ABSTRACT
Nine analogues of thymic humoral factor (THF)-gamma-2 were prepared by the solid-phase method, and their in vitro restoring effect on the impaired blastogenic response of phytohemagglutinin(PHA)-stimulated T-lymphocytes of uremic patients with infectious diseases were examined. The results were as follows: [Arg6]-THF-gamma-2 exhibited higher restoring activity than that of our synthetic THF-gamma-2. [Sar4]-, [Val1]-, [Arg3]-, [Gly5]-, and [Asn3]-THF-gamma-2 were also active but less potent than that of our synthetic THF-gamma-2. Three other peptides, [beta-Ala4]-, [Arg2]-, and [Gln2]-THF-gamma-2, did not show any restoring activity on the impaired blastogenic response of uremic patients with infectious disease.
Subject(s)
Oligopeptides/chemical synthesis , Oligopeptides/immunology , Amino Acid Sequence , Humans , Immunity, Cellular , Indicators and Reagents , Peptide Fragments/chemical synthesis , Peptide Fragments/chemistry , Thymus Hormones/chemical synthesis , Thymus Hormones/immunology , Uremia/immunologyABSTRACT
The peptide analogs of thymic humoral factor-gamma 2 (THF-gamma 2) in which phenylalanine residue at the 7th position are replaced by phenylglycine (Phg), homophenylalanine (Hph), and 1-naphthylalanine (1-Nal) were synthesized by a solid-phase method and the immunological significance of the aromatic amino acid of this position was comparatively investigated. The in vitro restoring effect of the synthetic peptides on the impaired phytohemagglutinin (PHA) response of T-lymphocytes from uremic patients was tested. The observed activities of these peptides were in order (1-Nal7) thymic humoral factor [THF]-gamma 2 > 4-Fluoro (Phe7) THF-gamma 2 > THF-gamma 2. However, the other two analogs, [Phg7] THF-gamma 2 and [Hph7] THF-gamma 2, had no restoring effect even at a higher concentration.
Subject(s)
Adjuvants, Immunologic/chemistry , Adjuvants, Immunologic/pharmacology , Lymphocyte Activation/drug effects , Oligopeptides/chemistry , Oligopeptides/pharmacology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Thymus Hormones/chemistry , Thymus Hormones/pharmacology , Uremia/immunology , Adjuvants, Immunologic/chemical synthesis , Amino Acid Sequence , Amino Acid Substitution , Humans , In Vitro Techniques , Oligopeptides/chemical synthesis , Phenylalanine/chemistry , Structure-Activity Relationship , Thymus Hormones/chemical synthesis , Uremia/therapyABSTRACT
Thymic humoral factor-gamma 2 and five analogues modified at position 7 with various phenylalanine derivatives were synthesized by a solid-phase method. The synthetic peptides were tested for their effects on the impaired blastogenic response of phytohemagglutinin-stimulated T lymphocytes of uremic patients with infectious diseases. The synthetic thymic humoral factor-gamma 2 enhanced the blastogenic response of T lymphocytes in the blood of the 2 patients tested. Of the synthetic peptides, [Phe(4F)7]thymic humoral factor-gamma 2 exhibited the most potent effect.
Subject(s)
Lymphocyte Activation/drug effects , Oligopeptides/pharmacology , T-Lymphocytes/drug effects , Thymus Hormones/pharmacology , Amino Acid Sequence , Communicable Diseases/drug therapy , Communicable Diseases/immunology , Fluorometry , Humans , Lymphocyte Activation/immunology , Oligopeptides/chemical synthesis , Oligopeptides/chemistry , Oligopeptides/therapeutic use , Phytohemagglutinins/toxicity , Structure-Activity Relationship , T-Lymphocytes/cytology , T-Lymphocytes/immunology , Thymidine/metabolism , Thymus Hormones/chemical synthesis , Thymus Hormones/chemistry , Thymus Hormones/therapeutic use , Uremia/immunology , Uremia/pathologyABSTRACT
Acetyl-thymic humoral factor-gamma 2 chloromethyl ketone [Ac-Leu-Glu-Asp-Gly-Pro-Lys-Phe-Leu-CH2Cl], an analog of thymic humoral factor-gamma 2, was synthesized and studied for its immunological effects on the impaired blastogenic response of T-lymphocytes isolated from uremic patients. Synthetic thymic humoral factor-gamma 2 and the synthetic acetyl-thymic humoral factor-gamma 2 chloromethyl ketone both restored the impaired blastogenic response of T-lymphocytes of uremic patients. However, the synthetic thymic humoral factor-gamma 2 is susceptible to proteolytic digestion. On the other hand, the synthetic acetylthymic humoral factor-gamma 2 chloromethyl ketone retained activity and was shown to exhibit a high degree of stability when incubated in human serum, These results indicate that N-terminal acetylation and the introduction of a chloromethyl ketone residue into the C-terminal residue of thymic humoral factor-gamma 2 increase resistance to proteolytic degradation by exopeptidases without loss of immunological activity.
Subject(s)
Thymus Hormones/chemical synthesis , Acetylation , Amino Acid Sequence , Drug Stability , Humans , In Vitro Techniques , Lymphocyte Activation/drug effects , Molecular Sequence Data , Structure-Activity Relationship , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Thymus Hormones/metabolism , Thymus Hormones/pharmacology , Uremia/immunologyABSTRACT
The impurities which formed in the large-scale synthesis of THF-gamma 2, an immunomodulatory peptide of formula H-Leu-Glu-Asp-Gly-Pro-Lys-Phe-Leu-OH, were identified by a combination of analytical methods, and their structure confirmed by synthesis. Most impurities originated from side-reactions involving the aspartyl residue (cyclization, beta-aspartyl formation and cleavage). Based on this knowledge, modifications were introduced into the work up and the purification procedure which resulted in a very pure final product (> 99% by RP-HPLC).
Subject(s)
Oligopeptides/chemical synthesis , Amino Acid Sequence , Chromatography, High Pressure Liquid , Hydrogen-Ion Concentration , Molecular Sequence Data , Oligopeptides/isolation & purification , Thymus Hormones/chemical synthesis , Thymus Hormones/isolation & purificationABSTRACT
Human splenin (hSP) was synthesized by assembling eight peptide fragments followed by deprotection with 1M trifluoromethanesulfonic acid-thioanisole (molar ratios, 1:1) in trifluoroacetic acid in the presence of m-cresol and dimethylselenium. Finally, the deprotected peptide was incubated with dithiothreitol to reduce sulfoxide on the methionine side chain. Incubation of peripheral lymphocytes isolated from uremic patients with the synthetic hSP showed an enhancing effect on the reduced B-lymphocytes, but had no restoring effect on the impaired blastogenic response of T-lymphocytes.
Subject(s)
B-Lymphocytes/drug effects , T-Lymphocytes/drug effects , Thymopoietins/chemical synthesis , Thymus Hormones/chemical synthesis , Uremia/immunology , Amino Acid Sequence , Humans , In Vitro Techniques , Molecular Sequence Data , Thymopoietins/pharmacologyABSTRACT
A heptadecapeptide, H-Arg-Lys-Ala-Val-Tyr-Val-Glu-Leu-Tyr-Leu-Gln-Ser-Leu-Thr-Ala-Glu-His-OH , corresponding to amino acids 32 to 48 of human splenin (hSP) and an analog in which the amino acid residue at position 34 is changed from Ala to Glu, were synthesized. These peptides were synthesized using conventional solution synthesis and were tested for their effect on reduced B-lymphocytes of uremic patients. Incubation of peripheral lymphocytes isolated from uremic patients with these two synthetic heptadecapeptides, hSP fragment 32-48 and [Glu34]hSP fragment 32-48, had an enhancing effect on the reduced B-lymphocytes, but synthetic bovine thymopoietin II (bTP-II) fragment 32-49 had no effect under the same conditions.
Subject(s)
B-Lymphocytes/drug effects , Peptide Fragments/chemical synthesis , Thymopoietins/chemical synthesis , Thymus Hormones/chemical synthesis , Uremia/immunology , Amino Acid Sequence , Humans , Molecular Sequence Data , Peptide Fragments/pharmacology , Thymopoietins/pharmacologyABSTRACT
[Glu34]human splenin (hSP) was synthesized in a conventional manner by assembling ten peptide fragments followed by deprotection with 1 M trifluoromethanesulfonic acid-thioanisole (molar ratio, 1:1) in trifluoroacetic acid in the presence of m-cresol and dimethylselenide. Finally, the deprotected peptide was incubated with dithiothreitol to reduce sulfoxide on the methionine side chain. Incubation of peripheral lymphocytes isolated from uremic patients with the synthetic [Glu34]hSP showed an enhancing effect on the reduced B-lymphocytes, but synthetic human thymopoietin (hTP) had no effect under the same conditions.
Subject(s)
B-Lymphocytes/drug effects , Thymopoietins/chemical synthesis , Thymus Hormones/chemical synthesis , Uremia/blood , Amino Acid Sequence , Amino Acids/analysis , Humans , In Vitro Techniques , Molecular Sequence Data , Thymopoietins/immunology , Thymopoietins/pharmacologyABSTRACT
This study reports on the synthesis of two fluorescent analogues of thymopentin (TP-5; Arg-Lys-Asp-Val-Tyr). A fluorescein isothiocyanate labeled analogue (FITC-TP-5) and a stilbene isothiocyanate labeled analogue (SITS-TP-5) were extensively purified by ion-exchange and gel filtration chromatography. Characterization of the coupling site through amino acid analysis, dansylation and N-terminal cleavage of the fluorescent amino acid yielded results which indicated that both were mono-labeled analogues derivatized at the N-terminal. These analogues were shown to be TP-5-like in nature by their ability to induce the expression of the Thy 1.2 surface marker on nude mouse prothymocytes in both in vivo and in vitro assays. In addition, these analogues were able to inhibit the specific binding of radiolabeled TP-5 to human lymphocytes. Initial studies describing the interaction of FITC-TP-5 with human lymphocytes are shown.
Subject(s)
4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid/chemical synthesis , Fluorescein-5-isothiocyanate/analogs & derivatives , Fluoresceins/chemical synthesis , Peptide Fragments/metabolism , Receptors, Peptide , Stilbenes/chemical synthesis , Thymopoietins/chemical synthesis , Thymopoietins/metabolism , Thymus Hormones/chemical synthesis , Thymus Hormones/metabolism , 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid/analogs & derivatives , 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid/pharmacology , Animals , Antigens, Surface/analysis , Fluoresceins/pharmacology , Indicators and Reagents , Mice , Mice, Nude , Receptors, Cell Surface/drug effects , Receptors, Cell Surface/metabolism , Structure-Activity Relationship , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Thy-1 Antigens , Thymopentin , Thymopoietins/pharmacologyABSTRACT
Thymopentin, a synthetic pentapeptide fragment of thymopoietin (residues 32-36, Arg-Lys-Asp-Val-Tyr) is biologically active but susceptible to proteolytic digestion. Analogs were synthesized and studied for biological activity and susceptibility to peptidases. Amino acid changes were incorporated at positions known to not affect activity adversely and N-terminal acetylation and C-terminal amidation were used to increase resistance to proteolytic degradation by exopeptidases. Ac-Pro2-TP5-NH2 and Aib2-TP5-NH2 retained activity and were shown to exhibit a high degree of stability when incubated in human serum.
Subject(s)
Cyclic GMP/metabolism , Peptide Fragments/chemical synthesis , Peptide Fragments/pharmacology , T-Lymphocytes/metabolism , Thymopoietins/chemical synthesis , Thymopoietins/pharmacology , Thymus Hormones/chemical synthesis , Thymus Hormones/pharmacology , Cell Line , Humans , Kinetics , Peptide Fragments/blood , Structure-Activity Relationship , T-Lymphocytes/drug effects , Thymopentin , Thymopoietins/bloodSubject(s)
Thymus Hormones/physiology , Aging , Animals , Autoimmune Diseases/drug therapy , Candida albicans/drug effects , Candidiasis/drug therapy , Candidiasis/immunology , Clinical Trials as Topic , Drug Evaluation , Herpesviridae Infections/drug therapy , Humans , Immunologic Deficiency Syndromes/blood , Immunologic Deficiency Syndromes/drug therapy , Immunotherapy , Lung Neoplasms/drug therapy , Lung Neoplasms/therapy , Mice , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/therapy , Prospective Studies , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Thymus Hormones/chemical synthesis , Thymus Hormones/immunology , Thymus Hormones/isolation & purification , Thymus Hormones/therapeutic useABSTRACT
Many different approaches have been used, over the last 15 years, for the design of potential immunostimulating drugs: Fractionation of crude natural substances (of eukaryotic or prokaryotic origin) already known to enhance immune functions, followed by chemical characterization and, in many cases, full synthesis of the active moiety: examples are provided by thymic hormones and the muramyldipeptide (MDP); Chemical modification of natural substances of known chemical structure in order to potentiate or change their biological activities or reduce their toxicity: murabutide, lipophilic MDP derivatives, lipopeptides (such as pimelautide), tuftsin analogs; Chemical synthesis (often without preconceived ideas about structure-activity relationship) of a great variety of molecules which are then screened in vitro and in vivo for immunopharmacological activity. The chemical structures and the biological profiles (in terms of possible primary cellular targets and mechanisms of immunostimulating activities) of representatives of class (b) and class (c) immunostimulants are reviewed in this paper.
Subject(s)
Adjuvants, Immunologic/chemical synthesis , Acetylmuramyl-Alanyl-Isoglutamine/chemical synthesis , Acetylmuramyl-Alanyl-Isoglutamine/pharmacology , Adjuvants, Immunologic/pharmacology , Animals , Humans , Thymus Hormones/chemical synthesis , Thymus Hormones/pharmacologyABSTRACT
Two chiral chromatographic procedures (HPLC and capillary GC) were developed to monitor the extent of epimerization of protected and unprotected amino acids used in a synthesis of thymopentin, an immunoregulatory peptide currently in clinical trials. The capillary GC method allowed the detection of the (R)-enantiomer in the presence of the (S)-enantiomer at levels of greater than or equal to 0.2%, while the HPLC method allowed similar detection at levels of greater than or equal to 0.1%.
Subject(s)
Amino Acids/analysis , Peptide Fragments/chemical synthesis , Thymopoietins/chemical synthesis , Thymus Hormones/chemical synthesis , Chromatography, Gas , Chromatography, High Pressure Liquid , Stereoisomerism , ThymopentinABSTRACT
To synthesize peptidic derivatives of polyamines, sequential reactions were designed for the polyfunctional peptides and polyfunctional amines which utilize specific protection and deprotection. Utilizing these reactions, a thymic nonapeptide, a lymphocyte-differentiating hormone, was coupled with spermidine and spermine to yield, respectively: Glp-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn-NH(CH2)3NH(CH2)4NH2; Glp-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn-NH(CH2)3NH-(CH2)4NH(CH2) 3NH2. Both conjugates inhibited rather than stimulated incorporation of [3H]-thymidine into DNA at levels 20-fold that of spermine. This activity is less than that based on the percent of the spermine moeity (ca. 20%) in the conjugate.
Subject(s)
Peptides/chemical synthesis , Spermidine , Spermine , Thymic Factor, Circulating/chemical synthesis , Thymus Hormones/chemical synthesis , Animals , Cell Division/drug effects , Chemical Phenomena , Chemistry , Lymphocytes/drug effects , Male , Mice , Mice, Inbred C57BL , Peptides/pharmacology , Thymic Factor, Circulating/analogs & derivativesABSTRACT
Seventeen peptides, related to thymopoietin pentapeptide, L-arginyl-L-lysyl-L-aspartyl-L-valyl-L-tyrosine (thymopentin) were synthesized by the stepwise strategy in solution. Of these, L-arginyl-L-lysyl-L-aspartic acid and L-arginyl-L-lysyl-L-aspartyl-L-valine, shortened from the C-terminus of the pentapeptide, exhibit significant immuno-stimulating potencies, exceeding those of thymopentin, both in vitro and in vivo immunological tests. Studies on the structure-activity relationships suggest that the potencial active site of thymopoietins is very sensitive to the N- and C-terminal elongations of the peptide chain. For thymic hormones, an active site carrying cumulative chemical signals is proposed instead of well-defined active centers.
Subject(s)
Oligopeptides/chemical synthesis , Thymopoietins/chemical synthesis , Thymus Hormones/chemical synthesis , Amino Acid Sequence , Animals , Antibody Formation/drug effects , Biological Assay , Immunoglobulin M/genetics , Indicators and Reagents , Oligopeptides/pharmacology , Rats , Structure-Activity Relationship , Thymopoietins/pharmacologyABSTRACT
In this report we further show the utility and efficiency of polymer-bound 1-hydroxybenzotriazole (PHBT) as an almost ideal support for the polymeric reagent method of peptide synthesis. This was demonstrated by the synthesis of thymosin alpha 1 (15-28), in which two suitably blocked segments, Boc-Asp (OtBu)-Leu-Lys (2Cz)-Glu (OBzl)-Lys (2Cz)-Lys (2Cz)-OH (3) and Boc-Glu (OBzl)-Val-Val-Glu (OBzl)-Glu (OBzl)-Ala-Glu (OBzl)-Asn-OBzl (2), were prepared entirely by utilizing PHBT activation for each coupling step. After appropriate deblocking of 2, segments 2 and 3 were coupled by the DCC-HOBT method, followed by complete deblocking and ion-exchange chromatographic purification, affording the C-terminal half of thymosin alpha 1, H-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH (1).