Development of a prodrug of hydantoin based TACE inhibitor.

Our research on hydantoin based TNF-α converting enzyme (TACE) inhibitors led to fused bi-heteroaryl hydantoin series that demonstrate sub-nanomolar potency (Ki) as well as excellent activity in human whole blood (hWBA). However, lead compound 2 posed some formulation challenges which prevented it for further development. A prodrug approach was investigated to address this issue. The pivalate prodrug 3 can be formulated as stable neutral form and demonstrated improved DMPK properties when compared with parent compound.

Fused bi-heteroaryl substituted hydantoin compounds as TACE inhibitors.

We have identified a series of hydantoin-derived TNF-a converting enzyme (TACE) inhibitors containing a pendant fused bi-heteroaryl group, which demonstrate sub-nanomolar potency (Ki), excellent activity in human whole blood assay, and improved DMPK profiles over prior series.

Pharmacological characterization of a novel α2C-adrenoceptor agonist N-[3,4-dihydro-4-(1H-imidazol-4-ylmethyl)-2H-1, 4-benzoxazin-6-yl]-N-ethyl-N'-methylurea (compound A).

We define the pharmacological and pharmacokinetic profiles of a novel α(2C)-adrenoceptor agonist, compound A [N-[3,4-dihydro-4-(1H-imidazol-4-ylmethyl)-2H-1,4-benzoxazin-6-yl]-N-ethyl-N'-methylurea]. This compound has high affinity (K(i)) for the human α(2C)-adrenoceptor (K(i) = 12 nM), and 190- to 260-fold selectivity over the α(2A)- and α(2B)-adrenoceptor subtypes. In cell-based functional assays, compound A produced good agonist (EC(50) = 166 nM) and efficacy (E(max) = 64%) responses at the α(2C)-adrenoceptor, much lower potency and efficacy at the α(2A)-adrenoceptor (EC(50) = 1525 nM; E(max) = 8%) and α(2B)-adrenoceptor (EC(50) = 5814 nM; E(max) = 21%) subtypes, and low or no affinity and functional activity at the α(1A)-, α(1B)-, and α(1D)-adrenoceptor subtypes. In the human saphenous vein postjunctional α(2C)-adrenoceptor bioassay, compound A functions as a potent agonist (pD(2) = 6.3). In a real-time contraction bioassay of pig nasal mucosa, compound A preferentially constricted the veins (EC(50) = 108 nM), and the magnitude of arteriolar contraction reached only 50% of the maximum venular responses. Compound A exhibited no effect on locomotor activity, sedation, and body temperature in mice (up to 100 mg/kg) and did not cause hypertension and mydriasis (30 mg/kg) in conscious rats. Compound A is orally bioavailable (24%) with good plasma exposure. This compound is a substrate for the efflux P-glycoprotein transporter, resulting in very low central nervous system (CNS) penetration. In summary, compound A is a highly selective, orally active, and non-CNS-penetrating α(2C)-adrenoceptor agonist with desirable in vitro and in vivo pharmacological properties suitable for the treatment of nasal congestion.

Novel TNF-α converting enzyme (TACE) inhibitors as potential treatment for inflammatory diseases.

Our research on hydantoin based TNF-α converting enzyme (TACE) inhibitors has led to an acetylene containing series that demonstrates sub-nanomolar potency (K(i)) as well as excellent activity in human whole blood. These studies led to the discovery of highly potent TACE inhibitors with good DMPK profiles.

Biaryl substituted hydantoin compounds as TACE inhibitors.

We disclose further optimization of hydantoin TNF-alpha convertase enzyme (TACE) inhibitors. SAR with respect to the non-prime region of TACE active site was explored. A series of biaryl substituted hydantoin compounds was shown to have sub-nanomolar K(i), good rat PK, and good selectivity versus MMP-1, -2, -3, -7, -9, and -13.

Discovery and SAR of hydantoin TACE inhibitors.

We disclose inhibitors of TNF-alpha converting enzyme (TACE) designed around a hydantoin zinc binding moiety. Crystal structures of inhibitors bound to TACE revealed monodentate coordination of the hydantoin to the zinc. SAR, X-ray, and modeling designs are described. To our knowledge, these are the first reported X-ray structures of TACE with a hydantoin zinc ligand.

ADAM10 is a principal 'sheddase' of the low-affinity immunoglobulin E receptor CD23.

CD23, the low-affinity immunoglobulin E receptor, is an important modulator of the allergic response and of diseases such as rheumatoid arthritis. The proteolytic release of CD23 from cells is considered a key event in the allergic response. Here we used loss-of-function and gain-of-function experiments with cells lacking or overexpressing candidate CD23-releasing enzymes (ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM19 and ADAM33), ADAM-knockout mice and a selective inhibitor to identify ADAM10 as the main CD23-releasing enzyme in vivo. Our findings provide a likely target for the treatment of allergic reactions and set the stage for further studies of the involvement of ADAM10 in CD23-dependent pathologies.

IK682, a tight binding inhibitor of TACE.

TNFalpha converting enzyme (TACE) is the major metalloproteinase for the processing of TNFalpha, a key inflammatory cytokine. IK682, a hydroxamate compound, was reported to be a potent and specific TACE inhibitor [J.J. Duan, L. Chen, Z.R. Wasserman, Z. Lu, R.Q. Liu, M.B. Covington, M. Qian, K.D. Hardman, R.L. Magolda, R.C. Newton, D.D. Christ, R.R. Wexler, C.P. Decicco, J. Med. Chem. 45 (2002) 4954-4957]. The binding kinetics of IK682 and the ectodomain of human TACE was examined. The k(on) of IK682 was determined as 1.1+/-0.3 x 10(8) M(-1) min(-1). No detectable dissociation of IK682 from TACE was observed following dialysis, dilution, and extensive washing over a maximum of 72 h. This was in contrast to the rapid dissociation of IK682 from ADAM10. LC/MS analysis of the TACE-IK682 complex after dissociation under denaturing conditions indicated that the tight binding is not due to covalent interaction. The X-ray crystal structure of TACE-IK682 complex revealed multiple binding points at the S1' and S3' sites and the movement of a loop (from Ala349 to Gly442) to accommodate the binding of the quinolinyl group of IK682 at the S3' pocket. The conformational changes of TACE may contribute significantly to the high affinity binding as a result of a more stable TACE-inhibitor complex.

The synthesis of substituted bipiperidine amide compounds as CCR3 ligands: antagonists versus agonists.

Structure-activity relationship study of bipiperidine amide 1 has identified the reverse bipiperidine amide 4a as a CC chemokine-3 (CCR3) receptor antagonist. Optimization of the structure-activity relationship of compound 4a has resulted in the identification of a CCR3 antagonist 4i as well as a CCR3 agonist 13.

ADAM33 enzyme properties and substrate specificity.

ADAM33 is an asthma susceptibility gene recently identified through a genetic study of asthmatic families [van Eerdewegh, et al. (2002) Nature 418, 426-430]. To understand the function of the gene product, the recombinant metalloproteinase domain of human ADAM33 was purified and tested for its substrate cleavage specificity using peptides derived from beta-amyloid precursor protein (APP). A single Ala substitution at the P2 position of a 10-residue APP peptide, YEVHHQKLVF, yielded a 20-fold more efficient substrate. Terminal truncation studies identified a minimal nine-residue core (P5-P4') important for ADAM33 recognition and cleavage. Full positional scanning of the 10-mer peptide using the 19 naturally occurring l-amino acids (excluding Cys) revealed a substrate specificity profile. A strong preference for Val or Ile at P3, Ala at P2, and Gln at P1' was observed. The substrate binding model based on the X-ray structure of the ADAM33-inhibitor complex supported the observed substrate specificity profile. On the basis of this, an improved substrate was designed and a fluorescence resonance energy transfer (FRET) assay was developed using a fluorogenic derivative of this substrate. Kinetic studies confirmed that the best substrate, FRET-P2 [K(Dabcyl)YRVAFQKLAE(Edans)K], was approximately 100-fold more efficient than the wild-type APP peptide substrate, with a k(cat)/K(m) value of (3.6 +/- 0.1) x 10(4) s(-)(1) M(-)(1). Using this substrate and the FRET assay, ADAM33 enzyme activity and thermal stability were characterized. ADAM33 dependence on buffer conditions, detergents, and temperature was examined, and optimal conditions were defined. Accurate K(i) values for tissue inhibitors of metalloproteinase and small molecule compounds were obtained.

The synthesis of substituted bipiperidine amide compounds as CCR3 antagonists.

Bipiperidine amide 1 has been identified as a CC chemokine receptor 3 (CCR3) antagonist. Optimization of its structure-activity relationship has resulted in the identification of cis (R,R)-4-[(3,4-dichlorophenyl)methyl]-3-hydroxymethyl-1'(6-quinolinylcarbonyl)-1,4'-bipiperidine 14n, which exhibits potent receptor affinity and inhibition of both calcium flux and eosinophil chemotaxis.

Cloning and pharmacological characterization of mouse TRPV1.

The Transient Receptor Potential cation channel V1 (TRPV1) is expressed in peripheral nociceptive neurons and is subject to polymodal activation via various agents including capsaicin, noxious heat, low extracellular pH, and direct phosphorylation by protein kinase C (PKC). We have cloned and heterologously expressed mouse TRPV1 (mTRPV1) and characterized its function utilizing FLIPR-based calcium imaging to measure functional responses to various small molecule agonists, low pH and direct phosphorylation via PKC. The various TRPV1 agonists activated mTRPV1 with a rank order of agonist potency of (resiniferatoxin (RTX) = arvanil > capsaicin = olvanil > OLDA > PPAHV) (EC50 values of 0.15+/-0.04 nM, 0.27+/-0.07 nM, 9.1+/-1.2 nM, 3.7+/-0.3 nM, 258+/-105 nM, and 667+/-151 nM, respectively). Additionally, mTRPV1 was activated by either low pH or with addition of the PKC activator phorbol 12-myristate 13-acetate (PMA). The TRPV1 antagonists iodinated-resiniferatoxin (I-RTX) or BCTC were both able to block capsaicin, pH and PKC-induced responses of mTRPV1 (IC50 (I-RTX) = 0.35+/-0.12 nM, 1.9+/-0.7 nM, and 0.80+/-0.68 nM, IC50 (BCTC) = 1.3+/-0.36 nM, 0.59+/-0.16 nM, and 0.37+/-0.15 nM, respectively). However, the antagonist capsazepine was only able to inhibit a capsaicin-evoked response of mTRPV1 with an IC50 of 1426+/-316 nM. Comparable results were achieved with rat TRPV1, while capsazepine blocked all modes of human TRPV1 activation. Thus, the mTRPV1 cation channel has a molecular pharmacological profile more akin to rat TRPV1 than either human or guinea pig TRPV1 and the molecular pharmacology suggests that capsazepine may be an ineffective TRPV1 antagonist for in vivo models of inflammatory pain in the mouse.

Catalytic activity of human ADAM33.

ADAM33 (a disintegrin and metalloproteinase) is an asthma susceptibility gene recently identified through a genetic study of asthmatic families (van Eerdewegh et al. (2002) Nature 418, 426-430). In order to characterize the catalytic properties of ADAM33, the metalloproteinase domain of human ADAM33 was expressed in Drosophila S2 cells and purified. The N-terminal sequence of the purified metalloproteinase was exclusively (204)EARR, indicating utilization of one of three furin recognition sites. Of many synthetic peptides tested as potential substrates, four peptides derived from beta-amyloid precursor protein (APP), Kit-ligand-1 (KL-1), tumor necrosis factor-related activation-induced cytokine, and insulin B chain were cleaved by ADAM33; mutation at the catalytic site, E346A, inactivated catalytic activity. Cleavage of APP occurred at His(14)/Gln(15), not at the alpha-secretase site and was inefficient (k(cat)/K(m) (1.6 +/- 0.3) x 10(2) m(-1) s(-1)). Cleavage of a juxtamembrane KL-1 peptide occurred at a site used physiologically with a similar efficiency. Mutagenesis of KL-1 peptide substrate indicated that the P3, P2, P1, and P3' residues were critical for activity. In a transfected cell-based sheddase assay, ADAM33 functioned as a negative regulator of APP shedding and mediated some constitutive shedding of KL-1, which was not regulated by phorbol 12-myristate 13-acetate activation. ADAM33 activity was sensitive to several hydroxamate inhibitors (IK682, K(i) = 23 +/- 7 nm) and to tissue inhibitors of metalloproteinase (TIMPs). Activity was inhibited moderately by TIMP-3 and TIMP-4 and weakly inhibited by TIMP-2 but not by TIMP-1, a profile distinct from other ADAMs. The identification of ADAM33 peptide substrates, cellular activity, and a distinct inhibitor profile provide the basis for further functional studies of ADAM33.

Mouse ADAM33: two splice variants differ in protein maturation and localization.

We compared the tissue mRNA prevalence and protein maturation of two splice variants of mouse ADAM33, a metalloprotease implicated in airway hyperresponsiveness. These variant cDNAs, designated 914 (alpha) and 906 (beta), encode membrane-bound forms that differ primarily in 26 residues (exon 17) between the cysteine-rich and epidermal growth factor-like domains. Proteins of approximately 120 and 103 kD, detectable by anti-ADAM33 antibodies, were expressed in 914-transfected HEK293 cells. The time-dependent appearance of the approximately 100-kD form and its inhibition by a peptidyl chloromethylketone, or the calcium ionophore, A23187, indicated that this was mature ADAM33, which was processed by a furin-like convertase. One form, approximately 110 kD, was detected in 906-transfected cell lysates. Trypsin and biotinylation treatment of transfected cells demonstrated that all of the mature approximately 100-kD, a minority of the approximately 120-kD pro-form, and none of the 906-expressed 110-kD form localized to the cell surface. The mature form was resistant to endoglycosidase H(f). The approximately 110-kD form was endoglycosidase H(f)-sensitive, indicating retention proximal to the trans-Golgi, consistent with a lack of maturation. Quantitation of transcripts demonstrated that those containing exon 17 predominate, whereas those lacking exon 17 are negligible in the mouse lung, although detectable at low levels in mouse testis, heart, and brain. Thus, potential dominant-negative effects exerted by the nonprocessed 906-encoded beta splice variant are unlikely to occur in mouse lung.

Human ADAM33 messenger RNA expression profile and post-transcriptional regulation.

We examined transcript expression and post-transcriptional regulation of human ADAM33, a recently identified asthma gene. A detailed messenger RNA (mRNA) expression profile was obtained using Northern, reverse transcription polymerase chain reaction, and in situ hybridization analyses. ADAM33 mRNA was expressed significantly in smooth muscle-containing organs, minimally in immune organs and hematopoietic cells, and highly in repairing duodenal granulation tissue. Expression was seen in asthmatic subepithelial fibroblasts and smooth muscle but not in respiratory epithelium. In all tissues, transcripts of approximately 5 kb predominated over those of approximately 3.5 kb by 2- to 5-fold. The effect of the 3' untranslated region (UTR) on ADAM33 protein expression and maturation was examined. The presence of the 3'UTR in untagged full-length constructs promoted prodomain removal, detected as mature approximately 100 kD protein by ADAM33-reactive antibodies; in its absence, maturation was 2- to 3-fold less in HEK293 cells. His-tagged and untagged constructs lacking the 3'UTR demonstrated that lack of maturation was not a result of tag-mediated effects. Minimal maturation of ADAM33 occurred in primary lung and MRC5 fibroblasts following adenoviral-mediated expression of ADAM33 lacking the 3'UTR. In contrast, prodomain removal was observed with plasmids and adenovirus encoding only the pro- and catalytic domains. Thus, the 3'UTR of ADAM33 and domains downstream of the catalytic domain regulate potential ADAM33 activity. Mechanisms of regulation of ADAM33, distinct from closely related ADAMs, thus include mRNA localization and processing and protein maturation.

Human ADAM33: protein maturation and localization.

ADAM33 (a disintegrin and metalloprotease) was recently found to be a novel asthma susceptibility gene. Domain-specific antibodies were used to study its expression and processing. When the pro-domain and catalytic domain were expressed by a stable-transfected cell line, the pro-domain was removed by cleavage within a putative furin cleavage site. The catalytic domain was active in an alpha(2)-macroglobulin complex formation assay and mutation of the catalytic site glutamic acid (E346A) eliminated activity. In transient transfections using the full-length protein, a pro-form and mature form were detectable and alternate glycosylation was demonstrated at sites within the catalytic domain. ADAM33 was detected on the cell surface, with the majority of protein detected intracellularly. The E346A mutation had no significant effect on protein processing. Endogenous ADAM33 was detected in bronchus tissue, bronchial smooth muscle cells, and MRC-5 fibroblasts, consistent with a role in the pathophysiology of asthma.

Identification of full, partial and inverse CC chemokine receptor 3 agonists using [35S]GTPgammaS binding.

Study of the CC chemokine receptor 3 (CCR3) has been limited to using radiolabeled agonist chemokines. A small molecule CCR3 antagonist, 2-[(6-amino-2-benzothiazolyl)thio]-N-[1-[(3,4-dichlorylphenyl)methyl]-4-piperidinyl]acetamide, Banyu (I), was tritiated and used for pharmacological studies. Banyu (I) has a K(d) of 5.0+/-0.4 and 4.3+/-1.8 nM on human CCR3 transfectants and eosinophils, and noncompetitively inhibits [125I]eotaxin binding and eotaxin-induced [35S]guanosine-5'-O-(3-thiotriphosphate) ([35S]GTPgammaS) binding. The proportion of [125I]eotaxin: [3H]Banyu (I) binding sites in eosinophils or transfectants was 35% or 13%, although both binding sites were overexpressed in transfectants. CCR3 spontaneously couples to G-proteins in CCR3 transfectants, demonstrated by changes in basal and eotaxin-induced [35S]GTPgammaS binding under reduced NaCl and GDP concentrations. Consequently, Banyu (I) was identified as an inverse agonist. In contrast, CCL18 and I-TAC (interferon-inducible T cell alpha-chemoattractant) were neutral antagonists, inhibiting eotaxin-induced [35S]GTPgammaS binding, with minimal effect on basal coupling of CCR3 to G proteins. Eotaxin, eotaxin-2 and monocyte chemoattractant protein (MCP)-4 are full agonists inducing [35S]GTPgammaS binding; eotaxin-3, MCP-3, RANTES (regulated on activation normal T cell expressed and secreted), vMIP-I (Kaposi's sarcoma-associated herpesvirus macrophage inflammatory protein-) and vMIP-II are partial agonists, indicating that this is a sensitive method to quantitate agonist efficacy.

Association of the ADAM33 gene with asthma and bronchial hyperresponsiveness.

Asthma is a common respiratory disorder characterized by recurrent episodes of coughing, wheezing and breathlessness. Although environmental factors such as allergen exposure are risk factors in the development of asthma, both twin and family studies point to a strong genetic component. To date, linkage studies have identified more than a dozen genomic regions linked to asthma. In this study, we performed a genome-wide scan on 460 Caucasian families and identified a locus on chromosome 20p13 that was linked to asthma (log(10) of the likelihood ratio (LOD), 2.94) and bronchial hyperresponsiveness (LOD, 3.93). A survey of 135 polymorphisms in 23 genes identified the ADAM33 gene as being significantly associated with asthma using case-control, transmission disequilibrium and haplotype analyses (P = 0.04 0.000003). ADAM proteins are membrane-anchored metalloproteases with diverse functions, which include the shedding of cell-surface proteins such as cytokines and cytokine receptors. The identification and characterization of ADAM33, a putative asthma susceptibility gene identified by positional cloning in an outbred population, should provide insights into the pathogenesis and natural history of this common disease.

Microarray profile of differentially expressed genes in a monkey model of allergic asthma.

BACKGROUND: Inhalation of Ascaris suum antigen by allergic monkeys causes an immediate bronchoconstriction and delayed allergic reaction, including a pulmonary inflammatory infiltrate. To identify genes involved in this process, the gene-expression pattern of allergic monkey lungs was profiled by microarrays. Monkeys were challenged by inhalation of A. suum antigen or given interleukin-4 (IL-4) treatment; lung tissue was collected at 4, 18 or 24 h after antigen challenge or 24 h after IL-4. Each challenged monkey lung was compared to a pool of normal, unchallenged monkey lungs. RESULTS: Of the approximately 40,000 cDNAs represented on the microarray, expression levels of 169 changed by more than 2.5-fold in at least one of the pairwise probe comparisons; these cDNAs encoded 149 genes, of which two thirds are known genes. The largest number of regulated genes was observed 4 h after challenge. Confirmation of differential expression in the original tissue was obtained for 95% of a set of these genes using real-time PCR. Cluster analysis revealed at least five groups of genes with unique expression patterns. One cluster contained genes for several chemokine mediators including eotaxin, PARC, MCP-1 and MCP-3. Genes involved in tissue remodeling and antioxidant responses were also identified as regulated by antigen and IL-4 or by antigen only. CONCLUSION: This study provides a large-scale profile of gene expression in the primate lung following allergen or IL-4 challenge. It shows that microarrays, with real-time PCR, are a powerful tool for identifying and validating differentially expressed genes in a disease model.

Review of the molecular and cellular mechanisms of action of glucocorticoids for use in asthma.

Asthma is characterized by inflammation in the lung and glucocorticoids (GCs) are the most clinically effective treatment available. The success of chronic GC therapy for asthma stems largely from the ability of the GC-GC receptor (GR) complex to alter transcription of a wide array of molecules involved in the inflammatory process. Many of the adverse effects of elevated systemic GC levels have been reduced through the use of inhalation as a method of administration, as opposed to oral GC. GCs exert their effects by binding to the wild-type GR, GR(alpha). The GR(alpha) complex can directly or indirectly alter gene transcription by binding to specific DNA sites or by activating transcription factors. There is also evidence to support GR(alpha) involvement in post-translational activities. In the management of asthma, the GR(alpha) down-regulates proinflammatory mediators such as interleukin-(IL)-1, 3, and 5, and up-regulates anti-inflammatory mediators such as IkappaB [inhibitory molecule for nuclear factor kappaB1 IL-10, and 12. Newer GCs are being designed to increase potency and topical activity. Mometasone furoate (MF), has recently been developed for the treatment of asthma and inhibits key anti-inflammatory processes with a potency equal to or greater than that of fluticasone propionate. A better understanding of the molecular mechanisms involved might provide strategies for optimizing the effectiveness of GC in the treatment of asthma.