We tested formyl MYVKWPWYVWL which we had previously shown

We tested formyl-MYVKWPWYVWL, which we had previously shown to have essentially identical Kis for W190/N192, R190/K192 and R190/N190 [8] of 130 nM, for its ability to downregulate surface receptor (Fig. 5A). R190/K192 exhibited the lowest EC50 of 44±4 nM about 3 fold below the observed Ki and a maximum downregulation very similar (89%) to that seen with formyl-Nle-Leu-Phe (Fig. 3A). R190/N192 had a 2 fold higher EC50 (P=0.005) than R190/K192 and maximum downregulation similar to that observed with formyl-Nle-Leu-Phe. W190/N192 had a EC50 14 fold greater than K192/N192 (P=0.0002) and 6 fold greater than R190/N192 (P=0.0006) and a maximum downregulation slightly reduced (74%) compared to that seen with formyl-Nle-Leu-Phe. This reduction is consistent with the reduced (65%) stimulation of GTPγS binding of W190/N192 FPR by 300 nM formyl-MYVKWPWYVWL compared to R190/K192 or R190/N192 FPRs reported previously [8]. We carried out a protein blastp search (http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi?PAGE=Proteins&PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on) with the sequence MMWEDAVGWI (Nle is a sch772984 resistant analog of M) to identify sequences which were most similar in sequence to formyl-NleNleWEDAVGWI. Bacteria (taxid:2) was used as the search organism. Two sequences of reasonable similarity to MMWEDAVGWI at their N-terminus were identified (Only N-termini would be formylated). One was protein MAP 4176 from Mycobacterium avium subsp. paratuberculosis and the other was protein JNB 01080 from Janibacter. Only the MAP 4176 sequence was investigated and it had MFEDAVAWF as its N-terminal sequence. Formyl-MFEDAVAWF was synthesized and evaluated for binding and downregulation. Formyl-MFEDAVAWF's Ki was 4 fold higher for W190/N192 than for R190/K192 or R190/N192 (Table 1). We also determined the EC50 for downregulation. Formyl-MFEDAVAWF behaved as a full agonist with all three FPRs (Fig. 5B) and this peptide activates W190/N190 ∼10 fold more weakly than either R190/K192 or R190/N192. EC50s for W190/N192, R190/K192 and R190/N192 were 13,000±1000 nM, 1000±80 nM, and 1500±100 nM, respectively (P<0.0001, W190/N192 vs. either R190/K192 or R190/N192). Formyl-MFEDAVAWF exhibited some similarity to the N-terminus of CHIPS so we synthesized the peptide FTFEPFPTN, which corresponds to the N-terminus of CHIPS, and also formyl-MFTFEPFPTN. We tested commercially available CHIPS for its ability to inhibit formyl-Nle-Leu-Phe-Nle-Tyr-Lys-FITC binding but could not detect any inhibition at 50 nM with any of the FPR variants. FTFEPFPTN produced very little inhibition of formyl-Nle-Leu-Phe-Nle-Tyr-Lys-FITC binding also with at most 30% inhibition at 100 μM with the three FPR variants. Formyl-MFTFEPFPTN was very effective at inhibiting all three variants (Table 1) and exhibited significantly different Kis for W190/N192, R190/K192 and R190/N192 with Kis of 71±4 nM, 16±0.7 nM, and 9.5±0.2 nM, respectively (P<0.0001 for each variant vs. each of the other two). We also tested formyl-MFTFEPFPTN for its ability to downregulate W190/N192, R190/K192 and R190/N192 (Fig. 5C). The EC50s were 19±1 nM, 3.1±0.2 nM, and 6.8±100 nM, respectively (P<0.0001 for each variant vs. each of the other two) and the maximum downregulation was very similar to that see with formyl-Nle-Leu-Phe. The addition of formyl-methionine to the N-terminus of FTFEPFPTN enhanced the affinity ∼10,000 fold, and formyl-MFTFEPFPTN exhibited an affinity similar to that seen with CHIPS (Kd ∼35 nM) [11]. It should be noted that while CHIPS was originally isolated as a secreted protein from S. aureus [35], many of the experiments were done using CHIPS expressed in Escherichia coli [10], [11], [36], and the properties of FLIPr were determined only on the E. coli expressed protein [12]. E. coli expressed proteins would be expected to have an N terminal formyl-methionine. However, the authors noted no difference with the E. coli expressed CHIPS [11].