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.     

Proteasome Inhibitors Alter Levels of Intracellular Peptides in HEK293T and SH-SY5Y Cells

The proteasome cleaves intracellular proteins into peptides. Earlier studies found that treatment of human embryonic kidney 293T (HEK293T) cells with epoxomicin (an irreversible proteasome inhibitor) generally caused a decrease in levels of intracellular peptides. However, bortezomib (an antitumor drug and proteasome inhibitor) caused an unexpected increase in the levels of most intracellular peptides in HEK293T and SH-SY5Y cells. To address this apparent paradox, quantitative peptidomics was used to study the effect of a variety of other proteasome inhibitors on peptide levels in HEK293T and SH-SY5Y cells. Inhibitors tested included carfilzomib, MG132, MG262, MLN2238, AM114, and clasto-Lactacystin β-lactone. Only MG262 caused a substantial elevation in peptide levels that was comparable to the effect of bortezomib, although carfilzomib and MLN2238 elevated the levels of some peptides. To explore off-target effects, the proteosome inhibitors were tested with various cellular peptidases. Bortezomib did not inhibit tripeptidyl peptidase 2 and only weakly inhibited cellular aminopeptidase activity, as did some of the other proteasome inhibitors. However, potent inhibitors of tripeptidyl peptidase 2 (butabindide) and cellular aminopeptidases (bestatin) did not substantially alter the peptidome, indicating that the increase in peptide levels due to proteasome inhibitors is not a result of peptidase inhibition. Although we cannot exclude other possibilities, we presume that the paradoxical increase in peptide levels upon treatment with bortezomib and other inhibitors is the result of allosteric effects of these compounds on the proteasome. Because intracellular peptides are likely to be functional, it is possible that some of the physiologic effects of bortezomib and carfilzomib arise from the perturbation of peptide levels inside the cell.     

A broad-spectrum peptide inhibitor of beta-lactamase identified using phage display and peptide arrays

Hydrolysis of beta-lactam antibiotics by beta-lactamase enzymes is the most common mechanism of bacterial resistance to these agents. Several small-molecule, mechanism-based inhibitors of beta-lactamases such as clavulanic acid are clinically available although resistance to these inhibitors has been increasing in bacterial populations. In addition, these inhibitors act only on class A beta-lactamases. Here we utilized phage display to identify peptides that bind to the class A beta-lactamase, TEM-1. The binding affinity of one of these peptides was further optimized by the synthesis of peptide arrays using SPOT synthesis technology. After two rounds of optimization, a linear 6-mer peptide with the sequence RRGHYY was obtained. A soluble version of this peptide was synthesized and found to inhibit TEM-1 beta-lactamase with a K(i) of 136 micro M. Surprisingly, the peptide inhibits the class A Bacillus anthracis Bla1 beta-lactamase with a K(i) of 42 micro M and the class C beta-lactamase, P99, with a K(i) of 140 micro M, despite the fact that it was not optimized to bind these enzymes. This peptide may be a useful starting point for the design of non-beta-lactam, broad-spectrum peptidomimetic inhibitors of beta-lactamases.     

胰凝乳蛋白酶短肽抑制剂的筛选和活性分析

报道了一种从噬菌体肽库中筛选胰凝乳蛋白酶短肽抑制剂的新方法．在通常的亲和富集筛选的基础上,利用胰凝乳蛋白酶自身的水解活力切割掉结合的底物噬菌体,再经抑制活力分析得到抑制性噬菌体克隆．这样筛得的噬菌体克隆具有明显的胰凝乳蛋白酶结合活力和抑制活力,ＤＮＡ序列分析发现其保守序列为（Ｓ／Ｔ）ＲＶＰＲ（Ｒ／Ｈ）．按此序列化学合成的短肽Ａｃ－ＡＳＲＶＰＲＲＧ－ＮＨ２、Ａｃ－ＡＳＲＶＰＲＨＧ－ＮＨ２同样表现出对胰凝乳蛋白酶的抑制作用．该方法为蛋白酶短肽抑制剂的筛选提供了一条有效途径     

Peptide gomesin triggers cell death through L-type channel calcium influx,MAPK/ERK,PKC and PI3K signaling and generation of reactive oxygen species

Gomesin is an antimicrobial peptide isolated from hemocytes of a common Brazilian tarantula spider named Acanthoscurria gomesiana. This peptide exerts antitumor activity in vitro and in vivo by an unknown mechanism. In this study, the cytotoxic mechanism of gomesin in human neuroblastoma SH-SY5Y and rat pheochromocytoma PC12 cells was investigated. Gomesin induced necrotic cell death and was cytotoxic to SH-SY5Y and PC12 cells. The peptide evoked a rapid and transient elevation of intracellular calcium levels in Fluo-4-AM loaded PC12 cells, which was inhibited by nimodipine, an L-type calcium channel blocker. Preincubation with nimodipine also inhibited cell death induced by gomesin in SH-SY5Y and PC12 cells. Gomesin-induced cell death was prevented by the pretreatment with MAPK/ERK, PKC or PI3K inhibitors, but not with PKA inhibitor. In addition, gomesin generated reactive oxygen species (ROS) in SH-SY5Y cells, which were blocked with nimodipine and MAPK/ERK, PKC or PI3K inhibitors. Taken together, these results suggest that gomesin could be a useful anticancer agent, which mechanism of cytotoxicity implicates calcium entry through L-type calcium channels, activation of MAPK/ERK, PKC and PI3K signaling as well as the generation of reactive oxygen species.     

Introduction of the new dipeptide isostere 7-endo-BtA as reverse turn inducer in a Bowman-Birk proteinase inhibitor: synthesis and conformational analysis.

Two dipeptide isosteres 7-exo-BTG (1) and 7-endo-BtA (2), belonging to the new class of gamma/delta-bicyclic amino acid BTAa, were inserted into an 11-residue peptide deriving from the Bowman Birk Inhibitor (BBI) class of serine protease inhibitors, and the conformational properties of these modified peptides have been studied by NMR and molecular modelling. The dipeptide isostere 7-endo-BtA [(1R,4S,5R,7R)-4-endo-methyl-6,8-dioxa-3-azabicyclo[3.2.1]octane-7-endo-carboxylic acid] (2), derived from L-alanine and meso tartaric acid, gave rise to the modified BBI peptide 5 whose structure was very similar to that of the original peptide 3, suggesting a possible reverse turn inducing property for this dipeptide isostere.     

Kinetic characterization of the calmodulin-activated catalytic subunit of phosphorylase kinase

The phosphorylase kinase holoenzyme from skeletal muscle is composed of a catalytic and three different regulatory subunits. Analysis of the kinetic mechanism of the holoenzyme is complicated because both the natural substrate phosphorylase b and also phosphorylase kinase itself have allosteric binding sites for adenine nucleotides. In the case of the kinase, these allosteric sites are not on the catalytic subunit. We have investigated the kinetic mechanism of phosphorylase kinase by using its isolated catalytic gamma-subunit (activated by calmodulin) and an alternative peptide substrate (SDQEKRKQISVRGL) corresponding to the convertible region of phosphorylase b, thus eliminating from our system all known allosteric binding sites for nucleotides. This peptide has been previously employed to study the kinetic mechanism of the kinase holoenzyme before the existence of the allosteric sites on the regulatory subunits was suspected [Tabatabai, L. B., & Graves, D. J. (1978) J. Biol. Chem. 253, 2196-2202]. This peptide was determined to be as good an alternative substrate for the isolated catalytic subunit as it was for the holoenzyme. Initial velocity data indicated a sequential kinetic mechanism with apparent Km's for MgATP and peptide of 0.07 and 0.47 mM, respectively. MgADP used as product inhibitor showed competitive inhibition against MgATP and noncompetitive inhibition against peptide, whereas with phosphopeptide as product inhibitor, the inhibition was competitive against both MgATP and peptide. The initial velocity and product inhibition studies were consistent with a rapid equilibrium random mechanism with one abortive complex, enzyme-MgADP-peptide. The substrate-directed, dead-end inhibitors 5'-adenylyl imidodiphosphate and Asp-peptide, in which the convertible Ser of the alternative peptide substrate was replaced with Asp, were competitive inhibitors toward their like substrates and noncompetitive inhibitors toward their unlike substrates, further supporting a random mechanism, which was also the conclusion from the report cited above that used the holoenzyme.     

Design of serine proteinase inhibitors by combinatorial chemistry using trypsin inhibitor SFTI-1 as a starting structure.

A small peptide library of monocyclic SFTI-1 trypsin inhibitor from sunflower seeds modified in positions P(1) and P(4)' was synthesized using a portioning-mixing method. The peptide library was deconvoluted by the iterative approach in solution. Two trypsin ([Met(9)]-SFTI-1 and [Arg(5), Abu(9)]-SFTI-1), one chymotrypsin ([Phe(5)]-SFTI-1) and one human elastase ([Leu(5), Trp(9)]-SFTI-1) inhibitors were selected and resynthesized. The values of their association equilibrium constants (K(a)) with target enzymes indicate that they are potent inhibitors. In addition, the last two analoges belong to the most active inhibitors of this size. The results obtained show that the conserved Pro(9) residue in the Bowman-Birk inhibitor (BBI)s is not essential for inhibitory activity.     

Peptide hydroxamic acids inhibit skin collagenase

A number of peptide hydroxamic acids have been synthesized and have been shown to be inhibitors of human skin collagenase. One of these, Z-Pro-Leu-Gly-NHOH, has an IC50 value of 4 X 10(-5)M. Corresponding peptides with different C-terminal functional groups, such as amide, carboxylate and aldehyde, showed little or no inhibition, indicating the importance of the hydroxamate functional group. In addition, the peptide sequence of this effective inhibitor corresponds closely to that of the cleavage site of native collagen, the substrate for the enzyme. Thus, substrate analogs incorporating a suitable metal coordinating group serve as potential inhibitors of human collagenase.     

A novel Lyn-binding peptide inhibitor blocks eosinophil differentiation, survival, and airway eosinophilic inflammation.

Receptor antagonists block all receptor-coupled signaling pathways indiscriminately. We introduce a novel class of peptide inhibitors that is designed to block a specific signal from a receptor while keeping other signals intact. This concept was tested in the model of IL-5 signaling via Lyn kinase. We have previously mapped the Lyn-binding site of the IL-5/GM-CSF receptor common beta (beta c) subunit. In the present study, we designed a peptide inhibitor using the Lyn-binding sequence. The peptide was N-stearated to enable cellular internalization. The stearated peptide blocked the binding of Lyn to the beta c receptor and the activation of Lyn. The lipopeptide did not affect the activation of Janus kinase 2 or its association with beta c. The inhibitor blocked the Lyn-dependent functions of IL-5 in vitro (e.g., eosinophil differentiation from stem cells and eosinophil survival). It did not affect eosinophil degranulation. When applied in vivo, the Lyn-binding peptide significantly inhibited airway eosinophil influx in a mouse model of asthma. The lipopeptide had no effect on basophil histamine release or on the proliferation of B cells and T cells. To our knowledge, this is the first report on an inhibitor of IL-5 that blocks eosinophil differentiation, survival, and airway eosinophilic inflammation. This novel strategy to develop peptide inhibitors can be applied to other receptors.     

Potential transition state analogue inhibitors for the penicillin-binding proteins

Penicillin-binding proteins (PBPs) are ubiquitous bacterial enzymes involved in cell wall biosynthesis. The development of new PBP inhibitors is a potentially viable strategy for developing new antibacterial agents. Several potential transition state analogue inhibitors for the PBPs were synthesized, including peptide chloromethyl ketones, trifluoromethyl ketones, aldehydes, and boronic acids. These agents were characterized chemically, stereochemically, and as inhibitors of a set of low molecular mass PBPs: Escherichia coli (EC) PBP 5, Neisseria gonorrhoeae (NG) PBP 3, and NG PBP 4. A peptide boronic acid was the most effective PBP inhibitor in the series, with a preference observed for a d-boroAla-based over an l-boroAla-based inhibitor, as expected given that physiological PBP substrates are based on d-Ala at the cleavage site. The lowest K(I) of 370 nM was obtained for NG PBP 3 inhibition by Boc-l-Lys(Cbz)-d-boroAla (10b). Competitive inhibition was observed for this enzyme-inhibitor pair, as expected for an active site-directed inhibitor. For the three PBPs included in this study, an inverse correlation was observed between the values for log K(I) with 10b and the values for log(k(cat)/K(m)) for activity against the analogous substrate, and K(m)/K(I) ratios were 90, 1900, and 9600 for NG PBP 4, EC PBP 5, and NG PBP 3, respectively. These results demonstrate that peptide boronic acids can be effective transition state analogue inhibitors for the PBPs and provide a basis for the use of these agents as probes of PBP structure, function, and mechanism, as well as a possible basis for the development of new PBP-targeted antibacterial agents.     

Synthesis and antibacterial activity of peptide deformylase inhibitors

Peptide deformylase catalyzes the removal of the N-terminal formyl group from newly synthesized polypeptides in eubacteria. Its essential character in bacterial cells makes it an attractive target for antibacterial drug design. In this work, we have rationally designed and synthesized a series of peptide thiols that act as potent, reversible inhibitors of purified recombinant peptide deformylase from Escherichia coli and Bacillus subtilis. The most potent inhibitor has a K(I) value of 11 nM toward the B. subtilis enzyme. These inhibitors showed antibacterial activity against both Gram-positive and Gram-negative bacteria, with minimal inhibitory concentrations (MIC) as low as 5 microM ( approximately 2 microg/mL). The PDF inhibitors induce bacterial cell lysis and are bactericidal toward all four bacterial strains that have been tested, B. subtilis, Staphylococcus epidermidis, Enterococcus faecalis, and E. coli. Resistance evaluation of one of the inhibitors (1b) against B. subtilis showed that no resistant clone could be found from >1 x 10(9) cells. Quantitative analysis using a set of inhibitors designed to possess varying potencies against the deformylase enzyme revealed a linear correlation between the MIC values and the K(I) values. These results suggest that peptide deformylase is the likely molecular target responsible for the antibacterial activity of these inhibitors and is therefore a viable target for antibacterial drug design.     

Rational design of tropoelastin peptide-based inhibitors of metalloproteinases

Abnormal production of matrix metalloproteinases (MMPs) has been observed in a variety of diseases, such as emphysema, atherosclerosis, and cancer metastasis. Destruction of connective tissue ensues and elastin is often a key target. Three of the main elastolytic MMPs are the gelatinases MMP-2 and MMP-9 and the metalloelastase MMP-12. To investigate the possibility of using peptides to inhibit the elastolytic activity of these enzymes, we mapped the sites within tropoelastin recognized by MMP-9 and MMP-12. Peptides that correspond to regions overlapping these sites were then tested for their ability to inhibit these MMPs. These included an unmodified peptide directed against MMP-9 (peptide PP), cysteine-containing peptides that mimicked either the MMP-9 (peptide NCP) or the MMP-12 (peptide lin24) cleavage sites in tropoelastin and their cyclized forms (CP and cyc24, respectively), and a peptide containing a zinc-chelating hydroxamate group directed against MMP-9 (HP). The presence of a free sulfhydryl or hydroxamate group capable of chelating the zinc ion in the active site of the MMPs was generally found to increase the inhibitory activity of the peptides. The specificity of the inhibitors varied, with some of the inhibitors showing activity against all of the MMPs examined. None of the inhibitors had any significant effect on the activity of the unrelated serine protease, plasmin. K(i) values for the inhibitors were in the micromolar range. Our results suggest ways of developing other MMP inhibitors based on substrate recognition sites that may provide greater levels of inhibition.     

Inhibition of Membrane-Active Peptides by Fatty Acid–Peptide Hybrids

Nine fatty acid–peptide hybrid molecules were constructed using the general formula CH₃(CH₂) _n CO-Phe Asp Cys-amide and tested for their ability to inhibit cell lysis induced by the membrane-active peptide melittin. All of these molecules, where n = 4–14, inhibited the action of melittin to some extent, but the longer carbon chains were most effective. Several potential inhibitors were also constructed with conservative substitutions in the peptide portion of the molecule. All were effective to varying degrees. We concluded that in the hexapeptide inhibitor published by Blondelle et al. (1993), the role of the first three residues is only to provide hydrophobic interaction with the melittin and has no particular amino acid sequence specificity. Some of these inhibitors were found to inhibit the lytic activity of a melittin analogue which had only superficial sequence similarity to melittin and also a truncated form of melittin, indicating the generality of the action of the inhibitors.Deceased 5/4/98     

Characterization of a cDNA for human protein C inhibitor. A new member of the plasma serine protease inhibitor superfamily

A cDNA library in lambda-phage lambda gt11 containing DNA inserts prepared from human liver mRNA was screened with monoclonal antibodies to human protein C inhibitor. Six positive clones were isolated from 6 X 10(6) phages and plaque purified. The cDNA in the phage containing the largest insert, which hybridized to a DNA probe prepared on the basis of the amino-terminal amino acid sequence of the mature inhibitor, was sequenced. This cDNA insert contained 2106 base pairs coding for a 5'-noncoding region, a 19-amino acid signal peptide, a 387-amino acid mature protein, a stop codon, and a long 3'-noncoding region of 839 base pairs. Based on the amino acid sequence of the carboxyl-terminal peptide released by cleavage of protein C inhibitor by activated protein C as well as by thrombin, the reactive site peptide bond of protein C inhibitor is Arg354-Ser355. Five potential carbohydrate-binding sites were found in the mature protein. The high homology of the amino acid sequence of protein C inhibitor to the other known inhibitors clearly demonstrates that protein C inhibitor is a member of the superfamily of serine protease inhibitors including alpha 1-antichymotrypsin, alpha 1-antitrypsin, antithrombin III, ovalbumin, and angiotensinogen. Based on the difference matrices for these proteins, we present possible phylogenetic trees for these proteins.     

Inhibition of the motility of the gastrointestinal tract by low-molecular peptides from k-casein

Two low-molecular peptides, peptide III and peptide IV-1, were isolated from peptic digest of k-casein by Sephadex G-50 and BIO-Gel P-2 chromatography. Intravenous administration of these peptides to dogs at a dose of 0.006 mg/kg inhibited food motility of the stomach and duodenum, but did not change or stimulate fasting motility. The duration of the inhibition was decreased with the diminution of the molecular weight of peptide fractions. Peptide III consisted of two inhibitors. Inhibitor I acted immediately after the injection, while inhibitor 2 acted 60-75 minutes later. Being the natural products of food protein proteolysis, these inhibitors may be apparently used in the clinical practice.     

Signal peptide digestion in Escherichia coli. Effect of protease inhibitors on hydrolysis of the cleaved signal peptide of the major outer-membrane lipoprotein

Upon incubation of the envelope fraction of Escherichia coli a precursor of the major outer membrane lipoprotein that accumulates in the cytoplasmic membrane of the globomycin-treated cell is processed to the mature form [Hussain, M., Ichihara, S., and Mizushima, S. (1980) J. Biol. Chem. 255, 3707-3712; (1982) J. Biol. Chem. 257, 5177-5182]. When this precursor-containing envelope fraction was incubated in the presence of protease inhibitors such as antipain, leupeptin, chymostatin and elastatinal, a new peptide appeared on a polyacrylamide gel at the position where the signal peptide was expected to appear. This was proved to be the signal peptide of the lipoprotein from the following facts: (a) its appearance is in proportion to the appearance of the lipoprotein and disappearance of the precursor; (b) when the cleavage of the signal peptide from the precursor was inhibited by globomycin, the peptide did not appear on the gel; and (c) the results of labeling of the peptide with [3H]leucine, [35S]methionine and [3H]arginine were consistent with the amino acid composition of the signal peptide. The signal peptide thus accumulated in the envelope fraction was hydrolyzed by an enzyme named 'signal peptide peptidase' when the envelope fraction was washed to remove the inhibitors. The hydrolysis was inhibited by re-addition of these inhibitors. The signal peptide peptidase hydrolyzed the signal peptide only after its cleavage from the lipoprotein precursor.     

Activity of recombinant dengue 2 virus NS3 protease in the presence of a truncated NS2B co-factor, small peptide substrates, and inhibitors

Recombinant forms of the dengue 2 virus NS3 protease linked to a 40-residue co-factor, corresponding to part of NS2B, have been expressed in Escherichia coli and shown to be active against para-nitroanilide substrates comprising the P6-P1 residues of four substrate cleavage sequences. The enzyme is inactive alone or after the addition of a putative 13-residue co-factor peptide but is active when fused to the 40-residue co-factor, by either a cleavable or a noncleavable glycine linker. The NS4B/NS5 cleavage site was processed most readily, with optimal processing conditions being pH 9, I = 10 mm, 1 mm CHAPS, 20% glycerol. A longer 10-residue peptide corresponding to the NS2B/NS3 cleavage site (P6-P4') was a poorer substrate than the hexapeptide (P6-P1) para-nitroanilide substrate under these conditions, suggesting that the prime side substrate residues did not contribute significantly to protease binding. We also report the first inhibitors of a co-factor-complexed, catalytically active flavivirus NS3 protease. Aprotinin was the only standard serine protease inhibitor to be active, whereas a number of peptide substrate analogues were found to be competitive inhibitors at micromolar concentrations.     

Identification of a linear peptide recognized by monoclonal antibody 2D7 capable of generating CCR5-specific antibodies with human immunodeficiency virus-neutralizing activity

CCR5 is the major coreceptor for human immunodeficiency virus (HIV) infection. The murine monoclonal antibody (MAb) 2D7, which recognizes a conformation-dependent epitope in the second extracellular loop of CCR5, is one of the most potent inhibitors of R5 virus cell entry. However, attempts to humanize 2D7 for in vivo human use have been unsuccessful so far. A filamentous phage library expressing random peptides was used to identify a peptide mimitope that is recognized by MAb 2D7. A synthetic peptide containing this sequence (2D7-2SK) bound to MAb 2D7 with high affinity and reversed its HIV type 1 (HIV-1) fusion inhibitory activity. The peptide contains sequence homologies to two distal regions of the second extracellular loop of human CCR5, both of which are required for MAb 2D7 binding. Rabbit anti-2D7-mimitope antibodies competed with MAb 2D7 for binding to the 2D7-2SK peptide in Biacore biosensor testing. Importantly, the rabbit anti-2D7-2SK antibodies bound to CCR5 on cells and specifically inhibited R5 (but not X4) envelope-mediated syncytium formation. These antibodies also neutralized infection of human peripheral blood mononuclear cells with R5 HIV isolates comparably to MAb 2D7. In summary, we have identified a novel peptide that closely mimics the MAb 2D7 epitope on CCR5. This peptide could be included as a potential vaccine candidate or to isolate 2D7-like human antibodies as entry inhibitors for R5 viruses.     

Regulation of cytosolic protein-tyrosine kinase from porcine spleen by polyamines and negative-charged polysaccharides

In vitro regulation of cytosolic protein-tyrosine kinase from porcine spleen (CPTK-40) by various positive or negative charged compounds was studied. Spermine and spermidine stimulated the activity of CPTK-40 about two-fold using (Val5)angiotensin II as a substrate. This stimulation was not specific for the peptide but was also observed in the case of tubulin phosphorylation indicating a direct effect of these compounds on the enzyme itself. On the contrary, negative-charged polysaccharides were shown to be strong inhibitors of CPTK-40. The possibility of the physiological regulation of CPTK-40 by these compounds is briefly discussed.