Pilgrim J. Jackson

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.

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.

Summary: 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.

Thalidomide and the immunomodulatory drug, lenalidomide, are therapeutically active in hematological malignancies. The ubiquitously expressed E3 ligase protein cereblon (CRBN) has been identified as the primary teratogenic target of thalidomide. Our studies demonstrate that thalidomide, lenalidomide and another immunomodulatory drug, pomalidomide, bound endogenous CRBN and recombinant CRBN–DNA damage binding protein-1 (DDB1) complexes. CRBN mediated antiproliferative activities of lenalidomide and pomalidomide in myeloma cells, as well as lenalidomide- and pomalidomide-induced cytokine production in T cells. Lenalidomide and pomalidomide inhibited autoubiquitination of CRBN in HEK293T cells expressing thalidomide-binding competent wild-type CRBN, but not thalidomide-binding defective CRBN^YW/AA. Overexpression of CRBN wild-type protein, but not CRBN^YW/AA mutant protein, in KMS12 myeloma cells, amplified pomalidomide-mediated reductions in c-myc and IRF4 expression and increases in p21^WAF-1 expression. Long-term selection for lenalidomide resistance in H929 myeloma cell lines was accompanied by a reduction in CRBN, while in DF15R myeloma cells resistant to both pomalidomide and lenalidomide, CRBN protein was undetectable. Our biophysical, biochemical and gene silencing studies show that CRBN is a proximate, therapeutically important molecular target of lenalidomide and pomalidomide.

Abstract 738 Thalidomide, lenalidomide and pomalidomide are therapeutically active in a number of hematological malignant and premalignant conditions including myelodysplastic syndromes, multiple myeloma, and lymphomas. Clinical efficacy is ascribed to a complement of overlapping activities including direct antitumor effects, immune system activation and inhibition of stromal support of tumor growth. Thalidomide has previously been shown to bind cereblon (CRBN) a protein required for the teratogenic effects of thalidomide in zebrafish and chicken embryos (Ito et al). CRBN forms an ubiquitin E3 ligase complex with DNA damage-binding protein 1 (DDB1), cullin 4 (CUL4) and protein Rbx1 and thalidomide treatment has been shown to inhibit the ubiquitin ligase activity of the complex (Ito et al). Using two independent biophysical methods, we demonstrate that lenalidomide and pomalidomide bind to CRBN-DDB1 complex. Fluorescence-based thermal shift analysis was carried out using purified ZZ-CRBN-DDB1. Phthalimide showed no appreciable binding to the CRBN-DDB1 complex while dose-dependent interaction of thalidomide, lenalidomide and pomalidomide was observed. Thalidomide binding showed approximately ten-fold less affinity (∼ 30 μM) than lenalidomide and pomalidomide (each ∼ 3 μM IC₅₀). Following the procedure of Ito et al, we used thalidomide analog-coupled beads (Thal-beads) and demonstrated binding of CRBN in complex with DDB1 from U266B1 myeloma cell extracts. Preincubation of the extracts with lenalidomide (100 μM) prevented CRBN binding to Thal-beads. Furthermore, the binding of CRBN was dose-dependently inhibited by preincubation with either lenalidomide or pomalidomide with IC₅₀s of 2.3 and 2.1 μM, respectively. We then investigated whether CRBN was required for lenalidomide and pomalidomide responses associated with efficacy. First, CRBN expression was reduced in activated human T cells using CRBN siRNAs. After T cell activation, incubation with lenalidomide (1 μM) or pomalidomide (1 μM) resulted in an 11 to 14- fold-increase in IL-2 and a 5 to 10-fold increase in TNF-α. This increase was reduced ∼60% in the presence of siCRBN. Since IL-2 and TNF-α are important cytokines for tumor surveillance by activated T cells, our results indicate that some of the immunomodulatory effects of lenalidomide and pomalidomide are mediated via CRBN. We next determined if CRBN was required for the antiproliferative effect of lenalidomide and pomalidomide in myeloma cells. Multiple siRNAs were used to silence the expression of CRBN in U266B1 cells resulting in the absence of CRBN protein as determined by immunoblot analysis. Propidium-iodide staining showed that depletion of CRBN affected neither cell cycle nor proliferation of U266B1 cells. However, knockdown of CRBN markedly abrogated lenalidomide- and pomalidomide-induced delay of cell cycle progression. In addition, using lentiviral vectors we produced U266B1 cell lines with either 60% or 75% less expression of CRBN and showed that relative to the parental cell line these cells were gene dose-dependently less responsive to inhibition of proliferation by lenalidomide. The U266B1 cells in which CRBN had been decreased were also less responsive to inhibition by pomalidomide, but this compound maintained greater inhibition of proliferation than lenalidomide in the context of decreasing CRBN. Moreover, gene profile changes by lenalidomide and pomalidomide were reversed in the presence of siCRBN. In particular, induction of p21^WAF1 cyclin –dependent kinase inhibitor protein was prevented in the absence of the expression of CRBN. Similar results on different myeloma cell lines and using multiple CRBN siRNAs, confirmed the same critical role of CRBN in the antiproliferative response to lenalidomide and pomalidomide of myeloma cells. Finally, we demonstrated that CRBN expression decreases concomitantly with the acquisition of lenalidomide resistance in H929 myeloma cells. Lenalidomide-resistant H929 cells remain responsive to inhibition by pomalidomide despite relatively reduced expression of CRBN. However, in pomalidomide-resistant DF15R myeloma cells, the complete absence of CRBN confers resistance to both lenalidomide and pomalidomide. Our data demonstrate that CRBN is a direct target of lenalidomide and pomalidomide and plays a crucial role in the antitumor efficacy of lenalidomide and pomalidomide.

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. Lys₈, 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.

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.

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 α‐melanocyte stimulating hormone (α‐MSH) that function as melanocortin 1 receptor (MC1R) agonists. Treatment of human melanocytes with α‐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 α‐MSH analogs, Ac‐His‐D‐Phe‐Arg‐Trp‐NH ₂ , 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 α‐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‐NH ₂ with prolonged and reversible effects, can potentially be developed into topical agents to prevent skin photocarcinogenesis, particularly melanoma.—Abdel‐Malek, Z. A., Kadekaro, A. L., Kavanagh, R. J., Todorovic, A., Koikov, L. N., McNulty, J. C., Jackson, P. J., Millhauser, G. L., Schwemberger, S., Babcock, G., Haskell‐Luevano, C., Knittel, J. J. Melanoma prevention strategy based on using tetrapeptide α‐MSH analogs that protect human melanocytes from UV‐induced DNA damage and cytotoxicity. FASEB J. 20, E888–E896 (2006)