N-(3-(Aminomethyl)benzyl)acetamidine (1400W) was a slow, tight binding inhibitor of human inducible nitricoxide synthase (iNOS). The slow onset of inhibition by 1400W showed saturation kinetics with a maximal rate constant of 0.028 s ؊1 and a binding constant of 2.0 M. Inhibition was dependent on the cofactor NADPH. LArginine was a competitive inhibitor of 1400W binding with a K s value of 3.0 M. Inhibited enzyme did not recover activity after 2 h. Thus, 1400W was either an irreversible inhibitor or an extremely slowly reversible inhibitor of human iNOS with a K d value 7 nM. In contrast, inhibition of human neuronal NOS and endothelial NOS (eNOS) was relatively weaker, rapidly reversible, and competitive with L-arginine, with K i values of 2 M and 50 M, respectively. Thus, 1400W was at least 5000-fold selective for iNOS versus eNOS. This selectivity was similar to that observed in rat aortic rings, in which 1400W was greater than 1000-fold more potent against rat iNOS than eNOS. Finally, 1400W was greater than 50-fold more potent against iNOS than eNOS in a rat model of endotoxin-induced vascular injury. Thus, the potency and selectivity of 1400W inhibition of iNOS both in vitro and in vivo were far greater than of any previously described iNOS inhibitor.

Protein farnesyltransferase (FTase) catalyzes the prenylation of Ras and several other key proteins involved in cell regulation. The mechanism of the FTase reaction was elucidated by pre-steady-state and steady-state kinetic analysis. FTase catalyzed the farnesylation of biotinylated peptide substrate (BiopepSH) by farnesyl pyrophosphate (FPP) to an S-farnesylated peptide (BiopepS-C15). The steady-state kinetic mechanism was ordered. FTase bound FPP in a two-step process with an effective dissociation rate constant of 0.013 s-1 and an overall Kd of 2.8 nM. BiopepSH reacted with FTase.FPP irreversibly, with a second-order rate constant of 2.2 x 10(5) M-1 s-1, to form FTase.BiopepS-C15. Because most of the FPP in FTase.FPP was trapped as FTase.BiopepS-C15 at high concentrations of BiopepSH, FPP dissociated slowly from the ternary complex relative to catalysis, so that the commitment to catalysis was high. The maximal rate constant for formation of FTase.BiopepS-C15 (enzyme-bound product) is much larger than kcat (0.06 s-1), indicating that product release is the rate-determining step in the reaction mechanism.

The long circulating half-life of serum albumin, the most abundant protein in mammalian plasma, derives from pH-dependent endosomal salvage from degradation, mediated by the neonatal Fc receptor (FcRn). Using yeast display, we identified human serum albumin (HSA) variants with increased affinity for human FcRn at endosomal pH, enabling us to solve the crystal structure of a variant HSA/FcRn complex. We find an extensive, primarily hydrophobic interface stabilized by hydrogen-bonding networks involving protonated histidines internal to each protein. The interface features two key FcRn tryptophan side chains inserting into deep hydrophobic pockets on HSA that overlap albumin ligand binding sites. We find that fatty acids (FAs) compete with FcRn, revealing a clash between ligand binding and recycling, and that our high-affinity HSA variants have significantly increased circulating half-lives in mice and monkeys. These observations open the way for the creation of biotherapeutics with significantly improved pharmacokinetics.

L-NG-Nitroarginine (NA) inhibited both the L-arginine oxidation and the L-arginine-independent NADPH oxidation reactions catalyzed by the calcium/calmodulin-dependent constitutive nitric oxide synthase (cNOS) from bovine brain. NA binding did not require calmodulin, calcium, or NADPH. The onset of inhibition was slow with a second-order association rate constant (k(on) of 4.4 x 10(4) M-1 s-1. The dissociation rate constant (k(off) was 6.5 x 10(-4) s-1. The Kd value (k(off)/k(on)) of bovine brain cNOS for NA was 15 nM. L-Arginine was a competitive inhibitor of NA binding with a Ks value of 0.8 microM. The Km for L-arginine in the cNOS reaction was 1.2 microM. The NA binding sites of cNOS were titrated with NA, which enabled a kcat of 0.7 s-1, for the oxidation of L-arginine, to be calculated. Finally, a brain cNOS-(3H)NA complex was isolated. In contrast to the potent and slow onset of NA inhibition of brain cNOS, NA inhibition of inducible mouse macrophage NOS (iNOS) was weaker (Ki = 4.4 microM) and rapidly reversible. Thus, NA was a 300-fold more potent inhibitor of bovine brain cNOS than mouse macrophage iNOS.

Human immunodeficiency virus type 1 (HIV-1) Gag protease cleavage sites (CS) undergo sequence changes during the development of resistance to several protease inhibitors (PIs). We have analyzed the association of sequence variation at the p7/p1 and p1/p6 CS in conjunction with amprenavir (APV)-specific protease mutations following PI combination therapy with APV. Querying a central resistance data repository resulted in the detection of significant associations (P < 0.001) between the presence of APV protease signature mutations and Gag L449F (p1/p6 LP1F) and P453L (p1/p6 PP5L) CS changes. In population-based sequence analyses the I50V mutant was invariably linked to either L449F or P453L. Clonal analysis revealed that both CS mutations were never present in the same genome. Sequential plasma samples from one patient revealed a transition from I50V M46L P453L viruses at early time points to I50V M46I L449F viruses in later samples. Various combinations of the protease and Gag mutations were introduced into the HXB2 laboratory strain of HIV-1. In both single-and multiple-cycle assay systems and in the context of I50V, the L449F and P453L changes consistently increased the 50% inhibitory concentration of APV, while the CS changes alone had no measurable effect on inhibitor sensitivity. The decreased in vitro fitness of the I50V mutant was only partially improved by addition of either CS change (I50V M46I L449F mutant replicative capacity Ϸ 16% of that of wild-type virus). Western blot analysis of Pr55 Gag precursor cleavage products from infected-cell cultures indicated accumulation of uncleaved Gag p1-p6 in all I50V viruses without coexisting CS changes. Purified I50V protease catalyzed cleavage of decapeptides incorporating the L449F or P453L change 10-fold and 22-fold more efficiently than cleavage of the wild-type substrate, respectively. HIV-1 protease CS changes are selected during PI therapy and can have effects on both viral fitness and phenotypic resistance to PIs.