Design Of Peptide Inhibitors Research Articles

622 POSTER Discovery and characterization of a novel multi-targeted tyrosine kinase inhibitor with activity against c-ret, pdgfr, c-kit and c-src

Protein kinases are now the second largest group of drug targets, and most protein kinase inhibitors in clinical development are directed towards the ATP-binding site. However, these inhibitors must compete with high intracellular ATP concentrations and they must discriminate between the ATP-binding sites of all protein kinases as well the other proteins that also utilise ATP. It would therefore be beneficial to target sites on protein kinases other than the ATP-binding site. This review describes the discovery, characterisation and use of peptide inhibitors of protein kinases. In many cases, the development of these peptides has resulted from an understanding of the specific protein-binding partners for a particular protein kinase. In addition, novel peptide sequences have been discovered in library screening approaches and have provided new leads in the discovery and/or design of peptide inhibitors of protein kinases. These approaches are therefore providing exciting new opportunities in the development of ATP non-competitive inhibitors of protein kinases.

Template‐assisted rational design of peptide inhibitors of furin using the lysine fragment of the mung bean trypsin inhibitor

Highly active, small‐molecule furin inhibitors are attractive drug candidates to fend off bacterial exotoxins and viral infection. Based on the 22‐residue, active Lys fragment of the mung bean trypsin inhibitor, a series of furin inhibitors were designed and synthesized, and their inhibitory activity towards furin and kexin was evaluated using enzyme kinetic analysis. The most potent inhibitor, containing 16 amino acid residues with a K i value of 2.45 × 10−9 m for furin and of 5.60 × 10−7 m for kexin, was designed with three incremental approaches. First, two nonessential Cys residues in the Lys fragment were deleted via a Cys‐to‐Ser mutation to minimize peptide misfolding. Second, residues in the reactive site of the inhibitor were replaced by the consensus substrate recognition sequence of furin, namely, Arg at P1, Lys at P2, Arg at P4 and Arg at P6. In addition, the P7 residue Asp was substituted with Ala to avoid possible electrostatic interference with furin inhibition. Finally, the extra N‐terminal and C‐terminal residues beyond the doubly conjugated disulfide loops were further truncated. However, all resultant synthetic peptides were found to be temporary inhibitors of furin and kexin during a prolonged incubation, with the scissile peptide bond between P1 and P1′ being cleaved to different extents by the enzymes. To enhance proteolytic resistance, the P1′ residue Ser was mutated to d‐Ser or N‐methyl‐Ser. The N‐methyl‐Ser mutant gave rise to a K i value of 4.70 × 10−8 m for furin, and retained over 80% inhibitory activity even after a 3 h incubation with the enzyme. By contrast, the d‐Ser mutant was resistant to cleavage, although its inhibitory activity against furin drastically decreased. Our findings identify a useful template for the design of potent, specific and stable peptide inhibitors of furin, shedding light on the molecular determinants that dictate the inhibition of furin and kexin.

Rational Design of Peptide Inhibitors of the Sarcoplasmic Reticulum Calcium Pump

The sequence of phospholamban (PLB) is practically invariant among mammalian species. The hydrophobic transmembrane domain has 10 leucine and 8 isoleucine residues. Two roles have been proposed for the leucines; one subset stabilizes PLB oligomers, while a second subset physically interacts with SERCA. On the basis of the sequence of the PLB transmembrane domain, we chemically synthesized a series of peptides and tested their ability to regulate SERCA in reconstituted membranes. In all, eight peptides were studied: a peptide corresponding to the null-cysteine transmembrane domain of PLB (TM-Ala-PLB), two polyleucine peptides (Leu18 and Leu24), polyalanine peptides containing 4, 7, and 12 leucine residues (Leu4, Leu7, and Leu12, respectively), and a polyalanine peptide containing the 9 leucine residues present in the transmembrane domain of PLB with and without the essential Asn34 residue (Asn1Leu9 and Leu9, respectively). With the exception of Leu18, co-reconstitution of the peptides revealed effects on the apparent calcium affinity of SERCA. The TM-Ala-PLB peptide possessed approximately 70% of the inhibitory function of wild-type PLB. The remaining peptides exhibited significant inhibitory activity decreasing in the following order: Leu12, Leu9, Leu24, Leu7, and Leu4. Replacing Asn34 of PLB in the Leu9 peptide resulted in superinhibition of SERCA. On the basis of these observations, we conclude that a partial requirement for SERCA inhibition is met by a simple hydrophobic surface on a transmembrane alpha-helix. In addition, the superinhibition observed for the Asn34-containing peptide suggests that the model peptides mimic the inhibitory properties of PLB. A model is presented in which surface complementarity around key amino acid positions is enhanced in the interaction with SERCA.

Inhibitors of Amyloid Toxicity Based on β-sheet Packing of Aβ40 and Aβ42

Amyloid fibrils associated with Alzheimer's disease and a wide range of other neurodegenerative diseases have a cross beta-sheet structure, where main chain hydrogen bonding occurs between beta-strands in the direction of the fibril axis. The surface of the beta-sheet has pronounced ridges and grooves when the individual beta-strands have a parallel orientation and the amino acids are in-register with one another. Here we show that in Abeta amyloid fibrils, Met35 packs against Gly33 in the C-terminus of Abeta40 and against Gly37 in the C-terminus of Abeta42. These packing interactions suggest that the protofilament subunits are displaced relative to one another in the Abeta40 and Abeta42 fibril structures. We take advantage of this corrugated structure to design a new class of inhibitors that prevent fibril formation by placing alternating glycine and aromatic residues on one face of a beta-strand. We show that peptide inhibitors based on a GxFxGxF framework disrupt sheet-to-sheet packing and inhibit the formation of mature Abeta fibrils as assayed by thioflavin T fluorescence, electron microscopy, and solid-state NMR spectroscopy. The alternating large and small amino acids in the GxFxGxF sequence are complementary to the corresponding amino acids in the IxGxMxG motif found in the C-terminal sequence of Abeta40 and Abeta42. Importantly, the designed peptide inhibitors significantly reduce the toxicity induced by Abeta42 on cultured rat cortical neurons.

Pseudosubstrate peptides inhibit Akt and induce cell growth inhibition.

We have designed peptide inhibitors that potently inhibit Akt both in vitro and inside cells. These peptide inhibitors are selective for Akt versus other closely related kinases. The peptides inhibit the in vitro phosphorylation of a biotinylated Bad peptide by Akt with potency up to 100 nM. We have shown that the binding between Akt1 and these peptide inhibitors requires MgATP. Mutating the two putative Akt phosphorylation sites to Ala (nonsubstrate) in these peptides increases the inhibitory potency while mutating the sites to aspartic acid (phosphorylation mimetic) reduces the potency. When delivered into cells, these peptide inhibitors can inhibit cellular Akt activity and cell growth. Thus, these Akt-specific peptide inhibitors provide prototypes for peptide mimetic drugs as well as very useful tools to dissect cellular functions of Akt.

Design of Specific Peptide Inhibitors for Group I Phospholipase A2: Structure of a Complex Formed between Phospholipase A2 from Naja naja sagittifera (Group I) and a Designed Peptide Inhibitor Val-Ala-Phe-Arg-Ser (VAFRS) at 1.9 Å Resolution Reveals Unique Features,

Phospholipase A(2) (PLA(2)) (E. C. 3.1.1.4) is a common enzyme in the two-way cascade mechanism leading to the production of proinflammatory compounds known as eicosanoids. The binding of phospholipase A(2) to the membrane surface and hydrolysis of phospholipids are thought to involve the formation of a hydrophobic channel into which a single substrate molecule diffuses before its cleavage. To regulate the production of proinflammatory compounds, a specific peptide inhibitor Val-Ala-Phe-Arg-Ser (VAFRS) for the group I PLA(2) enzymes has been designed and synthesized. PLA(2) was isolated from Indian cobra (Naja naja sagittifera) venom and purified to homogeneity. The binding studies indicated the K(i) value of 1.02 +/- 0.10 x 10(-8) M. The purified PLA(2) samples and the designed inhibitor VAFRS were cocrystallized. The crystal structure of the complex was determined and refined to 1.9 A resolution. The peptide binds to PLA(2) at the active site and fills the hydrophobic channel completely. However, its placement with respect to the channel is in the opposite direction as compared to those observed in group II PLA(2)'s. Furthermore, the predominant intermolecular interactions involve strong electrostatic interactions between the side chains of peptide Arg and Asp 49 of PLA(2) together with a number of van der Waals interactions with other residues. A good number of observed interactions between the peptide and the protein indicate the significance of a structure-based drug design approach. The novel factor in the present sequence of the peptide is related to the introduction of a positively charged residue at the C-terminal part of the peptide.

An evolutionary approach for structure-based design of natural and non-natural peptidic ligands.

A new computational approach (PEP) is presented for the structure-based design of linear polymeric ligands consisting of any type of amino acid. Ligands are grown from a seed by iteratively adding amino acids to the growing construct. The search in chemical space is performed by a build-up approach which employs all of the residues of a user-defined library. At every growing step, a genetic algorithm is used for conformational optimization of the last added monomer inside the binding site of a rigid target protein. The binding energy with electrostatic solvation is evaluated to select sequences for further growing. PEP is tested on the peptide substrate binding site of the insulin receptor tyrosine kinase and farnesyltransferase. In both test cases, the peptides designed by PEP correspond to the sequence motifs of known substrates. For tyrosine kinase, tyrosine residues are suggested at position P and P+2. While the tyrosine at P is in agreement with the experimental data, the one at P+2 is a prediction which awaits experimental validation. For farnesyltransferase, it is shown that electrostatic solvation is necessary for the correct design of peptidic inhibitors.

Monoclonal antibodies to mycobacterial DNA gyrase A inhibit DNA supercoiling activity.

DNA gyrase is an essential type II topoisomerase found in bacteria. We have previously characterized DNA gyrase from Mycobacterium tuberculosis and Mycobacterium smegmatis. In this study, several monoclonal antibodies were generated against the gyrase A subunit (GyrA) of M. smegmatis. Three, MsGyrA:C3, MsGyrA:H11 and MsGyrA:E9, were further analyzed for their interaction with the enzyme. The monoclonal antibodies showed high degree of cross-reactivity with both fast-growing and slow-growing mycobacteria. In contrast, none recognized Escherichia coli GyrA. All the three monoclonal antibodies were of IgG1 isotype falling into two distinct types with respect to epitope recognition and interaction with the enzyme. MsGyrA:C3 and MsGyrA:H11 IgG, and their respective Fab fragments, inhibited the DNA supercoiling activity catalyzed by mycobacterial DNA gyrase. The epitope for the neutralizing monoclonal antibodies appeared to involve the region towards the N-terminus (residues 351-415) of the enzyme in a conformation-dependent manner. These monoclonal antibodies would serve as valuable tools for structure-function analysis and immunocytological studies of mycobacterial DNA gyrase. In addition, they would be useful for designing peptide inhibitors against DNA gyrase.

Toward a rational design of peptide inhibitors of ribonucleotide reductase: structure-function and modeling studies.

Mammalian ribonucleotide reductase, a chemotherapeutic target, has two subunits, mR1 and mR2, and is inhibited by AcF(1)TLDADF(7), denoted P7. P7 corresponds to the C-terminus of mR2 and competes with mR2 for binding to mR1. We report results of a structure-function analysis of P7, obtained using a new assay measuring peptide ligand binding to mR1, that demonstrate stringent specificity for Phe at F(7), high specificity for Phe at F(1), and little specificity for the N-acyl group. They support a structural model in which the dominant interactions of P7 occur at two mR1 sites, the F(1) and F(7) subsites. The model is constructed from the structure of Escherichia coli R1 (eR1) complexed with the C-terminal peptide from eR2, aligned sequences of mR1 and eR1, and the trNOE-derived structure of mR1-bound P7. Comparison of this model with similar models constructed for mR1 complexed with other inhibitory ligands indicates that increased F(1) subsite interaction can offset lower F(7) subsite interaction and suggests strategies for the design of new, higher affinity inhibitors.

Bmi-1 collaborates with c-Myc in tumorigenesis by inhibiting c-Myc-induced apoptosis via INK4a/ARF.

The bmi-1 and myc oncogenes collaborate strongly in murine lymphomagenesis, but the basis for this collaboration was not understood. We recently identified the ink4a-ARF tumor suppressor locus as a critical downstream target of the Polycomb-group transcriptional repressor Bmi-1. Others have shown that part of Myc's ability to induce apoptosis depends on induction of p19arf. Here we demonstrate that down-regulation of ink4a-ARF by Bmi-1 underlies its ability to cooperate with Myc in tumorigenesis. Heterozygosity for bmi-1 inhibits lymphomagenesis in Emu-myc mice by enhancing c-Myc-induced apoptosis. We observe increased apoptosis in bmi-1(-/-) lymphoid organs, which can be rescued by deletion of ink4a-ARF or overexpression of bcl2. Furthermore, Bmi-1 collaborates with Myc in enhancing proliferation and transformation of primary embryo fibroblasts (MEFs) in an ink4a-ARF dependent manner, by prohibiting Myc-mediated induction of p19arf and apoptosis. We observe strong collaboration between the Emu-myc transgene and heterozygosity for ink4a-ARF, which is accompanied by loss of the wild-type ink4a-ARF allele and formation of highly aggressive B-cell lymphomas. Together, these results reinforce the critical role of Bmi-1 as a dose-dependent regulator of ink4a-ARF, which on its turn acts to prevent tumorigenesis on activation of oncogenes such as c-myc.

Quantum chemical modelling of the effect of proline residues on peptide conformation

Semiempirical AM1 calculations were performed for quantum chemically optimized minimum-energy conformations of L-alanine oligomers (A)n at n = 7 and their derivatives containing one, two, or three proline residues at various positions along the peptide chain. The effect of proline residues on the peptide conformation was quantified in terms of the conformational “strain energy” and also analyzed in terms of the spatial compatibility of peptides. The defined “strain energy” corresponds to the transformation of the polyalanine peptide from its minimum conformation to the conformation corresponding to that of the proline-containing peptide. The results of calculations indicate that the “strain effect” of proline residues is additive at all locations along the peptide chain, except at the first and the second positions of its N-terminal part. Also, the regular α-helical polyalanine structure was most significantly altered by the presence of some specific motifs around the proline location in the peptide. This, in turn, has its implications in the prediction of the protein secondary structure, as well as in the design of peptide inhibitors and substrates for enzymes and receptors. © 1998 John Wiley & Sons, Inc. Int J Quant Chem 66: 391–396, 1998

A neutralizing epitope of human papillomavirus type 11 is principally described by a continuous set of residues which overlap a distinct linear, surface-exposed epitope.

A panel of monoclonal antibodies (MAbs) which neutralize human papillomavirus type 11 (HPV11) in the athymic mouse xenograph neutralization assay and bind HPV11 virus-like particles (VLPs) has been described. We recently presented evidence that the Gly131-Tyr132 residues of the major capsid protein L1 confer type 11-specific binding. However, residues distally located on the primary L1 sequence also were shown to affect binding. This poses the question whether the epitope is principally centered in the region of Gly131-Tyr132 or, alternatively, is comprised of diversely located residues which come into proximity only upon proper assembly. We analyzed the result of numerous substitutions located between Tyr123 and Val142 of the HPV11 L1 sequence. We show that substitutions at five positions result in loss of binding for one or more of these MAbs by an enzyme-linked immunosorbent assay which measures antibody binding to VLPs. We demonstrate that binding of these MAbs is redirected to HPV16 VLPs which harbor eight type 11-like substitutions within the homologous region. Three of these substitutions did not affect binding when individually substituted in HPV11 but yet were still required to transfer binding to substituted HPV16 VLPs. The results demonstrate that the epitope for this class of neutralizing MAbs, although conformational and requiring VLP assembly for presentation, principally lies along a 20-residue stretch of the L1 major capsid protein. This targets the region for evaluation of the possibility of receptor binding and suggests possibilities for the design of peptide inhibitors of virus infectivity.

Strategy to design peptide inhibitors: structure of a complex of proteinase K with a designed octapeptide inhibitor N-Ac-Pro-Ala-Pro-Phe-DAla-Ala-Ala-Ala-NH2 at 2.5 A resolution.

The crystal structure of a complex formed by the interaction between proteinase K and a designed octapeptide amide, N-Ac-Pro-Ala-Pro-Phe-DAla-Ala-Ala-Ala-NH2, has been determined at 2.5 A resolution and refined to an R-factor of 16.7% for 7,430 reflections in the resolution range of 8.0-2.50 A. The inhibitor forms a stable complex through a series of hydrogen bonds and hydrophobic interactions with the protein atoms and water molecules. The inhibitor is hydrolyzed between Phe4I and DAla5I (I indicates the inhibitor). The two fragments are separated by a distance of 3.2 A between the carbonyl carbon of Phe4I and the main-chain nitrogen of DAla5I. The N-terminal tetrapeptide occupies subsites S1-S5 (S5 for acetyl group), whereas the C-terminal part fits into S1'-S5' region (S5' for amide group). It is the first time that such an extended electron density for a designed synthetic peptide inhibitor has been observed in the prime region of an enzyme of the subtilisin family. In fact, the inhibitor fills the recognition site completely. There is only a slight rearrangement of the protein residues to accommodate the inhibitor. Superposition of the present octapeptide inhibitor on the hexapeptide inhibitor studied previously shows an overall homology of the two inhibitors, although the individual atoms are displaced significantly. It suggests the existence of a recognition site with flexible dimensions. Kinetic studies indicate an inhibition rate of 100% by this specifically designed peptide inhibitor.

Substrate specificity and inhibitor profile of human recombinant p56lck from a baculovirus expression vector.

p56lck, a member of the src family of non-receptor protein receptor kinases, is required for normal signal transduction through the T cell receptor. Inappropriate T cell activation and proliferation has been identified as an early event in auto-immune disease-agents which control T cell activation through modulation of p56lck kinase activity could therefore be potential therapeutic agents for a range of pathological conditions. To identify p56lck inhibitors, we have established an assay system suitable for the high throughput screening of compound libraries. The assay uses enzyme purified from baculovirus infected SF9 cells, and a novel peptidic substrate identified by L. Cantley from a degenerate combinatorial peptide library. We have used this assay system to screen a number of different compounds as potential inhibitors of p56lck. In addition, peptides based on the substrate sequence were also tested to identify a sequence that could be used in the rational design of peptide inhibitors of p56lck.

Kinetics and sequence specificity of processing of prepilin by PilD, the type IV leader peptidase of Pseudomonas aeruginosa

PilD, originally isolated as an essential component for the biogenesis of the type IV pili of Pseudomonas aeruginosa, is a unique endopeptidase responsible for processing the precursors of the P. aeruginosa pilin subunits. It is also required for the cleavage of the leader peptides from the Pdd proteins, which are essential components of an extracellular secretion pathway specific for the export of a number of P. aeruginosa hydrolytic enzymes and toxins. Substrates for PilD are initially synthesized with short, i.e., 6- to 8-amino-acid-long, leader peptides with a net basic charge and share a high degree of amino acid homology through the first 16 to 30 residues at the amino terminus. In addition, they all have a phenylalanine residue at the +1 site relative to the cleavage site, which is N methylated prior to assembly into the oligomeric structures. In this study, the kinetics of leader peptide cleavage from the precursor of the P. aeruginosa pilin subunit by PilD was determined in vitro. The rates of cleavage were compared for purified enzyme and substrate as well as for enzyme and substrate contained within total membranes extracted from P. aeruginosa strains overexpressing the cloned pilD or pilA genes. Optimal conditions were obtained only when both PilD and substrate were contained within total membranes. PilD catalysis of P. aeruginosa prepilin followed normal Michaelis-Menten kinetics, with a measured apparent Km of approximately 650 microM, and a kcat of 180 min-1. The kinetics of PilD processing of another type IV pilin precursor, that from Neisseria gonorrhoeae with a 7-amino-acid-long leader peptide, were essentially the same as that measured for wild-type P. aeruginosa prepilin. Quite different results were obtained for a number of prepilin substrates containing substitutions at the conserved phenylalanine at the +1 position relative to the cleavage site, which were previously shown to be well tolerated in vivo. Substitutions of methionine, serine, and cysteine for phenylalanine show that Km values remain close to that measured for wild-type substrate, while kcat and kcat/Km values were significantly decreased. This indicates that while the affinity of enzyme for substrate is relatively unaffected by the substitutions, the maximum rate of catalysis favors a phenylalanine at this position. Interesting, PilD cleavage of one mutated pillin (asparagine) resulted in a lower Km value of 52.5 microM, which indicates a higher affinity for the enzyme, as well as a lower kcat value of 6.1 min m(-1). This suggests that it may be feasible to design peptide inhibitors of PilD.

Thermodynamics of the interaction of inhibitors with the binding site of recombinant human renin

The independent subsite model is widely used for the design of peptide inhibitors of enzymes with extended active sites. This model assumes that the subsites are independent of each other and that the free energies of binding contributed by the several subsites are additive. We questioned the strict application of this model for structure-activity studies, since one can, a priori, conceive of likely deviations from this model. Accordingly, we tested the independent subsite model by measuring the thermodynamic binding parameters of a series of peptide inhibitors of human renin. This enzyme-inhibitor system was chosen as a model by virtue of the high degree of specificity of renin for its natural substrate, angiotensinogen, and the availability of a large number of structurally similar peptide inhibitors. Although we found the general mode of binding of these renin inhibitors to be primarily hydrophobic, serious deviations from additivity and independent subsite model constraints were observed. We conclude that an important determinant of binding is most probably the conformation assumed by the peptide inhibitor in solution. Thus, we suggest that caution be exercised in using affinity constants to assess the interactions of peptide inhibitors with human renin and possibly with other enzymes having extended binding sites. Furthermore, the thermodynamic parameters of a class of compounds provide more information as to the mode of binding of ligands to their respective receptors than do dissociation constants.

Stereospecific inhibitors of the gelation of sickle hemoglobin.

During the last decade there have been major advances in understanding the structure of the gel of deoxyhemoglobin S and the mechanism of its formation. These advances have allowed the development of a new strategy for the inhibition of gelation, i.e., stereospecific competitive inhibitors of the polymerization process. For this purpose we have used equilibrium solubility measurements to study the effects of amino acids and peptides on deoxyhemoglobin S solubility. We have found that aromatic amino acids and short peptides containing these amino acids significantly increase solubility; longer peptides, however, decrease solubility by an excluded-volume related effect. Recently we have used computer graphics projections of the surface of the hemoglobin molecules in the crystal form to design peptide inhibitors. In addition, we have developed 13C nuclear magnetic resonance spectroscopic methods to quantitate the gel of deoxyhemoglobin S in hemoglobin solutions an in cells. These spectroscopic methods allow us to study the mechanism of intracellular gelation and to test the effects of inhibitors on sickle cells.