Design and synthesis of a new type of non steroidal human aromatase inhibitors

The structure-activity relationship study of one of recently described aromatase inhibitors, compound 1 (MR20814), allowed us to design some related derivatives as potential new inhibitors. Among those we synthesized, chlorophenylpyridylmethylenetetrahydroindolizinone 5 (MR20492) exhibited in vitro a ten-fold higher inhibition of the enzyme (IC50 = 0.2 +/- 0.0 microM and Ki = 10.3 +/- 3.3 nM).     

Uridine phosphorylase from Hymenolepis diminuta (Cestoda): kinetics and inhibition by pyrimidine nucleoside analogs

A single pyrimidine nucleoside phosphorylase was found in the cytoplasmic extract from Hymenolepis diminuta. This enzyme preferentially cleaves uridine and, to a much lesser extent, thymidine. Its presence directly indicates the existence of pyrimidine nucleoside salvage pathway in this parasite. Detailed kinetic studies in the phosphorolytic and synthetic direction pointed to the sequential mechanism of these reactions. For phosphorolysis, Kurd = 33 microM and Kp = 806 microM. For synthesis of uridine, Kura = 204 microM and K1-P-rib. = 50 microM. Over six times higher K(m) for uracil than for uridine indicates that phosphorolysis is the favoured reaction in this tapeworm. Well known inhibitors of mammalian uridine phosphorylase: 2,2'-anhydro-5-ethyluridine and 1-(1,3-dihydroxy-2-propoxymethyl)-5-benzyluracil (DHPBU), both with Ki = 0.07 microM were potent competitive inhibitors of the enzyme from H. diminuta. The newly synthesized 2,3'-anhydro-5-ethyluridine (K. Felczak, unpublished) showed only moderate inhibitory activity (Ki = 14 microM) similarly as 1-(1,3-dihydroxy-2-propoxy-methyl)-5-benzyluracil. The same order of Ki values obtained for the investigated inhibitors vs uridine phosphorylase, irrespective whether the enzyme was isolated from rat intestinal mucosa (Drabikowska et al., 1987, Biochem. Pharmacol. 36, 4125-4128) or H. diminuta may point to a great similarity between binding sites on the parasite and the host enzyme.     

Optimizing the binding of fullerene inhibitors of the HIV-1 protease through predicted increases in hydrophobic desolvation

We have developed and applied a computational strategy to increase the affinity of fullerene-based inhibitors of the HIV protease. The result is a approximately 50-fold increase in affinity from previously tested fullerene compounds. The strategy is based on the design of derivatives which may potentially increase hydrophobic desolvation upon complex formation, followed by the docking of the hypothetical derivatives into the HIV protease active site and assessment of the model complexes so formed. The model complexes are generated by the program DOCK and then analyzed for desolvated hydrophobic surface. The amount of hydrophobic surface desolvated was compared with a previously tested compound, and if this amount was significantly greater, it was selected as a target. Using this approach, two targets were identified and synthesized, using two different synthetic approaches: a diphenyl C60 alcohol (5) based on a cyclopropyl derivative of Bingel (Chem.Ber. 1993, 126, 1957-1959) and a diisopropyl cyclohexyl C60 alcohol (4a) as synthesized by Ganapathi et al. (J. Org.Chem. 1995, 60, 2954-2955). Both showed tighter binding than the originally tested compound (diphenethylaminosuccinate methano-C60, Ki = 5 microM) with Ki values of 103 and 150 nM, respectively. In addition to demonstrating the utility of this approach, it shows that simple modification of fullerenes can result in high-affinity ligands of the HIV protease, for which they are highly complementary in structure and chemical nature.     

Nucleon-nucleon pion-exchange tested in three-body reactions

The proposed mechanistic pathway for the reaction catalyzed by 3-deoxy-D-manno-2-octulosonate-8-phosphate (Kdo8P) synthase was examined in terms of the structure of the putative bisphosphate intermediate. Two 2-deoxy analogues of the product Kdo8P, having been structurally prohibited from undergoing the ring-opening and possessing the stereochemistry of either the alpha-pyranase (compound 1) or the beta-pyranose form (compound 2) of the product, were synthesized and probed as inhibitors for the synthase. It was found that both analogues bind to the enzyme and are competitive inhibitors with respect to phosphoenolpyruvate binding, having Ki values of 470 microM and 303 microM, respectively. Comparison of this data to the Ki value of the tautomeric mixture of the product Kdo8P (Ki = 590 microM) suggests that both the alpha- and the beta-pyranose anomers (65.8% and 3.1%, respectively at neutral pH) bind to the enzyme with a slight (1.13 kJ/mol) preference for the beta-anomer, and that the C2 hydroxyl does not contribute to the binding. This uncertain stereochemical preference exhibited by the enzyme for the stereoisomers at the anomeric carbon suggests that the carboxylate binding site of the product is indistinct, while the hydroxyl and carboxylate binding sites may be interchangeable. More importantly, however, the isosteric phosphonate analogue 2,6-anhydro-3-deoxy-2 beta-phosphonylmethyl-8-phosphate-D-glycero-D-talo-octonate (3), which mimics the topological and electrostatic properties of the proposed cyclic intermediate, was found to be the most potent inhibitor of the enzyme with a Ki value of 5 microM. Two hitherto unrecognized aspects of the mechanism of the synthase were identified. First, the results showing that the cyclic analogues 1, 2 and 3 are inhibitors of the enzyme whereas the previously reported acyclic analogue, which contains no carbonyl group at C2 and may thus resemble the open-chain form of Kdo8P, is not an inhibitor, suggest that the pyranose form and not the open-chain acyclic form of the putative bisphosphate intermediate is handled by the enzyme. Second, since the overall stereochemical course of the transformation mediated by the synthase has been shown to involve si face addition of phosphoenolpyruvate to the re face of the carbonyl of arabinose 5-phosphate, the present observation involving analogue 3 suggest that the bisphosphate intermediate formed during the initial steps of synthesis may have the pyranose structure with the anomeric phosphate located in the beta-configuration.     

Pharmacological characterization of novel tissue kallikrein inhibitors in vivo

In this study we have investigated the effect of novel tissue kallikreins on the plasma protein exudation induced by porcine pancreatic kallikrein (PPK) in the rabbit skin in vivo. The tissue kallikrein inhibitors here described were synthesized based on analogues of peptide substrates for tissue kallikreins. The intradermal injection of PPK and rabbit urinary kallikrein, but not of rabbit plasma kallikrein, significantly increased the microvascular permeability leading to local oedema formation in the rabbit skin. At the dose of 3-200 nmol/site, the intradermal co-administration of the tissue kallikrein inhibitors Bz-F-F-S-R-EDDnp (Ki = 0.1 microM; ESP5), PAC-F-S-R-EDDnp (Ki = 0.7 microM; ESP6), Bz-F-F-A-P-R-NH2 (Ki = 7.8 microM; ESP8), PAC-F-F-R-P-R-NH2 (Ki = 0.3 microM; ESP9) and Bz-F-F-S-R-NH2 (Ki = 0.3 microM; ESP11) dose-dependently inhibited the plasma protein exudation induced by PPK. The most potent compound was ESP6 (IC25 = 7.8 nmol/site) followed by ESP5 (IC25 = 14.2 nmol/site), ESP8 (IC25 = 25 nmol/site), ESP9 (IC25 = 30 nmol/site) and ESP11 (IC25 = 50.4 nmol/site). The compounds Bz-F-F-R-P-R-NH2 (Ki = 0.5 microM; ESP1), Bz-F-F-pNa (Ki = 0.4 microM; ESP3), Bz-F(NH2)-F-R-P-R-NH2 (Ki = 1.1 microM; ESP7) and Bz-F-F-S-P-R-NH2 (Ki = 4.6 microM; ESP10) had no significant effect on the PPK-induced plasma protein exudation in doses up to 200 nmol/site. ESP6 also inhibited the PPK-induced plasma protein exudation when administered systemically. This compound may constitute a useful tool to further investigate both the physiological and pathological role of tissue kallikreins.     

Gliclazide hydroxylation by rat liver microsomes

1. The metabolism of gliclazide to hydroxygliclazide has been investigated in Sprague-Dawley rat liver microsomes. 2. The kinetics of hydroxygliclazide formation are consistent with Michaelis-Menten kinetics (mean (+/- SD, n = 3) apparent K(m) and Vmax = 256 +/- 27 microM and 1.85 +/- 0.10 nmol/ min/mg respectively). 3. Tolbutamide competitively inhibited hydroxygliclazide formation (Ki = 840 microM) and gliclazide competitively inhibited hydroxytolbutamide formation (Ki = 240 microM) with Ki similar to K(m). Therefore gliclazide and tolbutamide may be metabolized by the same enzyme in the rat. In nine livers the formation of hydroxygliclazide correlated with the formation of hydroxytolbutamide (rs = 0.82, p < 0.01). 4. Diclofenac (Ki = 64 microM), phenytoin (Ki = 38 microM), mephenytoin (Ki = 66 microM), glibenclamide (Ki = 14 microM) and glipizide (Ki = 189 microM) were fully competitive inhibitors of gliclazide hydroxylation. The rank order of Ki constants differed for gliclazide and tolbutamide suggesting that gliclazide and tolbutamide hydroxylases are not identical enzymes. 5. Quinine (Ki = 0.3 microM) and quinidine (Ki = 4.3 microM) were partially competitive inhibitors of hydroxygliclazide formation. Hydroxylation of gliclazide was related to the activity of CYP2D1 as assessed by dextrorphan production from dextromethorphan (rs = 0.83, p = 0.01). 6. In the rat gliclazide is metabolized to hydroxygliclazide by at least two cytochrome P450 isoforms, including tolbutamide hydroxylase and 2D1, which have similar affinities for gliclazide.     

Prejudice toward fat people: the development and validation of the antifat attitudes test

BACKGROUND: The identification of potent small molecule ligands to receptors and enzymes is one of the major goals of chemical and biological research. Two powerful new tools that can be used in these efforts are combinatorial chemistry and structure-based design. Here we address how to join these methods in a design protocol that produces libraries of compounds that are directed against specific macromolecular targets. The aspartyl class of proteases, which is involved in numerous biological processes, was chosen to demonstrate this effective procedure. RESULTS: Using cathepsin D, a prototypical aspartyl protease, a number of low nanomolar inhibitors were rapidly identified. Although cathepsin D is implicated in a number of therapeutically relevant processes, potent nonpeptide inhibitors have not been reported previously. The libraries, synthesized on solid support, displayed nonpeptide functionality about the (hydroxyethyl)amine isostere. The (hydroxyethyl)amine isostere, which targets the aspartyl protease class, is a stable mimetic of the tetrahedral intermediate of amide hydrolysis. Structure-based design, using the crystal structure of cathepsin D complexed with the peptide-based natural product pepstatin, was used to select the building blocks for the library synthesis. The library yielded a 'hit rate' of 6-7% at 1 microM inhibitor concentrations, with the most potent compound having a Ki value of 73 nM. More potent, nonpeptide inhibitors (Ki = 9-15 nM) of cathepsin D were rapidly identified by synthesizing and screening a small second generation library. CONCLUSIONS: The success of these studies clearly demonstrates the power of coupling the complementary methods of combinatorial chemistry and structure-based design. We anticipate that the general approaches described here will be successful for other members of the aspartyl protease class and for many other enzyme classes.     

Resistance to HIV protease inhibitors: a comparison of enzyme inhibition and antiviral potency

Resistance of HIV-1 to protease inhibitors has been associated with changes at residues Val82 and Ile84 of HIV-1 protease (HIV PR). Using both an enzyme assay with a peptide substrate and a cell-based infectivity assay, we examined the correlation between the inhibition constants for enzyme activity (Ki values) and viral replication (IC90 values) for 5 active site mutants and 19 protease inhibitors. Four of the five mutations studied (V82F, V82A, I84V, and V82F/I84V) had been identified as conferring resistance during in vitro selection using a protease inhibitor. The mutant protease genes were expressed in Escherichia coli for preparation of enzyme, and inserted into the HXB2 strain of HIV for test of antiviral activity. The inhibitors included saquinavir, indinavir, nelfinavir, 141W94, ritonavir (all in clinical use), and 14 cyclic ureas with a constant core structure and varying P2, P2' and P3, P3' groups. The single mutations V82F and I84V caused changes with various inhibitors ranging from 0.3- to 86-fold in Ki and from 0.1- to 11-fold in IC90. Much larger changes compared to wild type were observed for the double mutation V82F/I84V both for Ki (10-2000-fold) and for IC90 (0.7-377-fold). However, there were low correlations (r2 = 0.017-0.53) between the mutant/wild-type ratio of Ki values (enzyme resistance) and the mutant/wild-type ratio of viral IC90 values (antiviral resistance) for each of the HIV proteases and the viruses containing the identical enzyme. Assessing enzyme resistance by "vitality values", which adjust the Ki values with the catalytic efficiencies (kcat/Km), caused no significant improvement in the correlation with antiviral resistance. Therefore, our data suggest that measurements of enzyme inhibition with mutant proteases may be poorly predictive of the antiviral effect in resistant viruses even when mutations are restricted to the protease gene.     

Inhibition of myo-inositol monophosphatase isoforms by aromatic phosphonates

alpha-Hydroxyphosphonates are moderately potent (Ki = 6-600 microM) inhibitors of the enzyme myo-inositol monophosphatase (McLeod et al., Med. Chem. Res. 1992, 2, 96). Hydroxy-[4-(5,6,7,8-tetrahydronaphtyl-1-oxy)phenyl]methyl phosphonate (3) was resynthesized and its inhibitory potency towards the recombinant bovine brain enzyme confirmed (Ki = 20 microM). Similar aromatic difluoro-, keto-, and ketodifluorophosphonates (5, 7, 9) were inactive. Compound 3 was 15-fold less active on the human as compared to the bovine enzyme. Molecular modeling suggested that the hydrophobic part of the inhibitor interacts with amino acid side chains that are located at the interface between the enzyme subunits in an area (amino acids 175-185) with low similarity between the two isozymes. Phe-183 in the human enzyme was replaced with leucine, the corresponding residue in the bovine isoform. The three isozymes (human wild-type, bovine wild-type and human F183L) had similar kinetic properties, except that the bovine enzyme was less effectively inhibited by high concentrations of the activator Mg2+. The F183L mutant enzyme had a twofold increased affinity for compound 3 as compared to the human wild-type form. We conclude that residue 183 contributes to the binding of aromatic hydroxyphosphonates to IMPase, but it is not the only determining factor for inhibitor specificity with respect to different isozymes.     

A novel, picomolar inhibitor of human immunodeficiency virus type 1 protease

The design, synthesis, and molecular modeling studies of a novel series of azacyclic ureas, which are inhibitors of human immunodeficiency virus type 1 (HIV-1) protease that incorporate different ligands for the S1', S2, and S2' substrate-binding sites of HIV-1 protease are described. The synthesis of this series is highly flexible in the sense that the P1', P2, and P2' residues of the inhibitors can be changed independently. Molecular modeling studies on the phenyl ring of the P2 and P2' ligand suggested incorporation of hydrogen-bonding donor/acceptor groups at the 3' and 4-positions of the phenyl ring should increase binding potency. This led to the discovery of compound 7f (A-98881), which possesses high potency in the HIV-1 protease inhibition assay and the in vitro MT-4 cell culture assay (Ki = approximately 5 pM and EC50 = 0.002 microM). This compares well with the symmetrical cyclic urea 1 pioneered at DuPont Merck.     

Rational drug design approach for overcoming drug resistance: application to pyrimethamine resistance in malaria

Pyrimethamine acts by selectively inhibiting malarial dihydrofolate reductase-thymidylate synthase (DHFR-TS). Resistance in the most important human parasite, Plasmodium falciparum, initially results from an S108N mutation in the DHFR domain, with additional mutation (most commonly C59R or N51I or both) imparting much greater resistance. From a homology model of the 3-D structure of DHFR-TS, rational drug design techniques have been used to design and subsequently synthesize inhibitors able to overcome malarial pyrimethamine resistance. Compared to pyrimethamine (Ki 1.5 nM) with purified recombinant DHFR fromP. falciparum, the Ki value of the m-methoxy analogue of pyrimethamine was 1.07 nM, but against the DHFR bearing the double mutation (C59R + S108N), the Ki values for pyrimethamine and the m-methoxy analogue were 71.7 and 14.0 nM, respectively. The m-chloro analogue of pyrimethamine was a stronger inhibitor of both wild-type DHFR (with Ki 0.30 nM) and the doubly mutant (C59R +S108N) purified enzyme (with Ki 2.40 nM). Growth of parasite cultures of P. falciparum in vitro was also strongly inhibited by these compounds with 50% inhibition of growth occurring at 3.7 microM for the m-methoxy and 0.6 microM for the m-chloro compounds with the K1 parasite line bearing the double mutation (S108N + C59R), compared to 10.2 microM for pyrimethamine. These inhibitors were also found in preliminary studies to retain antimalarial activity in vivo in P. berghei-infected mice.     

10-Formyl-5,8,10-trideazafolic acid (10-formyl-TDAF): a potent inhibitor of glycinamide ribonucleotide transformylase

The synthesis of 10-formyl-5,8,10-trideazafolic acid (3) as a potential inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) is reported. The target compound was prepared by a convergent synthesis utilizing the alkylation of hydrazone 5 with benzylic bromide 6 to construct the core heterocycle 7. The aldehyde 3 and related agents were evaluated as inhibitors of purN GAR Tfase and avian AICAR Tfase. Compound 3 exhibited potent inhibition of GAR Tfase with a Ki of 0.26 +/- 0.05 microM. In contrast, 3 exhibited more moderate inhibition of aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase), with Ki of 7.6 +/- 1.5 microM.     

Viracept (nelfinavir mesylate, AG1343): a potent, orally bioavailable inhibitor of HIV-1 protease

Using a combination of iterative structure-based design and an analysis of oral pharmacokinetics and antiviral activity, AG1343 (Viracept, nelfinavir mesylate), a nonpeptidic inhibitor of HIV-1 protease, was identified. AG1343 is a potent enzyme inhibitor (Ki = 2 nM) and antiviral agent (HIV-1 ED50 = 14 nM). An X-ray cocrystal structure of the enzyme-AG1343 complex reveals how the novel thiophenyl ether and phenol-amide substituents of the inhibitor interact with the S1 and S2 subsites of HIV-1 protease, respectively. In vivo studies indicate that AG1343 is well absorbed orally in a variety of species and possesses favorable pharmacokinetic properties in humans. AG1343 (Viracept) has recently been approved for marketing for the treatment of AIDS.     

Leukotriene C4 is a tight-binding inhibitor of microsomal glutathione transferase-1. Effects of leukotriene pathway modifiers

Microsomal glutathione transferase-1 (MGST-1) is an abundant protein that catalyzes the conjugation of electrophilic compounds with glutathione, as well as the reduction of lipid hydroperoxides. Here we report that leukotriene C4 is a potent inhibitor of MGST-1. Leukotriene C4 was found to be a tight-binding inhibitor, with a Ki of 5.4 nM for the unactivated enzyme, and 9.2 nM for the N-ethylmaleimide activated enzyme. This is the first tight-binding inhibitor characterized for this enzyme. Leukotriene C4 was competitive with respect to glutathione and non-competitive toward the second substrate, CDNB. Analysis of stoichiometry supports binding of one molecule of inhibitor per homotrimer. Leukotrienes A4, D4, and E4 were much weaker inhibitors of the purified enzyme (by at least 3 orders of magnitude). Leukotriene C4 analogues, which have been developed as antagonists of leukotriene receptors, were found to display varying degrees of inhibition of MGST-1. In particular, the cysteinyl-leukotriene analogues SKF 104,353, ONO-1078, and BAYu9773 were strong inhibitors (IC50 values: 0.13, 3. 7, and 7.6 microM, respectively). In view of the partial structural similarity between MGST-1, leukotriene C4 synthase, and 5-lipoxygenase activating protein (FLAP), it was of interest that leukotriene C4 synthesis inhibitors (which antagonize FLAP) also displayed significant inhibition (e.g. IC50 for BAYx1005 was 58 microM). In contrast, selective 5-lipoxygenase inhibitors such as zileuton only marginally inhibited activity at high concentrations (500 microM). Our discovery that leukotriene C4 and drugs developed based on its structure are potent inhibitors of MGST-1 raises the possibility that MGST-1 influences the cellular processing of leukotrienes. These findings may also have implications for the effects and side-effects of drugs developed to manipulate leukotrienes.     

Tipranavir (PNU-140690): a potent, orally bioavailable nonpeptidic HIV protease inhibitor of the 5,6-dihydro-4-hydroxy-2-pyrone sulfonamide class

A broad screening program previously identified phenprocoumon (1) as a small molecule template for inhibition of HIV protease. Subsequent modification of this lead through iterative cycles of structure-based design led to the activity enhancements of pyrone and dihydropyrone ring systems (II and V) and amide-based substitution (III). Incorporation of sulfonamide substitution within the dihydropyrone template provided a series of highly potent HIV protease inhibitors, with structure-activity relationships described in this paper. Crystallographic studies provided further information on important binding interactions responsible for high enzymatic binding. These studies culminated in compound VI, which inhibits HIV protease with a Ki value of 8 pM and shows an IC90 value of 100 nM in antiviral cell culture. Clinical trials of this compound (PNU-140690, Tipranavir) for treatment of HIV infection are currently underway.     

Design and synthesis of new potent C2-symmetric HIV-1 protease inhibitors. Use of L-mannaric acid as a peptidomimetic scaffold

A study on the use of derivatized carbohydrates as C2-symmetric HIV-1 protease inhibitors has been undertaken. L-Mannaric acid (6) was bis-O-benzylated at C-2 and C-5 and subsequently coupled with amino acids and amines to give C2-symmetric products based on C-terminal duplication. Potent HIV protease inhibitors, 28 Ki = 0.4 nM and 43 Ki = 0.2 nM, have been discovered, and two synthetic methodologies have been developed, one whereby these inhibitors can be prepared in just three chemical steps from commercially available materials. A remarkable increase in potency going from IC50 = 5000 nM (23) to IC50 = 15 nM (28) was observed upon exchanging -COOMe for -CONHMe in the inhibitor, resulting in the net addition of one hydrogen bond interaction between each of the two -NH- groups and the HIV protease backbone (Gly 48/148). The X-ray crystal structures of 43 and of 48 have been determined (Figures 5 and 6), revealing the binding mode of these inhibitors which will aid further design.     

Human globin chain separation by capillary electrophoresis in acidic isoelectric buffers

The potency of a series of anticholinesterase (anti-ChE) agents and serotonin-related amines as inhibitors of the aryl acylamidase (AAA) activity associated with electric eel acetylcholinesterase (AChE) (EC 3.1.1.7) and horse serum butyrylcholinesterase (BuChE) (EC 3.1.1.8) was examined and compared with the potency of the same compounds as ChE inhibitors. Neostigmine, physostigmine, BW 284C51, (+/-)-huperzine A, E2020, tacrine, edrophonium and heptyl-physostigmine were, in that order, the most potent in inhibiting eel AChE-associated AAA activity, their inhibitor constant (Ki) values being in the range 0.02-0.37 microM. The rank order of the same compounds as AChE inhibitors basically paralleled that of AAA, although they were in general stronger on AChE (Ki = 0.001-0.05). The peripheral anionic site inhibitors propidium and gallamine were inactive on AChE-associated AAA. Serotonin and its derivatives were slightly stronger on AAA (Ki = 7.5-30 microM) than on AChE (Ki = 20-140 microM). Tacrine (IC50 = 0.03 microM), diisopropylfluorophosphate (IC50 = 0.04 microM), heptyl-physostigmine (IC50 = 0.11 microM), physostigmine (IC50 = 0.15 microM) and tetra-iso-propylpyrophosphoramide (iso-OMPA) (IC50 = 0.75 microM) were the most potent in inhibiting horse serum BuChE-associated AAA activity. Serotonin and related amines were very weak on BuChE-associated AAA activity. These results indicate that the inhibitory potencies of the active site anti-ChE agents on the AAA activity associated with eel AChE and horse serum BuChE are closely correlated with their action on the respective ChE. In addition, the efficacy of tacrine, E2020, heptyl-physostigmine and (+/-)-huperzine A in the treatment of Alzheimer's disease is unlikely to be related to the action of these drugs on ChE-associated AAA.     

1,2-Benzisothiazol-3-one 1,1-dioxide inhibitors of human mast cell tryptase

A library of compounds were prepared by reacting 2-(bromomethyl)-1, 2-benzisothiazol-3(2H)-one 1,1-dioxide (5) with commercially available carboxylic acids in the presence of potassium carbonate or a tertiary amine base. From this library, (1,1-dioxido-3-oxo-1, 2-benzisothiazol-2(3H)-yl)methyl N-[(phenylmethoxy)carbonyl]-beta-alanate (7b) emerged as a potent inhibitor of human mast cell tryptase (IC50 = 0.85 microM). Extension of the side chain of 7b by two carbons gave (1, 1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)methyl 5-[[(phenylmethoxy)carbonyl]amino]pentanoate (7d) which was an 8-fold more potent inhibitor (IC50 = 0.1 microM). Further modification of this series produced benzoic acid derivative (1, 1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)methyl 4-[[(phenylmethoxy)carbonyl]amino]benzoate (7n) which is the most potent inhibitor identified in this series (IC50 = 0.064 microM). These compounds exhibit time-dependent inhibition consistent with mechanism-based inhibition. For 7b, the initial enzyme velocity is not a saturable function of the inhibitor concentration and the initial Ki could not be determined (Ki > 10 microM). The steady-state rate constant, Ki, was determined to be 396 nM. On the other hand, compounds 7d and 7n are time-dependent inhibitors with a saturable initial complex. From these studies, an initial rate constant, Ki, for 7d and 7n was found to be 345 and 465 nM, respectively. The steady-state inhibition constants, Ki, for 7d and 7n were calculated to be 60 and 52 nM, respectively. Compound 7n is a 13-fold more potent inhibitor than 7b, and these kinetic studies indicate that the increase in inhibitory activity is due to an increase in initial affinity toward the enzyme and not an increase in chemical reactivity. These inhibitors generally show high selectivity for tryptase, being 40-fold weaker inhibitors of elastase, being 100-fold weaker against trypsin, and showing no inhibition against thrombin. These compounds are not inhibitors of thrombin, plasmin t-PA, urokinase, and factor Xa (IC50 > 33 microM). In the delayed-type hypersensitivity (DTH) mouse model, a model of skin inflammation, a 5% solution of 7d reduced edema by 69% compared to control animals.     

Rational design of novel antimicrobials: blocking purine salvage in a parasitic protozoan

All parasitic protozoa obtain purine nucleotides solely by salvaging purine bases and/or nucleosides from their host. This observation suggests that inhibiting purine salvage may be a good way of killing these organisms. To explore this idea, we attempted to block the purine salvage pathway of the parasitic protozoan Tritrichomonas foetus. T. foetus is a good organism to study because its purine salvage depends primarily on a single enzyme, hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase), and could provide a good model for rational drug design through specific enzyme inhibition. Guided by the crystal structure of T. foetus HGXPRTase, we used structure-based drug design to identify several non-purine compounds that inhibited this enzyme without any detectable effect on human HGPRTase. One of these compounds, 4-[N-(3, 4-dichlorophenyl)carbamoyl]phthalic anhydride (referred to as TF1), was selected for further characterization. TF1 was shown to be a competitive inhibitor of T. foetus HGXPRTase with respect to both guanine (in the forward reaction; Ki = 13 microM) and GMP (in the reverse reaction; Ki = 10 microM), but showed no effect on the homologous human enzyme at concentrations of up to 1 mM. TF1 inhibited the in vitro growth of T. foetus with an EC50 of approximately 40 microM. This inhibitory effect was associated with a decrease in the incorporation of exogenous guanine into nucleic acids, and could be reversed by supplementing the growth medium with excess exogenous hypoxanthine or guanine. Thus, rationally targeting an essential enzyme in a parasitic organism has yielded specific enzyme inhibitors capable of suppressing that parasite's growth.     

Structure-based inhibitor design by using protein models for the development of antiparasitic agents

The lack of an experimentally determined structure of a target protein frequently limits the application of structure-based drug design methods. In an effort to overcome this limitation, we have investigated the use of computer model-built structures for the identification of previously unknown inhibitors of enzymes from two major protease families, serine and cysteine proteases. We have successfully used our model-built structures to identify computationally and to confirm experimentally the activity of nonpeptidic inhibitors directed against important enzymes in the schistosome [2-(4-methoxybenzoyl)-1-naphthoic acid, Ki = 3 microM] and malaria (oxalic bis[(2-hydroxy-1-naphthylmethylene)hydrazide], IC50 = 6 microM) parasite life cycles.