy and survival benefit than selective B-RAF inhibitors or selective PI3K pathway inhibitors alone. (Disclosure: Funding for this research was provided by Endo Pharmaceuticals Inc.) Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B274. Citation Format: Sanjeeva P. Reddy, Mahaboobi Jaleel, Vijay K. Nyavanandi, Murali Ramachandra, Hosahalli Subramanya, Aravind Basavaraju, Vaibhav Sihorkar, Roger A. Smith, Sandeep Gupta, Scott K. Thompson. Discovery and characterization of a highly selective inhibitor of B-RAF, PI3K, and mTOR kinases with antitumor activity in B-RAF and B-RAF/PI3K pathway double mutant xenograft models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B274.