A Novel Triazolopyridine-Based Spleen Tyrosine Kinase Inhibitor That Arrests Joint Inflammation.

Autoantibodies and the immunoreceptors to which they bind can contribute to the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA). Spleen Tyrosine Kinase (Syk) is a non-receptor tyrosine kinase with a central role in immunoreceptor (FcR) signaling and immune cell functionality. Syk kinase inhibitors have activity in antibody-dependent immune cell activation assays, in preclinical models of arthritis, and have progressed into clinical trials for RA and other autoimmune diseases. Here we describe the characterization of a novel triazolopyridine-based Syk kinase inhibitor, CC-509. This compound is a potent inhibitor of purified Syk enzyme, FcR-dependent and FcR-independent signaling in primary immune cells, and basophil activation in human whole blood. CC-509 is moderately selective across the kinome and against other non-kinase enzymes or receptors. Importantly, CC-509 was optimized away from and has modest activity against cellular KDR and Jak2, kinases that when inhibited in a preclinical and clinical setting may promote hypertension and neutropenia, respectively. In addition, CC-509 is orally bioavailable and displays dose-dependent efficacy in two rodent models of immune-inflammatory disease. In passive cutaneous anaphylaxis (PCA), CC-509 significantly inhibited skin edema. Moreover, CC-509 significantly reduced paw swelling and the tissue levels of pro-inflammatory cytokines RANTES and MIP-1α in the collagen-induced arthritis (CIA) model. In summary, CC-509 is a potent, moderately selective, and efficacious inhibitor of Syk that has a differentiated profile when compared to other Syk compounds that have progressed into the clinic for RA.

Crystal structures of PRK1 in complex with the clinical compounds lestaurtinib and tofacitinib reveal ligand induced conformational changes.

Protein kinase C related kinase 1 (PRK1) is a component of Rho-GTPase, androgen receptor, histone demethylase and histone deacetylase signaling pathways implicated in prostate and ovarian cancer. Herein we describe the crystal structure of PRK1 in apo form, and also in complex with a panel of literature inhibitors including the clinical candidates lestaurtinib and tofacitinib, as well as the staurosporine analog Ro-31-8220. PRK1 is a member of the AGC-kinase class, and as such exhibits the characteristic regulatory sequence at the C-terminus of the catalytic domain--the 'C-tail'. The C-tail fully encircles the catalytic domain placing a phenylalanine in the ATP-binding site. Our inhibitor structures include examples of molecules which both interact with, and displace the C-tail from the active site. This information may assist in the design of inhibitors targeting both PRK and other members of the AGC kinase family.

Measuring cereblon as a biomarker of response or resistance to lenalidomide and pomalidomide requires use of standardized reagents and understanding of gene complexity.

Cereblon, a member of the cullin 4 ring ligase complex (CRL4), is the molecular target of the immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide and is required for the antiproliferative activity of these agents in multiple myeloma (MM) and immunomodulatory activity in T cells. Cereblon's central role as a target of lenalidomide and pomalidomide suggests potential utility as a predictive biomarker of response or resistance to IMiD therapy. Our studies characterized a cereblon monoclonal antibody CRBN65, with high sensitivity and specificity in Western analysis and immunohistochemistry that is superior to commercially available antibodies. We identified multiple cereblon splice variants in both MM cell lines and primary cells, highlighting challenges with conventional gene expression assays given this gene complexity. Using CRBN65 antibody and TaqMan quantitative reverse transcription polymerase chain reaction assays, we showed lack of correlation between cereblon protein and mRNA levels. Furthermore, lack of correlation between cereblon expression in MM cell lines and sensitivity to lenalidomide was shown. In cell lines made resistant to lenalidomide and pomalidomide, cereblon protein is greatly reduced. These studies show limitations to the current approaches of cereblon measurement that rely on commercial reagents and assays. Standardized reagents and validated assays are needed to accurately assess the role of cereblon as a predictive biomarker.

Chemical and genetic wrappers for improved phage and RNA display.

An Achilles heel inherent to all molecular display formats, background binding between target and display system introduces false positives into screens and selections. For example, the negatively charged surfaces of phage, mRNA, and ribosome display systems bind with unacceptably high nonspecificity to positively charged target molecules, which represent an estimated 35% of proteins in the human proteome. Here we report the first systematic attempt to understand why a broad class of molecular display selections fail, and then solve the underlying problem for both phage and RNA display. Firstly, a genetic strategy was used to introduce a short, charge-neutralizing peptide into the solvent-exposed, negatively charged phage coat. The modified phage (KO7(+)) reduced or eliminated nonspecific binding to the problematic high-pI proteins. In the second, chemical approach, nonspecific interactions were blocked by oligolysine wrappers in the cases of phage and total RNA. For phage display applications, the peptides Lys(n) (where n=16 to 24) emerged as optimal for wrapping the phage. Lys(8), however, provided effective wrappers for RNA binding in assays against the RNA binding protein HIV-1 Vif. The oligolysine peptides blocked nonspecific binding to allow successful selections, screens, and assays with five previously unworkable protein targets.

Double barrel shotgun scanning of the caveolin-1 scaffolding domain.

In the postgenomic era, a major challenge remains, elucidating the thermodynamic forces governing receptor-ligand specificity and promiscuity. We report a straightforward approach for mapping side-chain contributions to binding for the multipartner interactions characteristic of the human proteome. Double barrel shotgun scanning dissects binding to two or more targets through combinatorial mutagenesis of one protein binding to multiple targets. Examined here, the caveolin-1 scaffolding domain (CSD) binds to and inhibits both endothelial nitric oxide synthase (eNOS) and protein kinase A (PKA). Homolog shotgun scanning of CSD highlights residues responsible for CSD oligomerization and binding to eNOS and PKA. The experiments uncover a general mechanism in which CSD oligomerizes and deoligomerizes to modulate binding affinity to partner proteins. The results provide a detailed look at a multipartner protein interaction, uncovering strategies for one protein binding to multiple partners.

Structural and molecular evolutionary analysis of Agouti and Agouti-related proteins.

Agouti (ASIP) and Agouti-related protein (AgRP) are endogenous antagonists of melanocortin receptors that play critical roles in the regulation of pigmentation and energy balance, respectively, and which arose from a common ancestral gene early in vertebrate evolution. The N-terminal domain of ASIP facilitates antagonism by binding to an accessory receptor, but here we show that the N-terminal domain of AgRP has the opposite effect and acts as a prodomain that negatively regulates antagonist function. Computational analysis reveals similar patterns of evolutionary constraint in the ASIP and AgRP C-terminal domains, but fundamental differences between the N-terminal domains. These studies shed light on the relationships between regulation of pigmentation and body weight, and they illustrate how evolutionary structure function analysis can reveal both unique and common mechanisms of action for paralogous gene products.

Melanoma prevention strategy based on using tetrapeptide alpha-MSH analogs that protect human melanocytes from UV-induced DNA damage and cytotoxicity.

Melanoma is the deadliest form of skin cancer, with no cure for advanced disease. We propose a strategy for melanoma prevention based on using analogs of alpha-melanocyte stimulating hormone (alpha-MSH) that function as melanocortin 1 receptor (MC1R) agonists. Treatment of human melanocytes with alpha-MSH results in stimulation of eumelanin synthesis, reduction of apoptosis that is attributable to reduced hydrogen peroxide generation and enhanced repair of DNA photoproducts. These effects should contribute to genomic stability of human melanocytes, thus preventing their malignant transformation to melanoma. Based on these findings, we synthesized and tested the effects of 3 tetrapeptide alpha-MSH analogs, Ac-His-D-Phe-Arg-Trp-NH2, n-Pentadecanoyl- and 4-Phenylbutyryl-His-D-Phe-Arg-Trp-NH2, on cultured human melanocytes. The latter two analogs were more potent than the former, or alpha-MSH, in stimulating the activity of tyrosinase, thus melanogenesis, reducing apoptosis and release of hydrogen peroxide and enhancing repair of DNA photoproducts in melanocytes exposed to UV radiation (UVR). The above analogs are MC1R agonists, as their effects were abrogated by an analog of agouti signaling protein, the physiological MC1R antagonist, and were absent in melanocytes expressing loss-of-function MC1R. Analogs, such as 4-Phenylbutyryl-His-D-Phe-Arg-Trp-NH2 with prolonged and reversible effects, can potentially be developed into topical agents to prevent skin photocarcinogenesis, particularly melanoma.

The octarepeat domain of the prion protein binds Cu(II) with three distinct coordination modes at pH 7.4.

The prion protein (PrP) binds Cu2+ in its N-terminal octarepeat domain. This unusual domain is comprised of four or more tandem repeats of the fundamental sequence PHGGGWGQ. Previous work from our laboratories demonstrates that at full copper occupancy, each HGGGW segment binds a single Cu2+. However, several recent studies suggest that low copper occupancy favors different coordination modes, possibly involving imidazoles from histidines in adjacent octapeptide segments. This is investigated here using a combination of X-band EPR, S-band EPR, and ESEEM, along with a library of modified peptides designed to favor different coordination interactions. At pH 7.4, three distinct coordination modes are identified. Each mode is fully characterized to reveal a series of copper-dependent octarepeat domain structures. Multiple His coordination is clearly identified at low copper stoichiometry. In addition, EPR detected copper-copper interactions at full occupancy suggest that the octarepeat domain partially collapses, perhaps stabilizing this specific binding mode and facilitating cooperative copper uptake. This work provides the first complete characterization of all dominant copper coordination modes at pH 7.4.

Chimeras of the agouti-related protein: insights into agonist and antagonist selectivity of melanocortin receptors.

The specific melanocortin receptors, MC3R and MC4R, are directly linked to metabolism and body weight control. These receptors are activated by the peptide hormone alpha-MSH and antagonized by the agouti-related protein (AGRP). Whereas alpha-MSH acts broadly on most members of the MCR family (with the exception of MC2R), AGRP is highly specific for only MC3R and MC4R. AGRP is a complex ligand of approximately 100 amino acids. Within AGRP, MCR recognition and antagonism is localized to a 34 residue, cysteine-rich domain that adopts an inhibitor cystine knot (ICK) fold. An oxidatively folded peptide corresponding to this domain, referred to as mini-AGRP, exhibits full antagonist function and selectivity for MC3R and MC4R. Here we investigate a series of chimera proteins based on the mini-AGRP scaffold. Amino acid sequences derived from peptide agonists are grafted into the mini-AGRP active loop, implicated in receptor recognition, with the goal of producing ICK based agonists specific for MC3R and MC4R. Several constructs indeed exhibited potent agonist activity; however, with all chimeras, receptor selectivity is significantly altered. Pharmacologic data indicate that the chimeras do not interact with MC receptors through native AGRP like contacts. A model to explain the data suggest that there is only partial overlap of the agonist versus antagonist binding surfaces within MC receptors. Moreover, accessibility to the binding pocket is highly receptor specific with MC3R being the least tolerant of ligand alterations.

Functional analysis of the Ala67Thr polymorphism in agouti related protein associated with anorexia nervosa and leanness.

AgRP is a neuropeptide that stimulates food intake through inhibition of central melanocortin receptors (MCRs). In humans, the non-conservative amino acid substitution Alanine (Ala) 67 Threonine (Thr) has been associated with Anorexia Nervosa and with leanness. In the present study, the cellular distribution, processing and in vitro and in vivo activities of Ala67 and Thr67 AgRP were investigated. Western blots of media and lysates of BHK cells stably transfected with Ala67 or Thr67 expression constructs showed identical AgRP bands. Both Ala67 and Thr67 AgRP colocalised with the Golgi apparatus, but not with the ER or lysosomes when expressed in Att20 D16V cells. Also, no differences were observed between the potencies of bacterially expressed Ala67 and Thr67 AgRP to stimulate MC4R in a reporter gene assay or inhibit food intake in rats. Taken together, no evidence was found for a functional defect of Thr67 AgRP related to MC4R interactions.

Structures of the agouti signaling protein.

Expression of the agouti signaling protein (ASIP) during hair growth produces the red/yellow pigment pheomelanin. ASIP, and its neuropeptide homolog the agouti-related protein (AgRP) involved in energy balance, are novel, paracrine signaling molecules that act as inverse agonists at distinct subsets of melanocortin receptors. Ubiquitous ASIP expression in mice gives rise to a pleiotropic phenotype characterized by a uniform yellow coat color, obesity, overgrowth, and metabolic derangements similar to type II diabetes in humans. Here we report the synthesis and NMR structure of ASIP's active, cysteine-rich, C-terminal domain. ASIP adopts the inhibitor cystine knot fold and, along with AgRP, are the only known mammalian proteins in this structure class. Moreover, ASIP populates two distinct conformers resulting from a cis peptide bond at Pro102-Pro103 and a coexistence of cis/trans isomers of Ala104-Pro105. Pharmacologic studies of Pro-->Ala mutants demonstrate that the minor conformation with two cis peptide bonds is responsible for activity at all MCRs. The loop containing the heterogeneous Ala-Pro peptide bond is conserved in mammals, and suggests that ASIP is either trapped by evolution in this unusual configuration or possesses function outside of strict MCR antagonism.

Loops and links: structural insights into the remarkable function of the agouti-related protein.

The agouti-related protein (AGRP) is an endogenous antagonist of the melanocortin receptors MC3R and MC4R found in the hypothalamus and exhibits potent orexigenic activity. The cysteine-rich C-terminal domain of this protein, corresponding to AGRP(87-132), exhibits receptor binding affinity and antagonism equivalent to that of the full-length protein. We recently determined the NMR structure of AGRP(87-132) and demonstrated that a portion of the domain adopts the inhibitor cystine-knot fold. Remarkably, this is the first identification of a mammalian protein with this specific architecture. Further analysis of the structure suggests that melanocortin receptor contacts are made primarily by two loops presented within the cystine knot. (10) To test this hypothesis we designed a 34-residue AGRP analogue corresponding to only the cystine knot. We found that this designed miniprotein folds to a homogeneous product, retains the desired cystine-knot architecture, functions as a potent antagonist, and maintains the melanocortin receptor pharmacological profile of AGRP(87-132). (26) The AGRP-like activity of this molecule supports the hypothesis that indeed the cystine-knot region possesses the melanocortin receptor contacts. Based on these design and structure studies, we propose that the N-terminal loop of AGRP(87-132) makes contact with a receptor exoloop and helps confer AGRP's selectivity for the central MCRs.

Inverse agonist activity of agouti and agouti-related protein.

Agouti and agouti-related protein (AgRP) are endogenous antagonists of the melanocortin receptors (MCxR). Previous data showed that recombinant full-length agouti and a synthetic fragment of AgRP, AgRP (83-132), are inverse agonists at the MC1R and MC4R, respectively. This study demonstrates the smaller analogs AgRP (87-120) and ASIP [90-132 (L89Y)], and short peptides Yc[CRFFNAFC]Y and Qc[CRFFRSAC]S are also MC4R inverse agonists. Furthermore, the relative affinity of the series of MC4R ligands for displacement of radiolabeled antagonist 125I-AgRP (86-132) versus radiolabeled agonist 125I-NDP-MSH did not correlate with ligand efficacy, which is more consistent with an induced-fit model than a simple two-state model of MC4R activation. These data shed new light on the determinants and mechanism of inverse agonism at the MC4R.

Design, pharmacology, and NMR structure of a minimized cystine knot with agouti-related protein activity.

The agouti-related protein (AGRP) is an endogenous antagonist of the melanocortin receptors MC3R and MC4R found in the hypothalamus and exhibits potent orexigenic activity. The cysteine-rich C-terminal domain of this protein, corresponding to AGRP(87-132), exhibits receptor binding affinity and antagonism equivalent to that of the full-length protein. The NMR structure of this active domain was recently determined and suggested that melanocortin receptor contacts were made primarily by two loops presented by a well-structured cystine knot domain within AGRP(87-132) [McNulty et al. (2001) Biochemistry 40, 15520-15527]. This hypothesis is tested here with NMR structure and activity studies of a 34-residue AGRP analogue designed to contain only the cystine knot domain. The designed miniprotein folds to a homogeneous product, retains the desired cystine knot architecture, functions as an antagonist, and maintains the melanocortin receptor pharmacological profile of AGRP(87-132). The AGRP-like activity of this molecule supports the hypothesis that indeed the cystine knot region possesses the melanocortin receptor contact points. Moreover, this potent AGRP analogue is synthetically accessible, may serve in the development of therapeutics for the treatment of diseases related to energy balance. and may also find use as a new reagent for probing melanocortin receptor structure and function.