Substrate competitive inhibitors of IGF-1 receptor kinase

IGF-1 and its receptor play a pivotal role in many cancers, and therefore, IGF-1R is an attractive target for the design of inhibitors. In this communication, we report on a number of lead compounds for inhibitors of the isolated IGF-1R kinase. The search for these compounds utilized two novel in vitro assays and was aided by the knowledge of the three-dimensional structure of the insulin receptor kinase domain, which is 84% homologous to the IGF-1R kinase domain. The most potent inhibitor found in these assays was tyrphostin AG 538, with an IC(50) = 400 nM. In computer modeling, AG 538 was placed in the kinase domain of the insulin receptor and was able to sit in place of tyrosines 1158 and 1162, which undergo autophosphorylation. Experimentally it is indeed found that AG 538 does not compete with ATP but competes with the IGF-1R substrate. We prepared I-OMe AG 538, which is more hydrophobic and less sensitive to oxidation than AG 538. Both AG 538 and I-OMe AG 538 inhibit IGR-1R autophosphorylation in intact cells in a dose-dependent manner but I-OMe-AG 538 is superior, probably because of its enhanced hydrophobic nature. Both compounds inhibit the activation of the downstream targets PKB and Erk2. These findings suggest that AG 538 and I-OMe-AG 538 can serve as a lead compound for the development of substrate competitive inhibitors of the IGF-1R. The possible advantage of substrate competitive inhibitors vis-à-vis ATP competitive inhibitors is discussed.     

Resistance of herpes simplex virus type 1 to peptidomimetic ribonucleotide reductase inhibitors: selection and characterization of mutant isolates.

Herpes simplex virus (HSV) encodes its own ribonucleotide reductase (RR), which provides the high levels of deoxynucleoside triphosphates required for viral DNA replication in infected cells. HSV RR is composed of two distinct subunits, R1 and R2, whose association is required for enzymatic activity. Peptidomimetic inhibitors that mimic the C-terminal amino acids of R2 inhibit HSV RR by preventing the association of R1 and R2. These compounds are candidate antiviral therapeutic agents. Here we describe the in vitro selection of HSV type 1 KOS variants with three- to ninefold-decreased sensitivity to the RR inhibitor BILD 733. The resistant isolates have growth properties in vitro similar to those of wild-type KOS but are more sensitive to acyclovir, possibly as a consequence of functional impairment of their RRs. A single amino acid substitution in R1 (Ala-1091 to Ser) was associated with threefold resistance to BILD 733, whereas an additional substitution (Pro-1090 to Leu) was required for higher levels of resistance. These mutations were reintroduced into HSV type 1 KOS and shown to be sufficient to confer the resistance phenotype. Studies in vitro with RRs isolated from cells infected with these mutant viruses demonstrated that these RRs bind BILD 733 more weakly than the wild-type enzyme and are also functionally impaired, exhibiting an elevated dissociation constant (Kd) for R1-R2 subunit association and/or reduced activity (kcat). This work provides evidence that the C-terminal end of HSV R1 (residues 1090 and 1091) is involved in R2 binding interactions and demonstrates that resistance to subunit association inhibitors may be associated with compromised activity of the target enzyme.     

Non-competitive inhibition of ornithine decarboxylase by a phosphopeptide and phosphoamino acids

In Tetrahymena pyriformis the cytosolic ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity is considerably inhibited by the presence of polyamines in the growth medium, while the nuclear ornithine decarboxylase is only slightly affected. Experimental evidence suggests that the presence of putrescine and/or spermidine elicits the appearance of non-competitive inhibitors of ornithine decarboxylase. One of the inhibitors has a molecular weight of 25,000 and properties of antizyme. In addition, two other low molecular weight inhibitors are extracted, one which is a phosphoserine oligopeptide, and the other which is phosphotyrosine. All inhibit non-competitively the homologous and heterologous (Escherichia coli and rat liver) ornithine decarboxylases. Similarly, non-competitive inhibition was obtained when the commercially available phosphoamino acids were tested against the already mentioned ornithine decarboxylases.     

Identification of highly potent and selective MMP2 inhibitors addressing the S1′ subsite with d-proline-based compounds

MMP2 and MMP9, also called gelatinases, play a primary role in the angiogenic switch, as a fundamental step of tumor progression, and show high degree of structural similarity. Clinically successful gelatinase inhibitors need to be highly selective as opposite effects have been found for the two enzymes, and the S1′ subsite is the major driver to attain selective and potent inhibitors. The synthesis of d-proline-derived hydroxamic acids containing diverse appendages at the amino group, varying in length and decoration allowed to give insight on the MMP2/MMP9 selectivity around the S1′ subsite, resulting in the identification of sub-nanomolar compounds with high selectivity up to 730. Molecular docking studies revealed the existence of an additional hydrophobic channel at the bottom of S1′ subsite for MMP2 enzyme useful to drive selectivity towards such gelatinase.     

Extended binding inhibitors of chymotrypsin that interact with leaving group subsites S1'-S3'

We have synthesized inhibitors of chymotrypsin, based on fluoromethyl ketones, that bind at S and S' subsites. "Small" inhibitors of serine proteases, which have previously been synthesized, only interact with S subsites. The parent compound is Ac-Leu-ambo-Phe-CF2H (1) (Ki = 25 X 10(-6) M). This inhibitor was modified by successively replacing H of the -CF2H group by -CH2CH2CONHCH3, (4), -CH2CH2CONH-Leu-NHMe (5), -CH2CH2CONH-Leu-Val-OEt (6), and -CH2CH2CONH-Leu-Arg-OMe (7). Corresponding Ki values are 7.8 (4), 0.23 (5), 0.21 (6), and 0.014 (7) microM. Extending 5 to 6 by addition of Val-OEt at P3' does not decrease Ki. In contrast, extension of 5 to 7 by incorporating Arg-OMe at P3' decreases Ki approximately 15-fold, suggesting interaction between Arg and the S3' subsite but no corresponding interaction at that subsite with Val. These results are in accordance with results obtained with the homologous family of avian ovomucoid third domain proteins. Proteins with Arg at the P3' position show highly favorable interactions with the protease at the S3' subsite [Park, S. J. (1985) Ph.D. Thesis, Purdue University; M. Laskowski, Jr., personal communication]. These results establish that incorporation of residues which interact with S' subsites significantly increases the efficacy of inhibitors and that valuable information concerning the most effective amino acid composition of small inhibitors can be obtained from the amino acid sequence of protein inhibitors.     

Non-competitive inhibitioof ornithine decarboxylase by a phosphopeptide and phosphoamino acids

In Tetrahymena pyriformis the cytosolic ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity is considerably inhibited by the presence of polyamines in the growth medium, while the nuclear ornithine decarboxylase is only slightly affected. Experimental evidence suggests that the presence of putrescine and/or spermidine elicits the appearance of non-competitive inhibitors of ornithine decarboxylase. One pf the inhibitors has a molecular weight of 25 000 and properties of antizume. In addition, two other low molecular weight inhibitors are extracted, one which is a phosphoserine oligopeptide, and other which is phosphotyrosine. All inhibit non-competitively the homologous and heterologous (Escherichia coli and rat liver) ornithine decarboxylases. Similarly, non-competitive inhibition was obtained when the commercially available phosphoamino acids wre tested against the already mentioned ornithine decarboxylases.     

13C NMR study of the stereospecificity of the thiohemiacetals formed on inhibition of papain by specific enantiomeric aldehydes

The inhibition of papain by N-acetyl-D- and N-acetyl-L-phenylalanyl[1-13C]glycinal was investigated by 13C nuclear magnetic resonance (NMR) spectroscopy. Both the L- and D-aldehyde enantiomers formed thiohemiacetals with papain. The 13C-enriched carbon of the thiohemiacetals formed with the L- and D-aldehydes has chemical shifts at 74.7 and 75.1 ppm, respectively. The difference in chemical shift for the two inhibitor complexes is attributed to each forming a different diastereomeric papain thiohemiacetal. Each enantiomeric inhibitor formed two diastereomeric thiohemiacetals with chiral thiols but produced a single diastereoisomer with papain. It is concluded that with papain thiohemiacetal formation is stereospecific. The D inhibitor is bound only 5-fold less tightly than the L inhibitor, which suggests that in both these inhibitor complexes the phenyl ring of the inhibitor phenylalanine is bound at the S2 hydrophobic pocket of papain. This is supported by computer modeling studies that show that both the N-acetyl-D- and N-acetyl-L-phenylalanine moieties can be separately fitted into the S2 subsite with the phenyl ring of phenylalanine in the S2 hydrophobic pocket. It is concluded that thiohemiacetal formation at S1 (S1 and S1' are the active center amino acid binding sites) is stereospecific with both D and L inhibitors. Computer modeling studies support this showing that, due to steric hindrance between the thiohemiacetal hydroxyl group and the backbone amide nitrogen of serine-24, only one of the two possible thiohemiacetal enantiomers can be formed at the S1 subsite. The thiohemiacetals formed from both the D- and L-aldehyde inhibitors therefore have only one permitted conformation at S1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Predicting subsite interactions of plasmin with substrates and inhibitors through computational docking analysis

Plasmin plays important roles in various physiological systems. The identification of inhibitors controlling its regulation represents a promising drug-discovery challenge. To develop selective inhibitors of plasmin, structural information of the binding modes is crucial. Here, a computational docking study was conducted to provide structural insight into plasmin subsite interactions with substrates/inhibitors. Predicted binding modes of two peptide-substrates (D/L-Ile-Phe-Lys), and potent and weak inhibitors (YO-2 and PKSI-527) suggested non-prime and prime subsite interactions relevant to recognition by plasmin. Predicted binding modes also correlated well with the experimental structure-activity relationships for plasmin substrates/inhibitors, namely the differences of K(M) values between the D- and L-peptide-substrates and inhibitory potencies of YO-2 and PKSI-527. In particular, interaction observed at a hydrophobic pocket near S2 and at a tunnel-shaped hydrophobic S1' was strongly suggested to be significantly involved in tight binding of inhibitors to plasmin. Our present findings may aid in the design of potent and selective plasmin inhibitors.     

A tryptophan in the bottleneck of the catalytic gorge of an invertebrate acetylcholinesterase confers relative resistance to carbamate and organophosphate inhibitors

Amphioxus, an invertebrate chordate, has two acetylcholinesterases (AChEs): cholinesterase 1 (ChE1) and cholinesterase 2 (ChE2). ChE1 is up to 329-fold more resistant to a variety of carbamate and organophosphate inhibitors, including a number of insecticides, when compared with ChE2. One difference between the two enzymes is at the position homologous to Phe331 in Torpedo AChE. In Torpedo AChE, this residue is a component of the hydrophobic subsite and defines one side of the bottleneck in the catalytic gorge of the enzyme. In ChE1, the homologous residue is Trp353; in ChE2, it is Phe353. We used site-directed mutagenesis to investigate the proposal that the resistance of ChE1 to inhibition by carbamates and organophosphates was due to this difference, creating a ChE1 W353F mutant to widen the bottleneck. The mutation virtually abolishes the difference in sensitivity to the inhibitors. The ChE1 W353F mutant is only 2- to 3-fold more resistant than ChE2 to carbamates and is actually 2.5- to 10-fold more sensitive to inhibition by organophosphates. The differences in resistance are due to different affinities of the enzymes for the inhibitors, not different reactivities. Molecular modeling supports the proposal that the difference in inhibition is due to the width of the bottleneck of the gorge. Our results have implications for insecticide resistance in insects, in particular mosquitoes and aphids.     

Structural requirements of pyrimidine, thienopyridine and ureido thiophene carboxamide-based inhibitors of the checkpoint kinase 1: QSAR, docking, molecular dynamics analysis

Our focus of current research is directed toward clarification of novel inhibitors (pyrazolo[1,5-a] pyrimidine (PP), thienopyridines (TP) and 2-ureido thiophene carboxamide (UTC) derivatives) targeting Checkpoint kinase 1 (CHK(1)), which is an oncology target of significant current interest. Our computational approaches include: (i) QSAR analysis was carried out on the computed steric/electrostatic/hydrophobic/hydrogen bond donor/hydrogen bond acceptor interactions with the pseudoreceptor surface, which yielded predictive models capable of explaining much of the variance of inhibitors. The resultant optimum QSAR/CoMFA models exhibited (N(training) = 51, N(test) = 16, R(cv) (2) = 0.47, R(pred) (2) = 0.7) for PP, (N(training) = 45, N(test) = 9, R(cv) (2) = 0.52, R(pred) (2) = 0.75) for TP and (N(training) = 58, N(test) = 15, R(cv) (2) = 0.67, R(pred) (2) = 0.88) for UTC. (ii) Molecular docking and molecular dynamics simulations experiments of the inhibitors into the binding site of CHK(1) aided the interpretation of the QSAR models and demonstrated the binding modes in the aspects of inhibitor's conformation, subsite interaction, and hydrogen bonding interactions, which indicated that a set of critical residues (Cys87, Glu91, Glu85, Ser147, Asp148, Glu17, Leu84 and Asn135) played a key role in the drug-target interactions. The obtained results in the present work will be fruitful for the design of new potent and selective inhibitors of CHK(1).     

Predicting subsite interactions of plasmin with substrates and inhibitors through computational docking analysis

Plasmin plays important roles in various physiological systems. The identification of inhibitors controlling its regulation represents a promising drug-discovery challenge. To develop selective inhibitors of plasmin, structural information of the binding modes is crucial. Here, a computational docking study was conducted to provide structural insight into plasmin subsite interactions with substrates/inhibitors. Predicted binding modes of two peptide-substrates (D/L-Ile-Phe-Lys), and potent and weak inhibitors (YO-2 and PKSI-527) suggested non-prime and prime subsite interactions relevant to recognition by plasmin. Predicted binding modes also correlated well with the experimental structure–activity relationships for plasmin substrates/inhibitors, namely the differences of K_M values between the D- and L-peptide-substrates and inhibitory potencies of YO-2 and PKSI-527. In particular, interaction observed at a hydrophobic pocket near S2 and at a tunnel-shaped hydrophobic S1′ was strongly suggested to be significantly involved in tight binding of inhibitors to plasmin. Our present findings may aid in the design of potent and selective plasmin inhibitors.     

Design of inhibitors for human tissue kallikrein using non-natural aromatic and basic amino acids

We explored the unique substrate specificity of the primary S, subsite of human urinary kallikrein (hK1), which accepts both Phe or Arg synthesizing and assaying peptides derived from Phenylacetyl-Phe-Ser-Arg-EDDnp, a previously described inhibitor with analgesic and anti-inflammatory activities [Emim et al., Br. J. Pharmacol. 130 (2000), 1099-1107]. Phe was substituted by amino acids containing larger aliphatic or aromatic side chains as well as by non-natural basic amino acids, which were designed to combine a large hydrophobic and/or aromatic group with a positively-charged group at their side chains. In general, all peptides with basic amino acids represented better inhibitors than those with hydrophobic amino acids. Furthermore, the S1 subsite specificity proved to be much more selective than the mere distinction between Phe and Arg, for minor differences in the side chains of the non-natural amino acids resulted in major differences in the Ki values. Finally, we present a series of peptides that were assayed as competitive inhibitors for human tissue kallikrein that may lead to the development of novel peptides, which are both more potent and selective.     

Exploration of the S(')(1) subsite of neprilysin: a joined molecular modeling and site-directed mutagenesis study

Based on the recently described three-dimensional model of the 507-749 region of neprilysin, which contains the catalytic site of the enzyme, experiments were performed to improve the proposed topology of its large and hydrophobic S(')(1) subsite. Docking studies, site-directed mutagenesis, and biochemical studies were combined. The mutations of various residues proposed to be part of the S(')(1) subsite (F563A, F564A, M579A, F716A, and I718A) did not induce major structural reorganization of the active site as demonstrated by the slight modification of the enzyme activity. The mutations were also analyzed by measuring the inhibitory potencies of thiol inhibitors containing P(')(1) moieties of increasing sizes. These results combined with molecular modeling studies support the proposed topology of the S(')(1) subsite. This, and the critical role of F563 and M579 in inhibitor binding, could facilitate the synthesis of new potent and selective inhibitors.     

Solution structure of the catalytic domain of human stromelysin complexed with a hydrophobic inhibitor.

Stromelysin, a representative matrix metalloproteinase and target of drug development efforts, plays a prominent role in the pathological proteolysis associated with arthritis and secondarily in that of cancer metastasis and invasion. To provide a structural template to aid the development of therapeutic inhibitors, we have determined a medium-resolution structure of a 20-kDa complex of human stromelysin's catalytic domain with a hydrophobic peptidic inhibitor using multinuclear, multidimensional NMR spectroscopy. This domain of this zinc hydrolase contains a mixed beta-sheet comprising one antiparallel strand and four parallel strands, three helices, and a methionine-containing turn near the catalytic center. The ensemble of 20 structures was calculated using, on average, 8 interresidue NOE restraints per residue for the 166-residue protein fragment complexed with a 4-residue substrate analogue. The mean RMS deviation (RMSD) to the average structure for backbone heavy atoms is 0.91 A and for all heavy atoms is 1.42 A. The structure has good stereochemical properties, including its backbone torsion angles. The beta-sheet and alpha-helices of the catalytic domains of human stromelysin (NMR model) and human fibroblast collagenase (X-ray crystallographic model of Lovejoy B et al., 1994b, Biochemistry 33:8207-8217) superimpose well, having a pairwise RMSD for backbone heavy atoms of 2.28 A when three loop segments are disregarded. The hydroxamate-substituted inhibitor binds across the hydrophobic active site of stromelysin in an extended conformation. The first hydrophobic side chain is deeply buried in the principal S'1 subsite, the second hydrophobic side chain is located on the opposite side of the inhibitor backbone in the hydrophobic S'2 surface subsite, and a third hydrophobic side chain (P'3) lies at the surface.     

Structure-directed discovery of potent non-peptidic inhibitors of human urokinase that access a novel binding subsite

BACKGROUND: Human urokinase-type plasminogen activator has been implicated in the regulation and control of basement membrane and interstitial protein degradation. Because of its role in tissue remodeling, urokinase is a central player in the disease progression of cancer, making it an attractive target for design of an anticancer clinical agent: Few urokinase inhibitors have been described, which suggests that discovery of such a compound is in the early stages. Towards integrating structural data into this process, a new human urokinase crystal form amenable to structure-based drug design has been used to discover potent urokinase inhibitors. RESULTS: On the basis of crystallographic data, 2-naphthamidine was chosen as the lead scaffold for structure-directed optimization. This co-crystal structure shows the compound binding at the primary specificity pocket of the trypsin-like protease and at a novel binding subsite that is accessible from the 8-position of 2-napthamidine. This novel subsite was characterized and used to design two compounds with very different 8-substituents that inhibit urokinase with K(i) values of 30-40 nM. CONCLUSIONS: Utilization of a novel subsite yielded two potent urokinase inhibitors even though this site has not been widely used in inhibitor optimization with other trypsin-like proteases, such as those reported for thrombin or factor Xa. The extensive binding pockets present at the substrate-binding groove of these other proteins are blocked by unique insertion loops in urokinase, thus necessitating the utilization of additional binding subsites. Successful implementation of this strategy and characterization of the novel site provides a significant step towards the discovery of an anticancer clinical agent.     

The acylaminoacyl peptidase from Aeropyrum pernix K1 thought to be an exopeptidase displays endopeptidase activity

Mammalian acylaminoacyl peptidase, a member of the prolyl oligopeptidase family of serine peptidases, is an exopeptidase, which removes acylated amino acid residues from the N terminus of oligopeptides. We have investigated the kinetics and inhibitor binding of the orthologous acylaminoacyl peptidase from the thermophile Aeropyrum pernix K1 (ApAAP). Complex pH-rate profiles were found with charged substrates, indicating a strong electrostatic effect in the surroundings of the active site. Unexpectedly, we have found that oligopeptides can be hydrolysed beyond the N-terminal peptide bond, demonstrating that ApAAP exhibits endopeptidase activity. It was thought that the enzyme is specific for hydrophobic amino acids, in particular phenylalanine, in accord with the non-polar S1 subsite of ApAAP. However, cleavage after an Ala residue contradicted this notion and demonstrated that P1 residues of different nature may bind to the S1 subsite depending on the remaining peptide residues. The crystal structures of the complexes formed between the enzyme and product-like inhibitors identified the oxyanion-binding site unambiguously and demonstrated that the phenylalanine ring of the P1 peptide residue assumes a position different from that established in a previous study, using 4-nitrophenylphosphate. We have found that the substrate-binding site extends beyond the S2 subsite, being capable of binding peptides with a longer N terminus. The S2 subsite displays a non-polar character, which is unique among the enzymes of this family. The S3 site was identified as a hydrophobic region that does not form hydrogen bonds with the inhibitor P3 residue. The enzyme-inhibitor complexes revealed that, upon ligand-binding, the S1 subsite undergoes significant conformational changes, demonstrating the plasticity of the specificity site.     

Structural basis of the unusual stability and substrate specificity of ervatamin C, a plant cysteine protease from Ervatamia coronaria

Ervatamin C is an unusually stable cysteine protease from the medicinal plant Ervatamia coronaria belonging to the papain family. Though it cleaves denatured natural proteins with high specific activity, its activity toward some small synthetic substrates is found to be insignificant. The three-dimensional structure and amino acid sequence of the protein have been determined from X-ray diffraction data at 1.9 A (R = 17.7% and R(free) = 19.0%). The overall structure of ervatamin C is similar to those of other homologous cysteine proteases of the family, folding into two distinct left and right domains separated by an active site cleft. However, substitution of a few amino acid residues, which are conserved in the other members of the family, has been observed in both the domains and also at the region of the interdomain cleft. Consequently, the number of intra- and interdomain hydrogen-bonding interactions is enhanced in the structure of ervatamin C. Moreover, a unique disulfide bond has been identified in the right domain of the structure, in addition to the three conserved disulfide bridges present in the papain family. All these factors contribute to an increase in the stability of ervatamin C. In this enzyme, the nature of the S2 subsite, which is the primary determinant of specificity of these proteases, is similar to that of papain, but at the S3 subsite, Ala67 replaces an aromatic residue, and has the effect of eliminating sufficient hydrophobic interactions required for S3-P3 stabilization. This provides the possible explanation for the lower activity of ervatamin C toward the small substrate/inhibitor. This substitution, however, does not affect the binding of denatured natural protein substrates to the enzyme significantly, as there exist a number of additional interactions at the enzyme-substrate interface outside the active site cleft.     

Kinetic and modeling studies of S3-S3' subsites of HIV proteinases.

Kinetic analysis and modeling studies of HIV-1 and HIV-2 proteinases were carried out using the oligopeptide substrate [formula: see text] and its analogs containing single amino acid substitutions in P3-P3' positions. The two proteinases acted similarly on the substrates except those having certain hydrophobic amino acids at P2, P1, P2', and P3' positions (Ala, Leu, Met, Phe). Various amino acids seemed to be acceptable at P3 and P3' positions, while the P2 and P2' positions seemed to be more restrictive. Polar uncharged residues resulted in relatively good binding at P3 and P2 positions, while at P2' and P3' positions they gave very high Km values, indicating substantial differences in the respective S and S' subsites of the enzyme. Lys prevented substrate hydrolysis at any of the P2-P2' positions. The large differences for subsite preference at P2 and P2' positions seem to be at least partially due to the different internal interactions of P2 residue with P1', and P2' residue with P1. As expected on the basis of amino acid frequency in the naturally occurring cleavage sites, hydrophobic residues at P1 position resulted in cleavable peptides, while polar and beta-branched amino acids prevented hydrolysis. On the other hand, changing the P1' Pro to other amino acids prevented substrate hydrolysis, even if the substituted amino acid had produced a good substrate in other oligopeptides representing naturally occurring cleavage sites. The results suggest that the subsite specificity of the HIV proteinases may strongly depend on the sequence context of the substrate.     

Adenosine-anchored triphosphate subsite probing: distinguishing between HER-2 and HER-4 tyrosine protein kinases

A strategy of full-site occupancy and stereospecific recognition in the triphosphate subsite was used to specifically inhibit two protein kinases HER-2 and HER-4 from the EGFR family. The SAR profiles of a panel of adenosine-anchored bicyclic heterocycles against HER-2 and HER-4 indicated that specificity can be derived for highly homologous protein kinases from stereospecific recognition in the triphosphate-subsite.