The presence of hydrogen bond acceptors in the warhead, rather than hydrogen bond donors, as is the case in the acetamidine warhead, could also account for the lack of affinity. In Vitro Characterization of H- and Cl-Amidine The detailed inhibitory properties of H-amidine and Cl-amidine were also determined (see below) in an effort to gain additional insights into both their potency and mechanism of inhibition. PAD4 substrates [ em k /em cat/ em K /em m = 1.1 104 M?1 s?1 ( em 14 /em )], and can be considered to consist of two major moieties, a fluoroacetamidine-based warhead and a specificity determinant that was expected to target the warhead to the active site of PAD4, where it will react with C645 to form a stable thioether adduct via one of two potential mechanisms (Figure 2B) ( em 14 /em ). To AS2717638 AS2717638 identify PAD4 inhibitors with enhanced potency and to gain insights into the steric and leaving group requirements for PAD4 AS2717638 inactivation, we synthesized a series of compounds in which both the length of the side chain and the leaving group were varied. The lengths of the side chains ranged from two to four methylene units, thereby allowing us to evaluate the importance of positioning to inactivation, and the fluoro group was replaced with a chloro group. The fluoro group was also replaced with hydrogen to evaluate the requirement for a leaving group.2 Three potential scenarios were envisioned for H2-, H-, and H4-amidine [compounds 6, MUC12 3, and 9, respectively (Figure 2A)], which are detailed here. (i) The iminium carbon of the acetamidine moiety would not possess sufficient reactivity with the active site thiolate, and the compounds would be competitive inhibitors. (ii) The iminium carbon would react with the active site thiolate to form the first tetrahedral intermediate and thereby generate a transition state analogue. (iii) The iminium carbon would react with the active site thiolate to form the first tetrahedral intermediate. Subsequently, the intermediate would collapse, resulting in the loss of benzoylated ornithine, and the formation of an irreversible imidothioic acid adduct. StructureCActivity Relationships The inhibitory properties of compounds 2C9 were initially evaluated by determining the concentration of compound that yielded the half-maximal activity, i.e., the IC50, and comparing the results of these studies to the IC50 value obtained for F-amidine; IC50 values were determined under conditions that were identical to those used to determine the IC50 for F-amidine. The results of these initial studies (Table 2) indicated that Cl-amidine is a significantly more potent inhibitor than F-amidine; the detailed inhibitory properties of Cl-amidine are discussed below. Interestingly, H-amidine, the acetamidine-containing isostere of F-amidine (and BAA), is a very poor inhibitor of PAD4 (IC50 1000 M). Time AS2717638 course experiments with H-amidine, and related compounds H2- and H4-amidine (compounds 6 and 9, respectively), were linear with respect to time (see Figure S1 of the Supporting Information), thereby indicating that on the time scale of these experiments the acetamidine-bearing compounds are reversible PAD4 inhibitors. Note that even extended incubations (2 h) of H-amidine with PAD4 did not result in PAD4 inactivation, and full activity could be restored after overnight dialysis (not shown). The reversible nature of the inhibition rules out the possibility that these compounds react with the active site Cys to form the postulated imidothioic acid adduct. The fact that these compounds are such poor inhibitors suggests that an additional electron-withdrawing group is required to promote reactions between Cys645 and the iminium carbon, i.e., form the first transition state. Table 2 IC50 Values of PAD4 Inhibitors and Inactivatorsa thead th align=”left” rowspan=”1″ colspan=”1″ /th th align=”center” rowspan=”1″ colspan=”1″ compound no. /th th align=”center” rowspan=”1″ colspan=”1″ IC50 (M) /th /thead F-amidine 1 21.6 2.1Cl-amidine 2 5.9 0.3H-amidine 3 1000F2-amidine 4 1000Cl2-amidine 5 585 65H2-amidine 6 1000F4-amidine 7 655 100Cl4-amidine 8 640 10H4-amidine 9 1000F-acetamideb 10 500Cl-acetamideb 11 500 Open in a separate window aIC50 is the concentration of the inhibitor or inactivator that yields half-maximal activity. IC50 values were determined by preincubating PAD4 and the inhibitor or inactivator in the presence of 10 mM calcium for 15 min prior to the addition of 10 mM BAEE to initiate the enzyme assay. See Experimental Procedures for assay details. bNo inhibition was noted with these compounds even at the highest concentration that was tested. Higher concentrations could not be tested because of solubility issues. Cl2-, F4-, and Cl4-amidine were also relatively poor PAD4 inhibitors with IC50 values in the 500 M range. To determine if.