Degradation of thymopentin by human lymphocytes: evidence for aminopeptidase activity

Thymopentin (Arg-Lys-Asp-Val-Tyr) was shown to be degraded in vitro by human lymphocytes into two main fragments; the tetrapeptide Lys-Asp-Val-Tyr and the tripeptide Asp-Val-Tyr. Degradation products were identified by HPLC and amino-acid analysis. Analysis of the time-course of degradation revealed a 'stepwise' degradative event beginning at the N-terminal. The degradation of thymopentin after the first 10 min, as well as the formation of the tetrapeptide (5-30 min) were essentially curvilinear. Degradation of the tripeptide, was linear. Upon screening a panel of compounds that inhibit enzymatic activity, bestatin, amastatin and 1,10-phenanthroline were shown to be the most effective. Bestatin and amastatin caused an 85-90% inhibition of thymopentin degrading activity with IC50 values of 7.1 x 10(-6) M and 4.5 x 10(-9) M, respectively. 1,10-Phenanthroline completely inhibited the degradative process with an IC50 of 2 x 10(-4) M. When the tetrapeptide Lys-Asp-Val-Tyr was used as the starting substrate, similar IC50 values were seen for amastatin, bestatin and 1,10-phenanthroline. The importance of divalent metal ions in the degradative event was demonstrated not only by the effect of 1,10-phenanthroline, but also by the ability of Zn2+ and Co2+ to reverse the inhibition of 1,10-phenanthroline (at its IC50) to activities near control values (no inhibitor). These data strongly suggest that an aminopeptidase(s) is responsible for the degradative activity.     

Pharmacokinetics, toxicity of nasal cilia and immunomodulating effects in Sprague-Dawley rats following intranasal delivery of thymopentin with or without absorption enhancers

Thymopentin (TP 5), a synthetic pentapeptide, has been used in clinic as a modulator for immnuodeficiencies through intramuscular administration. The objectives of this study was to investigate the pharmacokinetics using normal rats and toxicity of nasal cilia as well as immunomodulating effects using immunosuppression rats after intranasal delivery of thymopentin with or without an absorption enhancer. The absorption extent of fluorescein isothiocyanate (FITC) labeled TP 5 via nasal delivery at a single dose is significantly improved by incorporating sodium deoxycholate, Brij 35 and chitosan, respectively. FITC-TP 5 can also be absorbed to such an extent ranging from 15 to 28% after intranasal administration of FITC-TP 5 alone, FITC-TP 5 with sodium caprylate, or with bacitracin, respectively. After seven consecutive days multiple dosing, TP 5 formulation with sodium deoxycholate or Brij 35 caused apparently injury to nasal cilia, indicating these two enhancers would not be appropriate for nasal delivery. Results from superoxide dismutase activity, maleic dialdehyde, T-lymphocyte subsets (CD3+, CD4+, CD8+ and CD4+/CD8+ ratio) analyses suggest that all the selected enhancers improve the modulating effects of TP 5 in the immunosuppression rats. On an overall evaluation, intranasal TP 5 alone, TP 5 with chitosan, or TP 5 with bacitracin formulation may be suitable for the future clinical application.     

Cooperativity of thymopoietin 32-36 (the active site) and thymopoietin 38-45 in receptor binding

Thymopoietin is a 49 amino acid polypeptide hormone of the thymus whose biological activity is reproduced by the synthetic pentapeptide thymopentin, corresponding to amino acids 32-36. Thymopentin requires the addition of an octapeptide corresponding to thymopoietin 38-45 for full competition with native thymopoietin in a radioreceptor assay with receptor derived from the human T-cell line CEM. Thus thymopoietin appears to bind to its receptor on T-cells by two regions (32-36 and 38-45). Thymopentin alone is biologically active and induces elevations of intracellular cyclic GMP. Whilst occupancy of the adjacent site by thymopoietin 37-45 does not of itself cause an elevation of intracellular cyclic GMP this peptide is not biologically silent as it does enhance the potency of thymopentin.     

Structural and dynamic properties of thymopoietin mimetics

We propose a hypothesis that the T-cell receptor is a possible target of thymic hormones. We modelled the conformational dynamics of thymopentin and its structural variants in solution, as well as the interactions of these short peptides with the proposed molecular target. Thymopentin is a five-amino-acid fragment of the thymic hormone thymopoietin (residues 32 to 36) that reproduces the immunomodulatory activity of the complete hormone. Using molecular dynamics and flexible docking methods, we demonstrated high-affinity binding of thymopentin and its prospective mimetics with the T-cell receptor. The calculated biological activity spectra of thymopentin and its two promising modifications can be used in immunomodulatory activity screenings with live systems.     

Structural basis for the inhibition of M1 family aminopeptidases by the natural product actinonin: Crystal structure in complex with E. coli aminopeptidase N

Actinonin is a pseudotripeptide that displays a high affinity towards metalloproteases including peptide deformylases (PDFs) and M1 family aminopeptidases. PDF and M1 family aminopeptidases belong to thermolysin-metzincin superfamily. One of the major differences in terms of substrate binding pockets between these families is presence (in M1 aminopeptidases) or absence (in PDFs) of an S1 substrate pocket. The binding mode of actinonin to PDFs has been established previously; however, it is not clear how the actinonin, without a P1 residue, would bind to the M1 aminopeptidases. Here we describe the crystal structure of Escherichia coli aminopeptidase N (ePepN), a model protein of the M1 family aminopeptidases in complex with actinonin. For comparison we have also determined the structure of ePepN in complex with a well-known tetrapeptide inhibitor, amastatin. From the comparison of the actinonin and amastatin ePepN complexes, it is clear that the P1 residue is not critical as long as strong metal chelating head groups, like hydroxamic acid or α-hydroxy ketone, are present. Results from this study will be useful for the design of selective and efficient hydroxamate inhibitors against M1 family aminopeptidases.     

The protective action of thymopentin on stress damage to the stomach

The influence of peptide thymopentin on ulcer effects of the stomach mucous membrane in rats and the state of its processes in acute and chronic stress as well as their combination influence were investigated. The intercommunication of frequency of the formation of ulcer and the activity of superoxide dismutase in stomach fibers was established. Thymopentin displayed a marked antiulcerogenic effect in all kinds of stress.     

Effect of thymopentin on the production of cytokines, heat shock proteins, and NF-κB signaling proteins

In vivo effects of thymopentin, an active fragment of the naturally occurring thymic hormone thymopoietin, on the production of cytokines, nitric oxide, heat shock proteins, and signaling proteins NF-κB, phNF-κB, and IκB-α in lymphoid cells of male NMRI mice was studied. Activation of production of several cytokines (IL-1α, IL-2, IL-6, IL-10, and IFN-γ), nitric oxide, and heat shock proteins (HSP70 and HSP90) was observed in peritoneal macrophages and spleen lymphocytes of mice that received intraperitoneal injections of thymopentin (15μg per 100 g body weight). Thymopentin apparently produces stress-like rather than damaging effects. A probable action mechanism of this hormone is activation of the NF-κB signaling pathway, which is most pronounced at the NF-κB phosphorylation stage.     

Degradation of cholecystokinin octapeptide,related fragments and analogs by human and rat plasma in vitro

The rate of degradation of cholecystokinin octapeptide, related fragments and analogs by human and rat plasma was investigated, using high pressure liquid chromatography for the separation and identification of the degradation products.CCK tetrapeptide showed a half-life of 13 min in human plasma while its cleavage in rat plasma occurred at a very high rate (half-life of less than 1 min).The kinetics of disappearance of both sulphated and unsulphated CCK-8 indicated more than a single rate of degradation; the faster degrading system showed a half-life of 18 min for unsulphated CCK-8 and of 50 min for the sulphated peptide in human plasma as compared respectively with 5 and 17 min in rat plasma. The cleavage of CCK-8 was inhibited by bestatin and puromycin, suggesting that aminopeptidases play a major role in the breakdown of this peptide.CCK-9 analogs were rapidly converted into their corresponding octapeptides (half-life of 2.7 min in human plasma). This conversion was inhibited by puromycin and bestatin, suggesting the participation of aminopeptidase(s) probably specific for basic amino acids.CCK decapeptide exhibited a greater stability than the nonapeptides (half-life of 30 and 45 min in human and rat plasma respectively) and also gave rise to CCK-8 as degradation product. This cleavage was not affected by aminopeptidase inhibitors but was decreased by aprotinin (Trasylol®), suggesting that trypsin-like and/or kallikrein-like enzyme(s) were involved in the plasma metabolism of this peptide.     

Non-histone chromatin proteins during various stages of activity of immunocompetent cells

Summary Some of the properties of the tetrapeptide tuftsin, Thr-Lys-Pro-Arg, are discussed. We describe three phases of tuftsin activation of the macrophage. Tuftsinyltuftsin, the octapeptide Thr-Lys-Pro-Arg-Thr-LysPro-Arg, was synthesized with a view of minimizing the formation of Lys-Pro-Arg, from tuftsin by tissue aminopeptidases. The tripeptide is a tuftsin inhibitor. The octapeptide proved to be quite effective in prolonging the life of syngeneic mice injected with L1210 leukemia cells. Its effect in our laboratory, was considerably better than we could obtain with tuftsin. A simple method for purifying tuftsin by high performance liquid chromatography is described using 0.75% trifluoroacetic acid in water.The tuftsin sequence Thr-Lys-Pro-Arg is present in P 12 protein of Rausher murine leukemia virus. A close analog Thr-Arg-Pro-Lys appears in yet another virus protein the haemagglutinin of influenza virus. A second close analog Thr-Arg-Pro-Arg forms the penultimate carboxyterminal of a pancreatic polypeptide found in human and several animals.     

Localization of the thiorphan-sensitive endopeptidase, termed enkephalinase A, on glial cells

Degradation of tritiated Leu-enkephalin was studied in cultures of primary astrocytes from rat brain. The incubation experiments with a cell suspension revealed Tyr as the main tritiated metabolite; however, Tyr-Gly-Gly and Tyr-Gly were detectable as well. Using a crude membrane preparation of the astrocytes, we found about equal amounts of Tyr and Tyr-Gly-Gly but only trace quantities of Tyr-Gly. The production of Tyr was completely inhibited by bestatin, an inhibitor of aminopeptidases, that of Tyr-Gly-Gly by thiorphan, a specific inhibitor of enkephalinase A. The results prove the ability of glial cells to degrade enkephalin by aminopeptidase and a membrane-bound enkephalinase A.     

Development of a new version of the Liverpool Malaria Model. I. Refining the parameter settings and mathematical formulation of basic processes based on a literature review

Artesunate (AS) is a clinically versatile artemisinin derivative utilized for the treatment of mild to severe malaria infection. Given the therapeutic significance of AS and the necessity of appropriate AS dosing, substantial research has been performed investigating the pharmacokinetics of AS and its active metabolite dihydroartemisinin (DHA). In this article, a comprehensive review is presented of AS clinical pharmacokinetics following administration of AS by the intravenous (IV), intramuscular (IM), oral or rectal routes. Intravenous AS is associated with high initial AS concentrations which subsequently decline rapidly, with typical AS half-life estimates of less than 15 minutes. AS clearance and volume estimates average 2 - 3 L/kg/hr and 0.1 - 0.3 L/kg, respectively. DHA concentrations peak within 25 minutes post-dose, and DHA is eliminated with a half-life of 30 - 60 minutes. DHA clearance and volume average between 0.5 - 1.5 L/kg/hr and 0.5 - 1.0 L/kg, respectively. Compared to IV administration, IM administration produces lower peaks, longer half-life values, and higher volumes of distribution for AS, as well as delayed peaks for DHA; other parameters are generally similar due to the high bioavailability, assessed by exposure to DHA, associated with IM AS administration (> 86%). Similarly high bioavailability of DHA (> 80%) is associated with oral administration. Following oral AS, peak AS concentrations (Cmax) are achieved within one hour, and AS is eliminated with a half-life of 20 - 45 minutes. DHA Cmax values are observed within two hours post-dose; DHA half-life values average 0.5 - 1.5 hours. AUC values reported for AS are often substantially lower than those reported for DHA following oral AS administration. Rectal AS administration yields pharmacokinetic results similar to those obtained from oral administration, with the exceptions of delayed AS Cmax and longer AS half-life. Drug interaction studies conducted with oral AS suggest that AS does not appreciably alter the pharmacokinetics of atovaquone/proguanil, chlorproguanil/dapsone, or sulphadoxine/pyrimethamine, and mefloquine and pyronaridine do not alter the pharmacokinetics of DHA. Finally, there is evidence suggesting that the pharmacokinetics of AS and/or DHA following AS administration may be altered by pregnancy and by acute malaria infection, but further investigation would be required to define those alterations precisely.     

Absorption, metabolism, and excretion studies of carbon 14- and tritium-labeled derivatives of a ketomethylene containing tripeptide

Tritium and Carbon 14 analogs of the angiotensin converting enzyme inhibitor ketoACE were synthesized and their oral absorption, metabolism and excretion in rats were investigated. KetoACE, a ketomethylene analog of the tripeptide Bz-Phe-Gly-Pro, was slowly absorbed at a 35% level upon oral administration. It is rapidly eliminated from the blood with a half-life of about 10 minutes. Its excretion is primarily via the bile duct and it is excreted as 80% unchanged drug. The only identified metabolite consisting of 5-10% of the excreted radioactivity was determined to be the reduced ketoACE in which the ketone group was reduced to a hydroxyl.     

High airway-to-blood transport of an opioid tetrapeptide in the isolated rat lung after aerosol delivery

The airway-to-blood absorption of the mu-selective opioid tetrapeptide agonist Tyr-D-Arg-Phe-Phe-NH(2) (MW 631) was investigated in the isolated, perfused, and ventilated rat lung model. The lung metabolism of the peptide was compared after airway and vascular delivery. The concentrations of the parent tetrapeptide and five of its metabolites in the perfusate and in bronchoalveolar lavage fluid were analyzed by LC-MS.The metabolism of the peptide was higher after delivery to the airways compared to vascular delivery. However, the tetrapeptide was highly transported from the air-to-blood side to an extent of 47.8 +/- 10.7% in 2 h. In conclusion, the results prompt investigations of the pulmonary route as a successful alternative to parenteral delivery for this tetrapeptide.     

Positioning of aminopeptidase inhibitors in next generation cancer therapy

Aminopeptidases represent a class of (zinc) metalloenzymes that catalyze the cleavage of amino acids nearby the N-terminus of polypeptides, resulting in hydrolysis of peptide bonds. Aminopeptidases operate downstream of the ubiquitin–proteasome pathway and are implicated in the final step of intracellular protein degradation either by trimming proteasome-generated peptides for antigen presentation or full hydrolysis into free amino acids for recycling in renewed protein synthesis. This review focuses on the function and subcellular location of five key aminopeptidases (aminopeptidase N, leucine aminopeptidase, puromycin-sensitive aminopeptidase, leukotriene A₄ hydrolase and endoplasmic reticulum aminopeptidase 1/2) and their association with different diseases, in particular cancer and their current position as target for therapeutic intervention by aminopeptidase inhibitors. Historically, bestatin was the first prototypical aminopeptidase inhibitor that entered the clinic 35 years ago and is still used for the treatment of lung cancer. More recently, new generation aminopeptidase inhibitors became available, including the aminopeptidase inhibitor prodrug tosedostat, which is currently tested in phase II clinical trials for acute myeloid leukemia. Beyond bestatin and tosedostat, medicinal chemistry has emerged with additional series of potential aminopeptidases inhibitors which are still in an early phase of (pre)clinical investigations. The expanded knowledge of the unique mechanism of action of aminopeptidases has revived interest in aminopeptidase inhibitors for drug combination regimens in anti-cancer treatment. In this context, this review will discuss relevant features and mechanisms of action of aminopeptidases and will also elaborate on factors contributing to aminopeptidase inhibitor efficacy and/or loss of efficacy due to drug resistance-related phenomena. Together, a growing body of data point to aminopeptidase inhibitors as attractive tools for combination chemotherapy, hence their implementation may be a step forward in a new era of personalized treatment of cancer patients.     

Studies on trypsin inhbitors. Part II. Synthesis of the protected tetrapeptide (sequence 11-14) of porcine pancreatic secretory trypsin inhibitor II (Kazal).

Synthesis is described of the protected tetrapeptide corresponding to positions 11-14 of the primary structure of the porcine pancreatic secretory trypsin inhibitor II (Kazal), in the form of free acid as well as protected hydrazide. The tetrapeptide tert-butyloxycarbonylglycyl-S-acetamidomethylcysteinylprolyl-Nepsilon-trifluoroacetyl-lysine was prepared by stepwise elongation from the C-terminal Nepsilon-trifluoroacetyllysine using successively 1-succinimidyl benzyloxycarbonylprolinate, p-nitrophenyl N-tert-butyloxycarbonyl-S-acetamidomethylcysteinate and 1-phenyl-3-methyl-4-(tert-butyloxycarbonylglycyl)-oximinyl-5-(benzyloxycarbonylglycyl)-imino-2-pyrazoline as acylating agents. Alternately, the dipeptide benzyloxycarbonylprolyl-Nepsilon-trifluoroacetyllsine was transformed into the corresponding tert-butyloxycarbonylhydrazide which was reacted, after catalytic hydrogenolysis, with tritylglycyl-S-acetamido-methylcysteine to give the tetrapeptide tritylglycyl-S-acetamidomethylcysteinylprolyl-Nepsilon-trifluoroacetyllsine tert-butyloxycarbonylhydrazide. The stereochemical homogeneity of the final products was assessed, after partial deprotection with aqueous 90% trifluoroacetic acid, by digestion with papain and aminopeptidase M, followed by quantitative amino acid analysis.     

Factors involved in the inactivation of vasopressin after intracerebroventricular injection in mice

Behavioral excitation induced by intracerebroventricularly administered vasopressin in mice is very short-lasting, suggesting a half-life of the injected peptide of only a few minutes. The results of the present study suggest that vasopressin and related peptides are too hydrophilic to penetrate lipid membranes readily by passive diffusion and that passive diffusion from the extracellular space into cells or the bloodstream is an unlikely mechanism of inactivation. Pharmacological desensitization (tachyphylaxis) occurs after higher doses, but does not seem to be the major factor responsible for the short duration of action. Some deaminoanalogs of vasopressin, however, show a prolonged action, suggesting that degradation by (an) aminopeptidase(s) is a major route of inactivation. These results also suggest that vasopressin-degrading aminopeptidases are accessible from the extracellular space.     

Regional differences in bioavailability of an opioid tetrapeptide in vivo in rats after administration to the respiratory tract

TArPP (Tyr-D-Arg-Phe-Phe-NH(2)), 1-10 micromol/kg, was administered to anesthetized rats by nasal microinfusion, intratracheal microinfusion, intratracheal nebulization, aerosol inhalation, and i.v. bolus and infusion. Plasma concentrations of TArPP and its deamidated metabolite were determined by LC-MS-MS.Regional differences in bioavailability (F), first-pass metabolism, and absorption rate were found for TArPP after delivery to the respiratory tract. Absorption was rapid after both pulmonary and nasal administration (t(max) approximately 10-20 min). After nasal microinfusion, F was 52 +/- 9%. For all the pulmonary groups, F was higher (72-114%). First-pass metabolism of TArPP was lower in the lung than in the nasal cavity. It is evident that the pulmonary route is attractive for successful systemic delivery of small, hydrophilic and enzymatic susceptible peptides.     

Functional Characterization of Two M42 Aminopeptidases Erroneously Annotated as Cellulases

Several aminopeptidases of the M42 family have been described as tetrahedral-shaped dodecameric (TET) aminopeptidases. A current hypothesis suggests that these enzymes are involved, along with the tricorn peptidase, in degrading peptides produced by the proteasome. Yet the M42 family remains ill defined, as some members have been annotated as cellulases because of their homology with CelM, formerly described as an endoglucanase of Clostridium thermocellum. Here we describe the catalytic functions and substrate profiles CelM and of TmPep1050, the latter having been annotated as an endoglucanase of Thermotoga maritima. Both enzymes were shown to catalyze hydrolysis of nonpolar aliphatic L-amino acid-pNA substrates, the L-leucine derivative appearing as the best substrate. No significant endoglucanase activity was measured, either for TmPep1050 or CelM. Addition of cobalt ions enhanced the activity of both enzymes significantly, while both the chelating agent EDTA and bestatin, a specific inhibitor of metalloaminopeptidases, proved inhibitory. Our results strongly suggest that one should avoid annotating members of the M42 aminopeptidase family as cellulases. In an updated assessment of the distribution of M42 aminopeptidases, we found TET aminopeptidases to be distributed widely amongst archaea and bacteria. We additionally observed that several phyla lack both TET and tricorn. This suggests that other complexes may act downstream from the proteasome.     

JM47, a cyclic tetrapeptide HC-toxin analogue from a marine Fusarium species

The known metabolite, enniatin B, and a cyclic tetrapeptide, JM47, which is a new natural product, were extracted from brown rice cultures of a marine fungus, identified as a Fusarium species, isolated from the marine alga Codium fragile. NMR studies, including 15N HMQC and 15N HMBC, established the structure of JM47 as cyclo(Ala-Ala-Aoh-Pro), where Aoh is the amino acid, (2S,9S)-2-amino-8-oxo-9-hydroxydecanoic acid. The absolute stereochemistry of the Aoh side chain carbinol centre was determined using Mosher ester methodology. Analysis of NOESY data assisted by molecular modelling revealed an alternating L-, D-, L-, D-configuration for the tetrapeptide core. The absolute stereochemistry of the core was determined by acidic hydrolysis and chiral TLC analysis of the proline residue. JM47 belongs to the HC-toxin family of cyclic tetrapeptides which possess a 2-amino-8-oxo-9,10-epoxydecanoic acid residue in place of the Aoh unit. This is the first report of an analogue of HC-toxin from a marine Fusarium species.