Journal Name Cite this: DOI: 10.1039/c0xx00000x www.rsc.org/xxxxxx Dynamic Article Links ► ARTICLE TYPE This journal is © The Royal Society of Chemistry [year] [journal], [year], [vol], 00–00 | 1 Structure-activity relationship studies of the aromatic positions in cyclopentapeptide CXCR4 antagonists† Jignesh Mungalpara,‡ a Zack G. Zachariassen,‡ a Stefanie Thiele, b Mette M. Rosenkilde, b and Jon Våbenø* a Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX 5 DOI: 10.1039/b000000x The cyclopentapeptide CXCR4 antagonist FC131 (cyclo(-Arg 1 -Arg 2 -2-Nal 3 -Gly 4 -D-Tyr 5 -), 2; 2-Nal = 3- (2-naphthyl)alanine) represents an excellent starting point for development of novel drug-like ligands with therapeutic potential in HIV, cancer, stem-cell mobilization, inflammation, and autoimmune diseases. While the structure-activity relationships for Arg 1 , Arg 2 , and Gly 4 are well established, less is 10 understood about the roles of the aromatic residues 2-Nal 3 and D-Tyr 5 . Here we report further structure- activity relationship studies of these two positions, which showed that (i) the distal aromatic ring of the 2- Nal 3 side chain is required in order to maintain high potency, and (ii) replacement of D-Tyr 5 with conformationally constrained analogues results in significantly reduced activity. However, a simplified analogue that contained Gly instead of D-Tyr 5 was only 13-fold less potent than 2, which means that the 15 D-Tyr 5 side chain is dispensable. These findings were rationalized based on molecular docking, and the collective structure-activity data for the cyclopentapeptides suggest that appropriately designed Arg 2 -2- Nal 3 dipeptidomimetics have potential as CXCR4 antagonists. Introduction By now, the role of the G protein-coupled C-X-C chemokine 20 receptor 4 (CXCR4) in HIV, cancer, stem-cell mobilization, inflammation, and autoimmune diseases is well established, 1 and several different antagonists for CXCR4 – both peptides and non- peptides – have been described in the literature over the last two decades. 2 The prototype non-peptide antagonist plerixafor 25 (AMD3100), which is administered by subcutaneous injection, was approved for stem-cell mobilization in 2008 and is currently the only marketed CXCR4 antagonist. The molecular pharmacology of AMD3100 3, 4 and the structurally related non- peptide antagonists AMD3465 5 and AMD11070 6 has been 30 extensively characterized. 7 The majority of the reported peptide antagonists has been developed by Fujii and co-workers, starting from the 18-mer synthetic polyphemusin II analogue T22. 8 Extensive structure- activity relationship (SAR) and downsizing studies first led to the 35 potent 14-mer antagonist T140 (1, Fig. 1), 9 and eventually to the discovery of the cyclopentapeptide FC131 (2, Fig. 1), 10 which we are currently using as lead compound for our ongoing efforts toward peptidomimetic CXCR4 antagonists. A 16-mer analogue of 1 that contains two additional C-terminal residues (CVX15, 3, 40 Fig. 1) was recently reported by Wu et al. as the ligand in an X- ray co-crystal structure of CXCR4. 11 Interestingly, the potent peptide antagonists 1–3 share an Arg 1 -Arg 2 -(n-Nal) 3 -Xaa 4 -L-/D- Tyr 5 pentapeptide motif (Fig. 1), a notable difference being that 1 and 2 both contain 3-(2-naphthyl)alanine (2-Nal) in position 3 45 while 3 contains the isomeric 3-(1-naphthyl)alanine (1-Nal). Arg 1 -Arg 2 -(2-Nal) 3 -Cys 4 -Tyr 5 -Arg 6 -Lys 7 -D-Lys 8 Arg 14 -Cys 13 -Cit 12 -Arg 11 -Tyr 10 -Pro 9 Arg 1 -Arg 2 -(1-Nal) 3 -Cys 4 -Tyr 5 -Gln 6 -Lys 7 -D-Pro 8 D-Pro 16 -Gly 15 -Arg 14 -Cys 13 -Cit 12 -Arg 11 -Tyr 10 -Pro 9 1 (T140) 3 (CVX15) 2 (FC131) N H N HN N NH O O O HN HN NH 2 NH H 2 N NH OH Gly 4 Arg 2 2-Nal 3 D-Tyr 5 Arg 1 O O Position 3 Position 5 H H Figure 1 Structure of the lead cyclopentapeptide CXCR4 antagonist FC131 (2) and sequences of the larger peptide antagonists T140 (1) and CVX15 (3). The conserved Arg 1 -Arg 2 -(n-Nal) 3 -Xaa 4 -L-/D-Tyr 5 50 pentapeptide motif is shown in bold, and the aromatic positions 3 and 5 are highlighted with grey background. Cit = citrulline; 1-Nal = 3-(1- naphthyl)alanine; 2-Nal = 3-(2-naphthyl)alanine.