Assaf Wool

Discovery and Validation of Novel Peptide Agonists for G-protein-coupled Receptors

G-protein-coupled receptors (GPCRs) represent an important group of targets for pharmaceutical therapeutics. The completion of the human genome revealed a large number of putative GPCRs. However, the identification of their natural ligands, and especially peptides, suffers from low discovery rates, thus impeding development of therapeutics based on these potential drug targets. We describe the discovery of novel GPCR ligands encrypted in the human proteome. Hundreds of potential peptide ligands were predicted by machine learning algorithms. In vitro screening of selected 33 peptides on a set of 152 GPCRs, including a group of designated orphan receptors, was conducted by intracellular calcium measurements and cAMP assays. The screening revealed eight novel peptides as potential agonists that specifically activated six different receptors in a dose-dependent manner. Most of the peptides showed distinct stimulatory patterns targeted at designated and orphan GPCRs. Further analysis demonstrated a significant in vivo effect for one of the peptides in a mouse inflammation model.

Prevention of Bleomycin-Induced Pulmonary Fibrosis by a Novel Antifibrotic Peptide with Relaxin-Like Activity

Pulmonary fibrosis is a progressive and lethal lung disease characterized by accumulation of extracellular matrix and loss of pulmonary function. No cure exists for this pathologic condition, and current treatments often fail to slow its progression or relieve its symptoms. Relaxin was previously shown to induce a matrix-degrading phenotype in human lung fibroblasts in vitro and to inhibit pulmonary fibrosis in vivo. A novel peptide that targets the relaxin RXFP1/LGR7 receptor was recently identified using our computational platform designed to predict novel G protein-coupled receptor peptide agonists. In this study, we examined the antifibrotic properties of this novel peptide, designated CGEN25009, in human cell-based assays and in a murine model of bleomycin-induced pulmonary fibrosis. Similar to relaxin, CGEN25009 was found to have an inhibitory effect on transforming growth factor-β1-induced collagen deposition in human dermal fibroblasts and to enhance MMP-2 expression. The peptides biological activity was also similar to relaxin in generating cellular stimulation of cAMP, cGMP, and NO in the THP-1 human cell line. In vivo, 2-week administration of CGEN25009 in a preventive or therapeutic mode (i.e., concurrently with or 7 days after bleomycin treatment, respectively) caused a significant reduction in lung inflammation and injury and ameliorated adverse airway remodeling and peribronchial fibrosis. The results of this study indicate that CGEN25009 displays antifibrotic and anti-inflammatory properties and may offer a new therapeutic option for the treatment of pulmonary fibrosis.

Peptides modulating conformational changes in secreted chaperones: From in silico design to preclinical proof of concept

Blocking conformational changes in biologically active proteins holds therapeutic promise. Inspired by the susceptibility of viral entry to inhibition by synthetic peptides that block the formation of helix–helix interactions in viral envelope proteins, we developed a computational approach for predicting interacting helices. Using this approach, which combines correlated mutations analysis and Fourier transform, we designed peptides that target gp96 and clusterin, 2 secreted chaperones known to shift between inactive and active conformations. In human blood mononuclear cells, the gp96-derived peptide inhibited the production of TNFα, IL-1β, IL-6, and IL-8 induced by endotoxin by >80%. When injected into mice, the peptide reduced circulating levels of endotoxin-induced TNFα, IL-6, and IFNγ by >50%. The clusterin-derived peptide arrested proliferation of several neoplastic cell lines, and significantly enhanced the cytostatic activity of taxol in vitro and in a xenograft model of lung cancer. Also, the predicted mode of action of the active peptides was experimentally verified. Both peptides bound to their parent proteins, and their biological activity was abolished in the presence of the peptides corresponding to the counterpart helices. These data demonstrate a previously uncharacterized method for rational design of protein antagonists.

Activation of Relaxin‐Related Receptors by Short, Linear Peptides Derived from a Collagen‐Containing Precursor

In a screening effort based on algorithmic predictions for novel G‐protein‐coupled receptor (GPCR) peptide activators, we were able to identify and examine two novel peptides (P59 and P74) which are short, linear, and derived from a natural, previously unidentified precursor protein containing a collagen‐like repeat. Both peptides seemed to show an apparent cAMP‐related effect on CHO‐K1 cells transiently transfected with either LGR7 or LGR8, usually after treatment with cAMP‐generating forskolin, compared to the same cells treated with forskolin plus relaxin. This activation was not found for the relaxin‐3 receptor (GPR135). In a set of follow‐up experiments, both peptides were found to stimulate cAMP production, mostly upon initial stimulation of cAMP production by 5 μM forskolin in cells transfected with either LGR7 or LGR8. In a dye‐free cell impedance GPCR activation assay, we were able to show that these peptides were also able to activate a cellular response mediated by these receptors. Although untransfected CHO‐K1 cells showed some cellular activation by both relaxin and at least one of our newly discovered peptides, both LGR7‐ and LGR8‐transfected cells showed a stronger response, indicating stimulation of a cellular pathway through activation of these receptors. In conclusion, we were able to show that these newly discovered peptides, which have no similarity to any member of the relaxin–insulin‐like peptide family, are potential ligands for the relaxin‐related family of receptors and as such might serve as novel candidates for relaxin‐related therapeutic indications. Both peptides are linear and were found to be active after being chemically synthesized.

CD6 and Syntaxin Binding Protein 6 Variants and Response to Tumor Necrosis Factor Alpha Inhibitors in Danish Patients with Rheumatoid Arthritis

Background TNFα inhibitor therapy has greatly improved the treatment of patients with rheumatoid arthritis, however at least 30% do not respond. We aimed to investigate insertions and deletions (INDELS) associated with response to TNFα inhibitors in patients with rheumatoid arthritis (RA). Methodology and Principal Findings In the DANBIO Registry we identified 237 TNFα inhibitor naïve patients with RA (81% women; median age 56 years; disease duration 6 years) who initiated treatment with infliximab (n = 160), adalimumab (n = 56) or etanercept (n = 21) between 1999 and 2008 according to national treatment guidelines. Clinical response was assessed at week 26 using EULAR response criteria. Based on literature, we selected 213 INDELS potentially related to RA and treatment response using the GeneVa® (Compugen) in silico database of 350,000 genetic variations in the human genome. Genomic segments were amplified by polymerase chain reaction (PCR), and genotyped by Sanger sequencing or fragment analysis. We tested the association between genotypes and EULAR good response versus no response, and EULAR good response versus moderate/no response using Fisher’s exact test. At baseline the median DAS28 was 5.1. At week 26, 68 (29%) patients were EULAR good responders, while 81 (34%) and 88 (37%) patients were moderate and non-responders, respectively. A 19 base pair insertion within the CD6 gene was associated with EULAR good response vs. no response (OR = 4.43, 95% CI: 1.99–10.09, p = 7.211×10−5) and with EULAR good response vs. moderate/no response (OR = 4.54, 95% CI: 2.29–8.99, p = 3.336×10−6). A microsatellite within the syntaxin binding protein 6 (STXBP6) was associated with EULAR good response vs. no response (OR = 4.01, 95% CI: 1.92–8.49, p = 5.067×10−5). Conclusion Genetic variations within CD6 and STXBP6 may influence response to TNFα inhibitors in patients with RA.

Peptides modulating conformational changes: From in silico design to in vivo proof of concept

From in-Silico Design to in-Vivo Proof of Concept Yossef Kliger, Ofer Levy, Anat Oren, Haim Ashkenazy, Zohar Tiran, Amit Novik, Avi Rosenberg, Anat Amir, Assaf Wool, Amir Toporik, Ehud Schreiber, Dani Eshel, Zurit Levine, Yossi Cohen, Claudia Nold-Petry, Charles A. Dinarello, Itamar Borukhov 1Compugen Ltd. Tel Aviv 69512, Israel. (www.cgen.com) *Correspondence: kliger@cgen.com, itamarb@cgen.com 2The Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat Gan, Israel 3University of Colorado Denver, Aurora, CO 80045, USA