Crystallographic analysis at 3.0-A resolution of the binding to human thrombin of four active site-directed inhibitors

The mode of binding of four active-site directed inhibitors to human thrombin has been determined by x-ray crystallographic analysis. The inhibitors studied are benzamidine, PPACK, NAPAP, and MD-805, of which the last three are compounds evolved specifically to inhibit thrombin. Crystal structures were determined in the presence of both the inhibitor and the undecapeptide [des-amino Asp55]hirudin(55-65) which binds distant from the active site. Despite having significantly different chemical structures, NAPAP and MD-805 bind to thrombin in a very similar "inhibitor binding mode" which is not that expected by direct analogy with the binding of substrate. Both inhibitors bind to thrombin in a similar way as to trypsin, but thrombin has an extra loop, the "Tyr-Pro-Pro-Trp loop," not present in trypsin, which gives further binding interactions and is seen to move somewhat to accommodate binding of the different inhibitors. The fact that NAPAP and MD-805 require different stereochemistry for potent inhibition is demonstrated, and its structural basis clarified. The wealth of data on analogs and variants of these lead compounds is shown to be compatible with this inhibitor binding mode.     

Protease inhibitors: Part 4. Synthesis of weakly basic thrombin inhibitors incorporating pyridinium-sulfanilylaminoguanidine moieties

Three series of derivatives have been prepared by reaction of sulfanilylaminoguanidine with pyrylium salts, with the pyridinium derivatives of glycine and with the pyridinium derivatives of beta-alanine, respectively. The new compounds were assayed as inhibitors of two serine proteases, thrombin and trypsin. The study showed that in contrast to the leads, possessing KI's around 100-300 nM against thrombin, and 450-1420 nM against trypsin, respectively, the new derivatives showed inhibition constants in the range of 15-50 nM against thrombin, whereas their affinity for trypsin remained relatively low. Derivatives of beta-alanine were more active than the corresponding glycine derivatives, which in turn were more inhibitory than the pyridinium derivatives of sulfanilylaminoguanidine possessing the same substitution pattern at the pyridinium ring. Thus, the present study proposes two novel approaches for the preparation of high affinity, specific thrombin inhibitors: a novel S1 anchoring moiety in the already large family of arginine/amidine-based inhibitors, i.e., the SO2NHNHC(=NH)NH2 group, and novel non-peptidomimetic scaffolds obtained by incorporating alkyl-/aryl-substituted-pyridinium moieties in the hydrophobic binding site(s). The first one is important for obtaining bioavailable thrombin inhibitors, devoid of the high basicity of the commonly used arginine/amidine-based inhibitors, whereas the second one may lead to improved water solubility of such compounds.     

Protease Inhibitors: Part 4. Synthesis of Weakly Basic Thrombin Inhibitors Incorporating Pyridinium-Sulfanilylaminoguanidine Moieties

Three series of derivatives have been prepared by reaction of sulfanilylaminoguanidine with pyrylium salts, with the pyridinium derivatives of glycine and with the pyridinium derivatives of β-alanine, respectively. The new compounds were assayed as inhibitors of two serine proteases, thrombin and trypsin. The study showed that in contrast to the leads, possessing K₁'s around 100–300nM against thrombin, and 450–1420nM against trypsin, respectively, the new derivdtives showed inhibition constants in the range of 15–50nM against thrombin, whereas their affinity for trypsin remained relatively low. Derivatives of β-alanine were more active than the corresponding glycine derivatives, which in turn were more inhibitory than the pyridinium derivatives of sulfanilylaminoguanidine possessing the same substitution pattern at the pyridinium ring. Thus, the present study proposes two novel approaches for the preparation of high affinity, specific thrombin inhibitors: a novel S1 anchoring moiety in the already large family of arginine/amidine-based inhibitors, i.e., the SO₂NHNHC(=NH)NH₂ group, and novel non-peptidomimetic scaffolds obtained by incorporating alkyl-/aryl-substituted-pyridinium moieties in the hydrophobic binding site(s). The first one is important for obtaining bioavailable thrombin inhibitors, devoid of the high basicity of the commonly used arginine/amidine-based inhibitors, whereas the second one may lead to improved water solubility of such compounds.     

Analogs of oxalacetate as potential substrates for phosphoenolpyruvate carboxykinase

Structural analogs of the substrate oxalacetate were examined as potential substrates and inhibitors for chicken liver mitochondrial phosphoenolpyruvate (P-enolpyruvate) carboxykinase. Steady-state kinetics were employed to characterize the inhibitory effects of these substrate analogs with the enzyme. Assays were carried out in both carboxylation and decarboxylation reaction directions. Pyruvate, beta-hydroxypyruvate, beta-mercaptopyruvate, beta-fluoropyruvate, DL-lactate, glycolate, glycoaldehyde, glyoxylate, glyphosate, and DL-aspartate showed no inhibitory effects by steady-state kinetics. Oxalate, acetopyruvate, and DL-, D-, and L-glycerate exhibited weak noncompetitive inhibition of the P-enolpyruvate carboxykinase-catalyzed reaction. DL-3-Nitro-2-hydroxypropionic acid, 3-nitro-2-oxopropionic acid, DL-malate, malonate, tartronate, and alpha-ketobutyrate all show weak inhibition with estimated inhibition constants greater than 20 nM. Several of these compounds were investigated by 31P NMR to determine if they function as phosphoryl acceptors for GTP. None of the compounds tested act as phosphoryl acceptors in the enzyme-catalyzed reaction. Chicken liver mitochondrial phosphoenolpyruvate carboxykinase shows a remarkably high degree of specificity at the binding site of oxalacetate.     

Refined 2.3 A X-ray crystal structure of bovine thrombin complexes formed with the benzamidine and arginine-based thrombin inhibitors NAPAP, 4-TAPAP and MQPA. A starting point for improving antithrombotics.

Well-diffracting crystals of bovine epsilon-thrombin in complex with several "non-peptidic" benzamidine and arginine-based thrombin inhibitors have been obtained by co-crystallization. The 2.3 A crystal structures of three complexes formed either with NAPAP, 4-TAPAP, or MQPA, were solved by Patterson search methods and refined to crystallographic R-values of 0.167 to 0.178. The active-site environment of thrombin is only slightly affected by binding of the different inhibitors; in particular, the exposed "60-insertion loop" essentially maintains its typical projecting structure. The D-stereoisomer of NAPAP and the L-stereoisomer of MQPA bind to thrombin with very similar conformations, as previously inferred from their binding to bovine trypsin; the arginine side-chain of the latter inserts into the specificity pocket in a "non-canonical" manner. The L-stereoisomer of 4-TAPAP, whose binding geometry towards trypsin was only poorly defined, is bound to the thrombin active-site in a compact conformation. In contrast to NAPAP, the distal p-amidino/guanidino groups of 4-TAPAP and MQPA do not interact with the carboxylate group of Asp189 in the thrombin specificity pocket in a "symmetrical" twin N-twin O manner, but through "lateral" single N-twin O contacts; in contrast to the p-amidino group of 4-TAPAP, however, the guanidyl group of MQPA packs favourably in the pocket due to an elaborate hydrogen bond network, which includes two entrapped water molecules. These thrombin structures confirm previous conclusions of the important role of the intermolecular hydrogen bonds formed with Gly216, and of the good sterical fit of the terminal bulky hydrophobic inhibitor groups with the hydrophobic aryl binding site and the S2-cavity, respectively, for tight thrombin active site binding of these non-peptidic inhibitors. These accurate crystal structures are presumed to be excellent starting points for the design and the elaboration of improved antithrombotics.     

Development of specific Rho-kinase inhibitors and their clinical application

Hexahydro-1-(isoquinoline-5-sulfonyl)-1H-1,4-diazepine, HA-1077, is a known selective inhibitor of Rho-kinase. Although its IC(50) value against Rho-kinase is more than 10 times lower than those for kinases such as PKA, PKB, PKC, PKG, MLCK, CaMKII and others, the molecule still retains relative potent inhibition activities against these kinases. In order to produce highly specific Rho-kinase inhibitors, several HA-1077 analogs were synthesized and their kinase inhibition properties evaluated. (S)-Hexahydro-1-(4-ethenylisoquinoline-5-sulfonyl)-2-methyl-1H-1,4-diazepine was found to be a potent Rho-kinase inhibitor. The IC50 value against Rho-kinase was 6 nM, while those against other kinases remained at almost the same level as that of HA-1077. Furthermore, we designed HA-1077 analogs on the basis of the complex structure of PKA and HA-1077. Amongst these, (S)-hexahydro-4-glycyl-2-methyl-1-(4-methylisoquinoline-5-sulfonyl)-1H-1,4-diazepine and other glycine derivatives were found to be highly specific Rho-kinase inhibitors. These Rho-kinase specific inhibitors were applied to rabbit ocular hypertensive models and were shown to reduce intraocular pressure. These results demonstrate that the new 5-isoquinolinesulfonylamides are not only potent ROCK selective compounds, but are also useful compounds for clinical applications.     

Inhibition effect of rosette formation between human lymphocytes and sheep erythrocytes by specific heptapeptide isolated from uremic fluid and its analogs.

One of middle molecular substances (H-His-Pro-Ala-Glu-Asn-Gly-Lys-OH) and its two analogs, in which the proline residue in position 2 was replaced by glycine and the alanine residue in position 3 was replaced by valine exert a inhibition effect on $\text{in vitro}$ E-rosette formation. Its synthetic two analogs showed diminished biological activity compared to native heptapeptide.     

Structure-based organic synthesis of unnatural aeruginosin hybrids as potent inhibitors of thrombin

Based on X-ray crystallographic data of complexes of chlorodysinosin A with the enzyme thrombin, a series of analogs were synthesized varying the nature of the P(1), P(2), and P(3) pharmacophoric sites and the central octahydroindole carboxyamide core. In general, introduction of a hydrophobic substituent on the d-leucine amide residue dramatically improved the inhibition of the enzyme. This is rationalized based on a better fit of the P(3) subunit in the hydrophobic S(3) enzyme site. Single digit nanomolar inhibition expressed as IC(50) was observed for several analogs.     

New bivalent thrombin inhibitors with N(alpha)(methyl)arginine at the P1-position

A series of bivalent thrombin inhibitors was synthesized, consisting of a d-phenylalanyl-prolyl-N(alpha)(methyl)arginyl active site blocking segment, a fibrinogen recognition exosite inhibitor part, and a peptidic linker connecting these fragments. The methylation of the P1 amino acid led to a moderate decrease in affinity compared with the unmethylated analog. In addition, it prevented the thrombin catalyzed proteolysis, independent of the P1' amino acid used. This is a significant advantage compared to the original hirulogs, which strictly require a proline as P1' amino acid to reduce the cleavage C-terminal to the arginyl residue. Several analogs were prepared by incorporation of different P1' amino acids found in natural thrombin substrates. The most potent inhibitor was I-11 [dCha-Pro-N(Me)Arg-Thr-(Gly)5-DYEPIPEEA-Cha-dGlu] with a Ki of 37 pM. I-11 is highly selective and no inhibition of the related serine proteases trypsin, factor Xa and plasmin was observed. The stability of I-11 in human plasma in vitro was strongly improved compared to hirulog-1. In addition, a significantly reduced plasma clearance of I-11 was observed after intravenous injection in rats. Results from molecular modeling suggest that a strong reorganization of the hydrogen bonds in the active site of thrombin may result in the proteolytic stability found in this inhibitor series.     

Resveratrol-salicylate derivatives as selective DNMT3 inhibitors and anticancer agents

Resveratrol is a natural polyphenol with plethora of biological activities. Resveratrol has previously shown to decrease DNA-methyltransferase (DNMT) enzymes expression and to reactivate silenced tumor suppressor genes. Currently, it seems that no resveratrol analogs have been developed as DNMT inhibitors. Recently, we reported the synthesis of resveratrol-salicylate derivatives and by examining the chemical structure of these analogs, we proposed that these compounds could exhibit DNMT inhibition especially that they resembled NSC 14778, a compound we previously identified as a DNMT inhibitor by virtual screening. Indeed, using in vitro DNMT inhibition assay, some of the resveratrol-salicylate analogs we screened in this work that showed selective inhibition against DNMT3 enzymes which were greater than resveratrol. A molecular docking study revealed key binding interactions with DNMT3A and DNMT3B enzymes. In addition, the most active analog, 10 showed considerable cytotoxicity against three human cancer cells; HT-29, HepG2 and SK-BR-3, which was greater than resveratrol. Further studies are needed to understand the anticancer mechanisms of these derivatives.     

Characterization of the interactions of a bifunctional inhibitor with alpha-thrombin by molecular modelling and peptide synthesis.

A potent thrombin inhibitor, [D-Phe45, Arg47] hirudin 45-65, that contains an active site-directed sequence D-Phe-Pro-Arg-Pro, an exosite specific fragment hirudin 55-65 (H55-65) and a linker portion hirudin 49-54, was designed based on the hirudin sequence [DiMaio et al. (1990) J. Biol. Chem., 265, 21698-21798]. A three-dimensional model of the complex between the B-chain of human thrombin and the inhibitor [D-Phe45, Arg47] hirudin 45-65 was constructed using molecular modelling starting from the X-ray C alpha coordinates of the thrombin-hirudin complex and the NMR-derived structure of the thrombin-bound hirudin 55-65. The contribution of the H49-54 fragment to the thrombin-inhibitor interaction was deduced by examining a series of analogs containing single glycine substitution and analogs with reduced number of residues within the linker. The results were consistent with the molecular modelling observations i.e. the H49-54 fragment serves the role of a spacer in the binding interaction and could be replaced by four glycine residues. The studies on the interaction of the exosite-directed portion of the inhibitor with thrombin using a series of synthetic H55-65 analogs demonstrated that residues AspH55 to ProH60 play a major role in binding to human thrombin where the side chains of PheH56, IleH59 and GluH57 showed critical contributions. Molecular modelling suggested that these side chains may contribute to inter- and intramolecular hydrophobic and electrostatic interactions, respectively.     

Alanine racemase of Pseudomonas. Observations on subtrate and inhibitor specificity

Systematic studies with purified alanine racemase and a number of substrate analogs permit the generalization that effective competitive inhibition is limited to 2- and 3-carbon compounds. A free α-amino group was not necessary for relatively tight binding; compounds lacking an amino group, or with an α-amino group acylated even by a bulky substituent, were bound as tightly as alanine. Substitution at the α-carbon of alanine (i.e., replacement of the α-H) eliminated binding, while substitution at the β-carbon generally reduced binding. Of several inhibitory compounds tested for substrate activity by H exchange with ³H₂O, only glycine appeared active. Covalent binding to the enzyme by halo analogs was not demonstrated.     

Antioxidation and tyrosinase inhibition of polyphenolic curcumin analogs

A series of polyphenolic curcumin analogs were synthesized and their inhibitory effects on mushroom tyrosinase and the inhibition of 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical formation were evaluated. The results indictated that the analogs possessing m-diphenols and o-diphenols exhibited more potent inhibitory activity on tyrosinase than reference compound rojic acid, and that the analogs with o-diphenols exhibited more potent inhibitory activity of DPPH free-radical formation than reference compound vitamin C. The inhibition kinetics, analyzed by Lineweaver-Burk plots, revealed that compounds B(2) and C(2) bearing o-diphenols were non-competitive inhibitors, while compounds B(11) and C(11) bearing m-diphenols were competitive inhibitors. In particular, representative compounds C(2) and B(11) showed no side effects at a dose of 2,000 mg/kg in a preliminary evaluation of acute toxicity in mice. These results suggest that such polyphenolic curcumin analogs might serve as lead compounds for further design of new potential tyrosinase inhibitors.     

1-Aminoisoquinoline as benzamidine isoster in the design and synthesis of orally active thrombin inhibitors

Replacement of the highly basic benzamidine moiety of NAPAP by the moderately basic 1-aminoisoquinoline moiety resulted in thrombin inhibitors with improved selectivity towards trypsin and enhanced Caco-2 cell permeability.     

Changes in the platelet phosphoinositides during the first minute after stimulation of washed rabbit platelets with thrombin.

Experiments with washed platelets from rabbits demonstrate that stimulation with a low concentration of thrombin (0.1 unit/ml) that causes maximal aggregation and partial release of granule contents does not significantly decrease the amount of phosphatidylinositol 4,5-bisphosphate [ PtdIns (4,5)P2] at 10s; this contrasts with ADP stimulation. The amount of PtdIns (4,5)P2 was significantly decreased by a higher concentration of thrombin (0.3 unit/ml). Increased turnover of the PtdIns (4,5)P2 at 60s was indicated by changes in labelling with [3H]glycerol in platelets stimulated with both concentrations of thrombin. An unexpected observation with the lower thrombin concentration was a significant increase in the amount of phosphatidylinositol ( PtdIns ) at 10s. This contrasts with data from other laboratories, which indicate that thrombin causes a significant decrease in PtdIns . At 60s, with the lower concentration of thrombin, PtdIns was significantly decreased. With the higher concentration of thrombin there was a significant decrease in the amount of PtdIns at 10s, in keeping with the data from other laboratories. The initial increase in PtdIns may not have been observed by other investigators because higher concentrations of thrombin were used. The reaction involved in this initial increase in the amount of PtdIns does not appear to be increased degradation of PtdIns4P or PtdIns (4,5)P2, since their total amount was unchanged at 10s. The magnitude of the increase in PtdIns is such that more than the existing pool of phosphatidic acid would have to be converted into PtdIns to account for the increase. It is suggested that synthesis of phosphatidic acid de novo from dihydroxyacetone phosphate and glycerol 3-phosphate might be the source of phosphatidic acid, which leads to increased PtdIns at 10s with the lower concentration of thrombin. Thus it appears that the initial response of platelets to thrombin does not require an early change in PtdIns (4,5)P2 and may involve stimulation of synthesis de novo of PtdIns via phosphatidic acid.     

Characterization of glycine betaine porter I from Listeria monocytogenes and its roles in salt and chill tolerance

Listeria monocytogenes is a pathogenic bacterium that can grow at low temperatures and elevated osmolarity. The organism survives these stresses by the intracellular accumulation of osmolytes: low-molecular-weight organic compounds which exert a counterbalancing force. The primary osmolyte in L. monocytogenes is glycine betaine, which is accumulated from the environment via two transport systems: glycine betaine porter I, an Na(+)-glycine betaine symporter; and glycine betaine porter II, an ATP-dependent transporter. The biochemical characteristics of glycine betaine porter I were investigated in a mutant strain (LTG59) lacking the ATP-dependent transporter. At 4% NaCl, glycine betaine uptake in LTG59 was about fivefold lower than in strain DP-L1044, which has both transporters, indicating that the ATP-dependent transporter is the primary means by which glycine betaine enters the cell. In the absence of osmotic stress, cold-activated uptake by both transporters was most rapid between 7 and 12 degrees C, but a larger fraction of the total uptake was via the ATP-dependent transporter than was observed under salt-stressed conditions. Twelve glycine betaine analogs were tested for their ability to inhibit glycine betaine uptake and growth of stressed cultures. Carnitine, dimethylglycine, and gamma-butyrobetaine appear to inhibit the ATP-dependent transporter, while trigonelline and triethylglycine primarily inhibit glycine betaine porter I. Triethylglycine was also able to retard the growth of osmotically stressed L. monocytogenes grown in the presence of glycine betaine.     

The long activations of α2 glycine channels can be described by a mechanism with reaction intermediates ("flip")

The α2 glycine receptor (GlyR) subunit, abundant in embryonic neurons, is replaced by α1 in the adult nervous system. The single-channel activity of homomeric α2 channels differs from that of α1-containing GlyRs, as even at the lowest glycine concentration (20 μM), openings occurred in long (>300-ms) groups with high open probability (P(open); 0.96; cell-attached recordings, HEK-expressed channels). Shut-time intervals within groups of openings were dominated by short shuttings of 5-10 μs. The lack of concentration dependence in the groups of openings suggests that they represent single activations, separated by very long shut times at low concentrations. Several putative mechanisms were fitted by maximizing the likelihood of the entire sequence of open and shut times, with exact missed-events allowance (program hjcfit). Records obtained at several glycine concentrations were fitted simultaneously. The adequacy of the different schemes was judged by the accuracy with which they predicted not only single-channel data but also the time course and concentration dependence of macroscopic responses elicited by rapid glycine applications to outside-out patches. The data were adequately described only with schemes incorporating a reaction intermediate in the activation, and the best was a flip mechanism with two binding sites and one open state. Fits with this mechanism showed that for α2 channels, the opening rate constant is very fast, ～130,000 s(-1), much as for α1β GlyRs (the receptor in mature synapses), but the estimated true mean open time is 20 times longer (around 3 ms). The efficacy for the flipping step and the binding affinity were lower for α2 than for α1β channels, but the overall efficacies were similar. As we previously showed for α1 homomeric receptors, in α2 glycine channels, maximum P(open) is achieved when fewer than all five of the putative binding sites in the pentamer are occupied by glycine.     

Search for new pharmacophores for antimalarial activity. Part I: Synthesis and antimalarial activity of new 2-methyl-6-ureido-4-quinolinamides

A total of 80 new 2-methyl-6-ureido-4-quinolinamides were synthesized and evaluated for their antimalarial activity. Several analogs elicited the antimalarial effect at MIC of 0.25 mg/mL against the chlooquine-sensitive P. falciparum strain. The IC₅₀ values of the active compounds were observed to be in ng/mL range and two of the analogs have better IC₅₀ value than the standard chloroquine. In the in vivo assay against mdr CQ resistant P. yoelii N67/P. yoelii nigeriensis, however, none of the compound showed complete suppression of parasitemia on day 7. One of the compounds displayed significant antibacterial effect against several strains of bacteria and was many-fold better than the standard drug gentamicin.     

Synthesis and evaluation of inhibitors of cytochrome P450 3A (CYP3A) for pharmacokinetic enhancement of drugs

The HIV protease inhibitor ritonavir (RTV) is also a potent inhibitor of the metabolizing enzyme cytochrome P450 3A (CYP3A) and is clinically useful in HIV therapy in its ability to enhance human plasma levels of other HIV protease inhibitors (PIs). A novel series of CYP3A inhibitors was designed around the structural elements of RTV believed to be important to CYP3A inhibition, with general design features being the attachment of groups that mimic the P2–P3 segment of RTV to a soluble core. Several analogs were found to strongly enhance plasma levels of lopinavir (LPV), including 8, which compares favorably with RTV in the same model. Interestingly, an inverse correlation between in vitro inhibition of CYP3A and elevation of LPV was observed. The compounds described in this study may be useful for enhancing the pharmacokinetics of drugs that are metabolized by CYP3A.     

Tubulin inhibitors. Synthesis and biological activity of HTI-286 analogs with B-segment heterosubstituents

Modifications of the B-segment of HTI-286 (2) produced a class of analogs incorporating heteroatom-substituents. The structure-activity relationship was studied. Analogs bearing methylsulfide and fluoride groups exhibited potency comparable to that of the parent compound HTI-286 and to paclitaxel in cytotoxicity assays against KB-3-1 cell lines. These analogs were more potent than paclitaxel against P-glycoprotein expressing KB-8-5 and KB-V1 cell lines. Several analogs showed strong inhibition of tubulin polymerization.