Edward P. Garvey

1400W Is a Slow, Tight Binding, and Highly Selective Inhibitor of Inducible Nitric-oxide Synthase in Vitro and in Vivo

N-(3-(Aminomethyl)benzyl)acetamidine (1400W) was a slow, tight binding inhibitor of human inducible nitric- oxide 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 μM. Inhibition was dependent on the cofactor NADPH. L-Arginine was a competitive inhibitor of 1400W binding with a Ks value of 3.0 μM. 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 Kd 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 Ki values of 2 μM and 50 μM, 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.

A bifunctional thymidylate synthetase-dihydrofolate reductase in protozoa

Thymidylate synthetase and dihydrofolate reductase exist as a bifunctional protein in a number of species of protozoa which span diverse groups of the subkingdom. The enzymes copurify upon gel filtration and on affinity chromatography columns specific for dihydrofolate reductase. The bifunctional protein has been found in species of Crithidia, Leishmania, Trypanosoma, Plasmodium, Eimeria, Tetrahymena and Euglena. For reasons unknown, neither enzyme could be detected in Entamoeba histolytica or E. invadens. Since neither enzyme has yet been found as a separate protein in protozoa, it is likely that the bifunctional protein is widespread among these primitive eukaryotes. In most cases, the apparent size of the native protein is approximately twice that of the subunit possessing thymidylate synthetase. Further, with one exception, the subunit sizes are close to the sum of the subunit sizes of the separate enzymes found in other sources.

Carbamoyl pyridone HIV-1 integrase inhibitors 3. A diastereomeric approach to chiral nonracemic tricyclic ring systems and the discovery of dolutegravir (S/GSK1349572) and (S/GSK1265744).

We report herein the discovery of the human immunodeficiency virus type-1 (HIV-1) integrase inhibitors dolutegravir (S/GSK1349572) (3) and S/GSK1265744 (4). These drugs stem from a series of carbamoyl pyridone analogues designed using a two-metal chelation model of the integrase catalytic active site. Structure-activity studies evolved a tricyclic series of carbamoyl pyridines that demonstrated properties indicative of once-daily dosing and superior potency against resistant viral strains. An inherent hemiaminal ring fusion stereocenter within the tricyclic carbamoyl pyridone scaffold led to a critical substrate controlled diastereoselective synthetic strategy whereby chiral information from small readily available amino alcohols was employed to control relative and absolute stereochemistry of the final drug candidates. Modest to extremely high levels of stereochemical control were observed depending on ring size and position of the stereocenter. This approach resulted in the discovery of 3 and 4, which are currently in clinical development.

Carbamoyl pyridone HIV-1 integrase inhibitors. 2. Bi- and tricyclic derivatives result in superior antiviral and pharmacokinetic profiles.

This work is a continuation of our initial discovery of a potent monocyclic carbamoyl pyridone human immunodeficiency virus type-1 (HIV-1) integrase inhibitor that displayed favorable antiviral and pharmacokinetic properties. We report herein a series of bicyclic carbamoyl pyridone analogues to address conformational issues from our initial SAR studies. This modification of the core unit succeeded to deliver low nanomolar potency in standard antiviral assays. An additional hydroxyl substituent on the bicyclic scaffold provides remarkable improvement of antiviral efficacies against clinically relevant resistant viruses. These findings led to additional cyclic tethering of the naked hydroxyl group resulting in tricyclic carbamoyl pyridone inhibitors to address remaining issues and deliver potential clinical candidates. The tricyclic carbamoyl pyridone derivatives described herein served as the immediate leads in molecules to the next generation integrase inhibitor dolutegravir which is currently in late stage clinical evaluation.

Potent inhibitors of HIV-1 integrase display a two-step, slow-binding inhibition mechanism which is absent in a drug-resistant T66I/M154I mutant.

Two-metal binding HIV-1 integrase inhibitors (INIs) are potent inhibitors of HIV-1 in vitro and in patients. We report here for the first time the kinetics of inhibition of integrase-catalyzed strand transfer. First, the IC(50) values for each of six structurally distinct INIs decreased when a preincubation was included: S-1360 (1.3 microM vs 0.12 microM), L-731,988 (130 nM vs 9 nM), L-870,810 (130 nM vs 4 nM), raltegravir (300 nM vs 9 nM), elvitegravir (90 nM vs 6 nM), and GSK364735 (90 nM vs 6 nM). When reactions with these INIs were initiated with integrase, progress curve analyses indicated time-dependent inhibition, which could be fitted to a two-step mechanism of binding. Overall fitted K(i) values matched the IC(50) values measured with a preincubation: S-1360 (0.17 microM), L-731,988 (34 nM), L-870,810 (2.4 nM), raltegravir (10 nM), elvitegravir (4.0 nM), and GSK364735 (2.5 nM). To begin to understand the mechanism for this slow onset of inhibition and its possible impact on drug resistance, studies of resistance mutations were initiated. T66I/M154I exhibited little if any time-dependent inhibition by any of the six INIs, as measured by differences in potency upon preincubation or by progress curve analysis. These data demonstrate that slow binding is a signature of two-metal binding INIs, and that the second slow step is required for full potency. We discuss a possible structural explanation of the second slow step of inhibition and also the relationship between loss of time-dependent inhibition and drug resistance of this important new class of HIV-1 antiretroviral drugs.

Synthesis and antiviral activity of 7-benzyl-4-hydroxy-1,5-naphthyridin-2(1H)-one HIV integrase inhibitors.

The medicinal chemistry and structure-activity relationships for a novel series of 7-benzyl-4-hydroxy-1,5-naphthyridin-2(1H)-one HIV-integrase inhibitors are disclosed. Substituent effects were evaluated at the N-1, C-3, and 7-benzyl positions of the naphthyridinone ring system. Low nanomolar IC(50) values were achieved in an HIV-integrase strand transfer assay with both carboxylic ester and carboxamide groups at C-3. More importantly, several carboxamide congeners showed potent antiviral activity in cellular assays. A 7-benzyl substituent was found to be critical for potent enzyme inhibition, and an N-(2-methoxyethyl)carboxamide moiety at C-3 significantly reduced plasma protein binding effects in vitro. Pharmacokinetic data in rats for one carboxamide analogue demonstrated oral bioavailability and reasonable in vivo clearance.

Selection and properties of Leishmania tropica resistant to 10-propargyl-5,8-dideazafolate, an inhibitor of thymidylate synthetase

Leishmania tropica promastigotes have been selected which are highly resistant to the thymidylate synthetase (TS) inhibitor, 10-propargyl-5,8-dideazafolate (CB3717). As reported for L. tropica resistant to methotrexate (MTX), an inhibitor of dihydrofolate reductase (DHFR), CB3717-resistant organisms have high levels of the bifunctional TS-DHFR and amplified DNA sequences. TS-DHFR represents up to 2% of the protein in cell extracts and does not appear to have a structural alteration that contributes to drug resistance. The amplified unit of DNA has a uniform restriction-site map throughout the selection and is nearly identical to the 30 kb amplified unit of R-region DNA found in MTX-resistant cells, except for a small increase in size of the fragment that contains a junction believed to be the site of DNA rearrangements generated during amplification. CB3717-resistant cells do not possess the amplified H-region DNA found in MTX-resistant cells. The amplified DNA in cells resistant to low levels of CB3717 appears as a 30-kb extrachromosomal circle, similar to the amplified DNA of MTX-resistant organisms. In cells resistant to higher levels of drug, the amplified DNA appeared as higher molecular weight forms. When resistant cells were grown in the absence of drug, the amplified DNA and levels of TS-DHFR gradually fell to approximately 10% of the resistant levels. These findings support the proposal that the R-region DNA possesses the sequences that encode the bifunctional protein.