Volume 8, Issue 11, Pages 1107-1121 (November 2001) Engineering inhibitors highly selective for the S1 sites of Ser190 trypsin-like serine protease drug targets  Bradley A Katz, Paul A Sprengeler, Christine Luong, Erik Verner, Kyle Elrod, Matt Kirtley, James Janc, Jeffrey R Spencer, J.Guy Breitenbucher, Hon Hui, Danny McGee, Darin Allen, Arnold Martelli, Richard L Mackman  Chemistry & Biology  Volume 8, Issue 11, Pages 1107-1121 (November 2001) DOI: 10.1016/S1074-5521(01)00084-9

Fig. 1 a: Ser190 protease–APC-8696; b: Ala190 protease–APC-8696; c: Ser190 protease–APC-10302; d: Ala190 protease–APC-10302. H2O1S1 is co-bound in all trypsin-like serine protease complexes of typical amidine inhibitors and in the apo-enzymes [1]. Chemistry & Biology 2001 8, 1107-1121DOI: (10.1016/S1074-5521(01)00084-9)

Fig. 2 a: APC-8696 scaffolds; b: APC-7136 scaffolds. Non-standard atom names are given in this and other figures. Standard names are given for the benzimidazole nitrogens in brackets. Chemistry & Biology 2001 8, 1107-1121DOI: (10.1016/S1074-5521(01)00084-9)

Fig. 3 a: Structure and associated (2|Fo|−|Fc|), αc map of P3121 (form c) trypsin–APC-8696, pH 7.07, 1.37 Å resolution, contoured at 1.0 σ (beige) and 2.4 σ (red). Residue names are labeled white, selected atom names light yellow, and H2O1S1 light blue. Short hydrogen bonds at the active site are cyan, and hydrogen bonds involving H2O1S1 at the S1 site are cyan. The directionalities of the hydrogen bonds involving OγSer190 and OηTyr228 can be inferred from the following: a well ordered water (not shown) donates one proton to OAla183, and the other to Oδ1Asp189. This water must therefore accept the hydrogen bond from OηTyr228. It also follows that OγSer190 donates a (partial) hydrogen bond to OηTyr228, and to H2O1S1. In addition to the hydrogen bonds accepted from H2O1S1, the carbonyl oxygens of residues 215 and 227 also accept β-sheet hydrogen bonds, with better angular components, from the peptide nitrogens of residues 227 and 215, respectively, in these and other structures. b: Structure and (2|Fo|−|Fc|), αc map for thrombin–APC-7806, pH 8.68, 1.75 Å resolution. Chemistry & Biology 2001 8, 1107-1121DOI: (10.1016/S1074-5521(01)00084-9)

Fig. 4 a: Structure and (2|Fo|−|Fc|), αc map for trypsin–APC-10302, pH 9.05, 1.50 Å resolution. b: Thrombin–APC-10302, pH 9.0, 1.81 Å resolution. Because of the poor Ki and limited solubility of APC-10302 in the Ala190 thrombin anti-target, the inhibitor is bound with partial occupancy (0.80). The chloro makes contacts with Cγ1Val213 (3.25 Å), CβAla190 (3.70 Å), OTrp215 (3.83 Å), and CαGly226 (4.14 Å). Hydrogen bonds involving the indole N3 atom are not short, as in thrombin–APC-7806. Chemistry & Biology 2001 8, 1107-1121DOI: (10.1016/S1074-5521(01)00084-9)

Fig. 5 Structure and (2|Fo|−|Fc|), αc map for (a) uPA–APC-8696, pH 6.50, 1.73 Å resolution, and (b) uPA–APC-10302, pH 6.50, 1.50 Å resolution. c: Superimposed structures of uPA–APC-8696 and uPA–APC-10302. Chemistry & Biology 2001 8, 1107-1121DOI: (10.1016/S1074-5521(01)00084-9)

Fig. 6 a: Structure and (2|Fo|−|Fc|), αc map for uPA–APC-11092, 1.80 Å resolution. The long N1–Oδ2Asp189 and N2–Oδ2Asp189 interactions (3.17 Å and 3.40 Å, respectively, at pH 6.5) are shown in yellow and transparent light yellow, respectively. b: Structure and (2|Fo|−|Fc|), αc map for uPA–APC-10950, 1.64 Å resolution. The H2O1S1–F hydrogen bond (2.76 Å) is formed at the expense of the H2O1S1–OTrp215 interaction, the length of which increases from 3.07 Å in uPA–APC-8696 to 3.52 Å in uPA–APC-10950 (Table 3c). Chemistry & Biology 2001 8, 1107-1121DOI: (10.1016/S1074-5521(01)00084-9)

Fig. 7 a: Structure and (2|Fo|−|Fc|), αc map of trypsin–APC-11922, 1.50 Å resolution. For the major inhibitor conformer (opaque sticks) there is a hydrogen bond between the inhibitor phenol (O6′) and Nϵ2His57, and a short O6′–OγSer195 hydrogen bond. b: Structure and (2|Fo|−|Fc|), αc map of uPA–APC-11421, 1.75 Å resolution. In this and all other uPA complexes of the APC-7136 analogs in Fig. 2b, the phenol hydroxyl is at or near the oxyanion hole, receiving hydrogen bonds from NGly193 and from the inhibitor NH amide group. In many trypsin and thrombin complexes the inhibitor is discretely disordered between the two binding modes in (a) and (b). Chemistry & Biology 2001 8, 1107-1121DOI: (10.1016/S1074-5521(01)00084-9)