Identification of a High-Affinity Phosphopeptide Inhibitor of Stat3 Zhiyong Ren, a Larry A. Cabell, b Timothy S. Schaefer a,y and John S. McMurray b, * a Department of Neuro-Surgery, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA b Department of Neuro-Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA Received 27 September 2002; accepted 25 November 2002 Abstract—Stat3 is a latent transcription factor that exhibits elevated activity in a variety of human cancers. To find a lead peptide for peptidomimetic drug development we synthesized and tested phosphopeptides derived from known receptor docking sites and found Y(p)LPQTV as the optimal sequence. SAR studies showed that each residue from pY to pY+3 provided binding energy. # 2003 Elsevier Science Ltd. All rights reserved. Stat3 (Signal Transduction and Activator of Transcrip- tion 3) is a member of the STAT family of latent, cyto- solic transcription factors that directly relate signals from the plasma membrane to the nucleus. 1,2 Stat3 mediates IL-6 signaling and has been shown to be con- stitutively activated in multiple myeloma and cancers of the head and neck, breast, prostate, and brain (reviewed in ref 3). Downstream targets of Stat3 include bcl-x L ,a member of the bcl-2 family of anti-apoptotic proteins, cell cycle regulators such as cyclin D1 and p21 WAF1/CIP1 , and other transcription factors including c-myc and c-fos. 1,3,4 Stat3 is recruited to phosphorylated receptors via its SH2 domain. It then becomes phosphorylated on Tyr 705 by JAK kinases, Src, Abl, or the kinase activity of the receptor. Upon tyrosine phosphorylation, Stat3 forms a dimer in which the SH2 domain of one protein molecule binds to the pTyr 705 residue of the other and vice versa. The dimer migrates to the nucleus, binds to specific DNA sequences and initiates transcription. As mentioned above, aberrantly activated Stat3 has been observed in a variety of tumors and is an attractive tar- get for therapeutic intervention. To this end, we have embarked on a program to impede Stat3 signaling by inhibiting receptor docking and/or dimer formation by the use of phosphotyrosine-based peptidomimetics targeted to the SH2 domain. To date we are aware of only one publication concerning the development of Stat3 inhibitors. 5 As part of its role in signaling, Stat3 has been shown to be recruited to phosphotyrosine residues on gp130, 6 leukemia inhibitory factor receptor (LIFR), 6 the epi- dermal growth factor receptor (EGFR), 7 interleukin 10 receptor (IL-10R), 8 and granulocyte colony stimulating factor receptor (G-CSFR), 9 presumably by binding via its SH2 domain. Phosphopeptides derived from putative binding sites were able to inhibit Stat3 binding to these receptors in cell free systems. 6 9 It was noted early on that in these receptors a consensus sequence for recognition was YXXQ. 6 Although phosphopeptides derived from Stat3 Tyr 705 have been assayed for their ability to inhibit dimeriza- tion and DNA binding by electrophoretic mobility shift assays (EMSAs), 5,10 to date, no reliable information is available on the affinity of this protein for the amino acid sequences surrounding the phosphotyrosine dock- ing sites of receptors. Haan et al. 11 assayed several phosphopeptides based on gp130 and LIFR as well as the sequence surrounding Stat3 Tyr 705 for binding to the isolated SH2 domain from Stat3. At pH 7.5 no complex formation was observed whereas binding occurred at pH 5.5. It was noted that at the higher pH the SH2 domain existed in a dimerized state, which appears to have precluded phosphopeptide binding. 0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0960-894X(02)01050-8 Bioorganic & Medicinal Chemistry Letters 13 (2003) 633–636 *Corresponding author. Tel.:+1-713-745-3763; fax:+1-713-745-1183; e-mail: jsmcmur@mdanderson.org y Current address: Meso Scale Discovery, 9238 Gaither Rd, Gaithers- burg, MD 20877, USA.