Solid-phase Peptide Synthesis Method Research Articles

Methods To Enhance The Metabolic Stability Of Peptide Drugs

Therapeutic peptides have high selectivity, efficacy, and low toxicity. These characteristics, combined with advances in solid-phase peptide synthesis and purification methods, have led to the widespread use of peptide-based drugs. Limitations in the research and development of peptide drugs are related to their short plasma half-life, rapid metabolism, proteolytic degradation and poor penetration through biological membranes. This mini-review examines the advantages of peptide drugs over small molecule drugs and biotechnological proteins, as well as various chemical methods for peptide modifications to increase the metabolic stability, bioavailability and pharmacokinetic properties of peptides.

Synthesis of Pentapeptide FWKVV (Phe-Trp-Lys-Val-Val) and Its Activity as Antioxidants

Antioxidant pentapeptides are pentapeptide compounds that have antioxidant activity. One of the pentapeptide compounds that have antioxidant activity is FWKVV. FWKVV is a linear pentapeptide with the amino acid sequence phenylalanine-tryptophan-lysine-valine-valine, which was first isolated to hydrolyzate the muscle protein of Miiuy croaker (Miichthysmiiuy). In addition to isolation, FWKVV compounds can be produced by the peptide synthesis method because this method requires a shorter time than the isolation method from natural materials. Synthesis methods commonly used are solution-phase peptide synthesis and solid-phase peptide synthesis (SPPS). However, the SPSS method is more efficient because it does not require purification in every process. The purpose of this study was to synthesize FWKVV compounds using the SPPS method and test their antioxidant activity. FWKVV has been synthesized using the SPPS method with HBTU/HOBt coupling reagent and Fmoc protective group. The FWKVV crud produced was 148.8 mg and had antioxidant activity against DPPH radicals with an IC50 value of 4.2 mg/mL.

Alginate-based microparticles coated with HPMCP/AS cellulose-derivatives enable the Ctx(Ile21)-Ha antimicrobial peptide application as a feed additive

Microencapsulation is a potential biotechnological tool, which can overcome antimicrobial peptides (AMP) instabilities and reduce toxic side effects. Thus, this study evaluates the antibacterial activities of the Ctx(Ile21)-Ha AMP against multidrug-resistant (MDR) and non-resistant bacteria and develop and characterize peptide-loaded microparticles coated with the enteric polymers hydroxypropylmethylcellulose acetate succinate (HPMCAS) and hydroxypropylmethylcellulose phthalate (HPMCP). Ctx(Ile21)-Ha was obtained by solid phase peptide synthesis (SPPS) method, purified and characterized by HPLC and Mass Spectrometry. The peptide exhibited potent antibiotic activities against Salmonella enteritidis, Salmonella typhimurium, Pseudomonas aeruginosa (MDR), Acinetobacter baumannii (MDR), and Staphylococcus aureus (MDR). Ctx(Ile21)-Ha microencapsulation was performed by ionic gelation with high efficiency, maintaining the physical-chemical stability. Ctx(Ile21)-Ha coated-microparticles were characterized by DSC, TGA, FTIR-Raman, XRD and SEM. Hemolytic activity assay demonstrated that hemolysis was decreased up to 95% compared to single molecule. In addition, in vitro release control profile simulating different portions of gastrointestinal tract was performed and showed the microcapsules' ability to protect the peptide and release it in the intestine, aiming pathogen's location, mainly by Salmonella sp. Therefore, use of microencapsulated Ctx(Ile21)-Ha can be allowed as an antimicrobial controller in monogastric animal production as an oral feed additive (antimicrobial controller), being a valuable option for molecules with low therapeutic indexes or high hemolytic rates.

Discovery of new inhibitor for the protein arginine deiminase type 4 (PAD4) by rational design of α-enolase-derived peptides

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease affecting about 0.24 % of the world population. Protein arginine deiminase type 4 (PAD4) is believed to be responsible for the occurrence of RA by catalyzing citrullination of proteins. The citrullinated proteins act as autoantigens by stimulating an immune response. Citrullinated α-enolase has been identified as one of the autoantigens for RA. Hence, α-enolase serves as a suitable template for design of potential peptide inhibitors against PAD4. The binding affinity of α-enolase-derived peptides and PAD4 was virtually determined using PatchDock and HADDOCK docking programs. Synthesis of the designed peptides was performed using a solid phase peptide synthesis method. The inhibitory potential of each peptide was determined experimentally by PAD4 inhibition assay and IC50 measurement. PAD4 assay data show that the N-P2 peptide is the most favourable substrate among all peptides. Further modification of N-P2 by changing the Arg residue to canavanine [P2 (Cav)] rendered it an inhibitor against PAD4 by reducing the PAD4 activity to 35 % with IC50 1.39 mM. We conclude that P2 (Cav) is a potential inhibitor against PAD4 and can serve as a starting point for the development of even more potent inhibitors.

Supramolecular Dipeptide-Based Near-Infrared Fluorescent Nanotubes for Cellular Mitochondria Targeted Imaging and Early Apoptosis.

Herein, we have designed and synthesized unsymmetrical visible Cy-3 and near-infrared (NIR) Cy-5 chromophores anchoring mitochondria targeting functional group conjugated with a Phe-Phe dipeptide by a microwave-assisted Fmoc solid phase peptide synthesis method on Wang resin. These dipeptide-based Cy-3-TPP/FF as well as Cy-5-TPP/FF molecules self-assemble to form fluorescent nanotubes in solution, and it has been confirmed by TEM, SEM, and AFM. The Cy-3-TPP/FF and Cy-5-TPP/FF molecules in solution exhibit narrow excitation as well as emission bands in the visible and NIR region, respectively. These lipophilic cationic fluorescent peptide molecules spontaneously and selectively accumulate inside the mitochondria of human carcinoma cells that have been experimentally validated by live cell confocal laser scanning microscopy and display a high Pearson's correlation coefficient in a colocalization assay. Live cell multicolor confocal imaging using the NIR Cy-5-TPP/FF in combination with other organelle specific dye is also accomplished. Moreover, these lipophilic dipeptide-based cationic molecules reach the critical aggregation concentration inside the mitochondria because of the extremely negative inner mitochondrial membrane potential [(ΔΨm)cancer ≈ -220 mV] and form supramolecular nanotubes which are accountable for malignant mitochondria targeted early apoptosis. The early apoptosis is arrested using Cy-5-TPP/FF and confirmed by annexin V-FITC/PI apoptosis detection assay.

Evaluation of Antibacterial Activity of Magainin and Mastoparan and Its Novel Hybrid Against MDR E. coli Isolates of Neonatal Calves

The extensive use of antibiotics in medical, as well as in veterinary practices has led to the development of antibiotic resistance which is of increasing global concern. The so called “superbugs” or the multi-drug resistant bacteria have made the present day antibiotics less effective. Therefore, search of an alternative is the need of hour, and for which antimicrobial peptides (AMPs) could be a good approach. AMPs are relatively short (12 to 100 amino acids), amphipathic peptides isolated from wide range of living organism. Four peptides were synthesized by solid phase peptide synthesis method; Magainin (Pep-1) & Mastoparan (Pep-2) and Pep-3 and Pep-4 (novel hybrid of Magainin and Mastoparan). MIC of these peptides on E. coli (ATCC-25922) strain was found to be 12 μM, 24 μM, 11 μM, and 40 μM respectively. 100 clinical E. coli isolates were screened against these peptides at their respective 1 and 2xMIC. Percentage of isolates inhibited at 2xMIC for different peptides were Pep-1(74%); Pep-2(91%); Pep-3(86%); Pep-4 (75%). The MBC of the peptides on ATCC-25922 was same as 1xMIC and for majority of the resistant strains it was at 2xMIC. The percentage of haemolysis even at 4xMIC was < 2% in cattle RBC and < 5% in dog RBC which is negligible. Owing to the low MIC and negligible chances of resistance development, AMPs can be a good therapeutic candidate to fight antimicrobial resistance.

Cytotoxicity and Immunogenicity Evaluation of Synthetic Cell-penetrating Peptides for Methotrexate Delivery.

Methotrexate (MTX) is one of the most effective therapeutics to treat different types of solid tumors; however, it suffers low permeability limiting its bioavailability and cellular uptake. To tackle this, we aim to design and fabricate different types of cell-penetrating peptides (CPPs) to improve the intracellular uptake of MTX without causing any immunogenic response. CPPs were synthesized by the solid-phase peptide synthesis method. Peptide-MTX conjugates were prepared via covalent binding of peptide and drug molecule. CPPs and peptide-E8 nanoparticles were characterized using zeta-sizer and scanning electron microscopy. Cytotoxicity of CPPs and peptide-MTX conjugates was evaluated by MTT assay. An enzyme-linked immunosorbent assay was employed to assess the IL-6 and TNF-α cytokine release profile. Amongst all sequences, W4R4-MTX possessed the highest loading efficiency (97%) and drug to peptide percentage (24.02%). The lowest loading efficiency (36%) and drug to peptide percentage (8.76%) were seen for NGRWK-MTX conjugates. The NGRWR peptide and NGRWR-E8 nanoparticles had acceptable size (~100 nm) with spherical and rod-like structures, respectively. The selected CPPs and peptide-MTX conjugates did not show any cytotoxicity or immunogenicity. The fabricated peptides are represented as promising carriers to improve the intracellular delivery of MTX to cancer cells with low immunogenic and cytotoxic effects on normal cells.

Tert-Butyloxycarbonyl-protected amino acid ionic liquids and their application to dipeptide synthesis.

Care should be taken when using amino acid ionic liquids (AAILs) for organic synthesis because of their multiple reactive groups. To expand the applicability of AAILs, we prepared a series of room-temperature ionic liquids derived from commercially available tert-butyloxycarbonyl-protected amino acids (Boc-AAILs). The resulting protected AAILs were used as the starting materials in dipeptide synthesis with commonly used coupling reagents. The distinctive coupling reagent N,N′-diethylene-N′′-2-chloroethyl thiophosphoramide was found to enhance amide formation in the Boc-AAILs without addition of base, giving the dipeptides in satisfactory yields in 15 min.

Design and Synthesis of a New Amphipathic Cyclic Decapeptide with Rapid, Stable, and Continuous Antibacterial Effects

Pathogens can acquire high resistance against even the most powerful antibiotics because of the long periods of treatment and high usage of antimicrobial agents. In addition, the severe side effects of commonly used antibiotics can initiate secondary diseases or may lead to death. Antimicrobial peptides (AMPs) have been reported to exhibit prokaryotic selectivity and low microbial resistance. Furthermore, AMPs show a good ability to penetrate the cell walls of microorganisms. In this study, a cyclic decapeptide and its linear counterpart were synthesized by a standard solid phase peptide synthesis method (SPPS) in a quantitative yield of the linear decapeptide (97%) and a good yield of the cyclic form (45%). Antibacterial studies were performed using Escherichia coli (a widespread Gram-negative pathogen) and Bacillus thuringiensis as a representative Gram-positive pathogen. The minimal inhibitory concentration (MIC) values were evaluated by the broth microdilution method. The cyclic peptide and its linear counterpart exhibited MIC values of 0.16 and 0.3 mg/mL, respectively, against Escherichia coli. Against Bacillus thuringiensis, the peptides had the same MIC value of 0.24 mg/mL. Time-kill studies were performed using E. coli, which indicated a fast killing effect of both peptides (≥ 99% of the bacterial cells) after 1 h of incubation using a concentration of two times the MIC value for each peptide. Moreover, bacterial cell viability studies against E. coli carried out using a high bacterial concentration showed that both peptides have a maximum killing effect of more than 80% of the tested bacterial cells.

Application of 2,2′‐dipyridyl disulfide‐mediated thiazolidine ring‐opening reaction to glycoprotein synthesis: Total chemical synthesis of evasin‐3

Thiazolidine ring-opening reaction is one of the key steps in protein chemical synthesis via sequential native chemical ligation strategy. We recently developed a novel thiazolidine ring-opening reaction with 2,2'-dipyridyl disulfide (DPDS). In order to investigate the applicability of this reaction to glycoprotein synthesis, we synthesized evasin-3, a cysteine-rich glycoprotein with chemokine-binding ability originally found in tick saliva. The sequence of evasin-3 was divided into three segments, and these segments were separately synthesized with the ordinary solid-phase peptide synthesis method. After the first ligation of middle and C-terminal segments, thiazolidine used as a protecting group of Cys residue at the N-terminus of the middle segment was converted to Cys with DPDS. In this thiazolidine ring-opening reaction, DPDS treatment did not affect the N-linked glycan moiety. After the second ligation with the N-terminal segment and the refolding reaction, evasin-3 could be obtained in good yield. The synthetic evasin-3 showed the binding ability specifically to CXCL chemokines. These results clearly indicate that this DPDS method is useful for glycoprotein synthesis.

Identification of β-conglycinin α' subunit antigenic epitopes destroyed by thermal treatments

In the present study, phage display technology (PDT) was used to examine β-conglycinin α' subunit epitopes destroyed by heat treatments. The overlapping gene fragments of the α' subunits were amplified using a polymerase chain reaction (PCR) method, and then the recombinant T7 phages containing the target genes were expressed on the phage surface. In addition, an indirect enzyme-linked immunosorbent assay (iELISA) method was adopted to screen the α' subunit epitopes which had been damaged by the heat treatments. Then, after three rounds of the expression and screening processes, the C1-Y fragments (488PHFNSKAIVVLVINEGEANIELVG511) were identified as α' subunit damaged epitopes. In order to confirm the types of epitopes, three peptides of the C1-Y fragments were synthesized using a solid-phase peptide synthesis method. The results of the iELISA showed that there was a linear epitope of 488PHFNSKAIVVLV499 in the C1-Y fragments. The results obtained in this study confirmed that the C1-Y fragments contained both linear and conformational epitopes, and that the conformational epitopes could be destroyed by thermal treatments. However, it was found that the linear epitopes could not be destroyed. The phage display protein Pt-C1-Y, coupling peptide BSA-Pep-Y, BSA-Pep-1, BSA-Pep-2, and soybean allergy patients' serum reaction results showed that only the Pt-C1-Y had reacted positively with the soybean allergy patients' serum. These findings further indicated that heat treatments can reduce not only the antigenicity, but also the allergenicity, of α' subunits in β-conglycinin.

Synthesis, biological evaluation and preclinical study of a novel 99mTc-peptide: A targeting probe of amyloid-β plaques as a possible diagnostic agent for Alzheimer's disease.

With respect to the main role of amyloid-β (Aβ) plaques as one of the pathological hallmarks in the brain of Alzheimer's patients, the development of new imaging probes for targeted detection of Aβ plaques has attracted considerable interests. In this study, a novel cyclopentadienyl tricarbonyl Technetium-99m (99mTc) agent with peptide scaffold, 99mTc-Cp-GABA-D-(FPLIAIMA)-NH2, for binding to the Aβ plaques was designed and successfully synthesized using the Fmoc solid-phase peptide synthesis method. This radiopeptide revealed a good affinity for Aβ42 aggregations (Kd=20µM) in binding affinity study and this result was confirmed by binding to Aβ plaques in brain sections of human Alzheimer's disease (AD) and rat models using in vitro autoradiography, fluorescent staining, and planar scintigraphy. Biodistribution studies of radiopeptide in AD and normal rats demonstrated a moderate initial brain uptake about 0.38 and 0.35% (ID/g) 2min post-injection, respectively. Whereas, AD rats showed a notable retention time in the brain (0.23% ID/g at 30min) in comparison with fast clearance in normal rat brains. Normal rats following treatment with cyclosporine A as a p-glycoprotein inhibitor showed a significant increase in the radiopeptide brain accumulation compared to non-treated ones. There was a good correlation between data gathered from single-photon emission computed tomography/computed tomography (SPECT/CT) imaging and biodistribution studies. Therefore, these findings showed that this novel radiopeptide could be a potential SPECT imaging agent for early detection of Aβ plaques in the brain of patients with AD.

Synthesis and characterization of cyclic PNC-28 Peptide, residues 17 – 26 (ETFSDLWKLL), a binding domain of p53

Abstract The present study reports the synthesis of cyclic PNC-28 peptide with solid phase peptide synthesis method. In first step we synthesize linear PNC-28 Peptide and in second step we cyclize (N-to-C or head-to-tail cyclization) the linear PNC-28 peptide. The molecular formula of cyclic PNC-28 peptide is C64H88N12O16 and its m/z mass is ≈1233.64. Elemental analysis of cyclic PNC-28 is C, 59.99; H, 6.92; N, 13.12; O, 19.98. The characterization of LC-MS, CD, FT-IR, 1HNMR has been done to confirm the successful synthesis and cyclization of linear PNC-28 peptide

Renoprotective Effects of Di- and Tri-peptides Containing Proline, Glycine and Leucine in Diabetes Model of Adult Mice: Enzymology and Histopathology

The small peptides, as multifunctional biomolecules, can affect some organs and metabolic disorders. The purpose of this experiment was to investigate the effects of some small peptides on the renal enzymes and histopathology in mice exposed to diabetes. Solid phase peptide synthesis method was used to synthesis di- and tri-peptides. Diabetes was induced in mice by multiple injection of low doses of streptozotocin. The effects of the di- and tri-peptides on renal aspartate aminotransferase, alanine transaminase, alkaline phosphatase, superoxide dismutase (SOD), glutathione peroxidase, total antioxidant (TAC), and catalase activities were evaluated by enzymatic biochemical methods and malondialdehyde (MDA) by thiobarbituric acid method. Hematoxylin and eosin and TUNEL stainings performed for assessing histopathology and apoptosis of the mice kidney. Hannaneh (Leu–Gly) and carnosine (Ala–His) exhibited suppression effects on the blood glucose elevation and the severity of the renal alterations in diabetic mice. They showed a decreasing effect on diabetes-induced DNA damage (P < 0.05). Carnosine significantly prevented from MDA elevation (P < 0.05). The Pro–Pro showed a supression effect on the blood glucose elevation (P < 0.05). Glutathione (Glu–Cys–Gly) and hannaneh caused to renal tissue TAC decreasing (P < 0.05). Mice receiving Pro–Gly and Pro–Gly–Pro have lesser renal SOD compared to diabetic mice (P < 0.05). Pro–Gly–Pro significantly decreased the diabetes-induced DNA damage. Although all the treatments had significant effects on alleviating the apoptosis, but carnosine and hannaneh dipeptides had the best protective effect on the kidney. The carnosine and hannaneh dipeptides had higher reno-protective effects against renal enzymes and histologic changes in diabetic mice.

Synthesis of Tetrapeptides and Screening of their Antioxidant Properties

Background: Tetrapeptide Pro-Ala-Gly-Tyr (PAGY) is an antioxidant peptide that was isolated by Nikoo et al. (2014) from hydrolysate of skin gelatin of amur sturgeon fish (Acipenser schrenckii). This research aims to synthesize PAGY and its analogues by a solid-phase method, and to screen their antioxidant activities. Methods: PAGY and its analogues, namely, Pro-Ser-Gly-Tyr (PSGY), Pro-Ala-Phe-Tyr (PAFY), Pro- Phe-Phe-Tyr (PFFY) and Pro-Ala-Ile-Tyr (PAIY), were synthesized via a solid phase peptide synthesis method with the Fmoc/t-Bu strategy. The synthesis was undertaken on 2-chlorotritylchloride resin as solid support, and all coupling reactions were facilitated by a combination of HBTU and HOBt reagents. All peptides were cleaved from the resin by using 95% TFA in water. Results: Through the solid-phase synthesis method, all peptides were obtained in 50-85% yields. Pure peptides were analysed by analytical RP-HPLC, and were characterized by HR-TOF-ESI-MS and 1HNMR. DPPH inhibition assay was applied to all of the peptides with IC50 of 1.750; 1.116; 1.177 ;1.437; and 1.642 mg/mL for PAGY, PSGY, PAFY, PFFY, and PAIY, respectively. Conclusion: PAGY and its four analogs were successfully synthesized by solid-phase method. PSGY compound was found to have the highest activity of all in DPPH assay.

Biomolecular Interactions of RAD51181–200 with BRCA1846–871 and Mutants and Molecular Docking Approach

Affecting the repair function of RAD51 can effectively reverse tumor resistance to chemotherapy and radiotherapy. There is interaction between RAD51181−200 and BRCA1846−871. Using the computer to simulate single mutation of lysine at 862 position of BRCA1846−871, six BRCA1846−871 mutants with significant interaction with RAD51181−200 were screened out. These mutant peptides were obtained by 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS) method. All peptides were purified and characterized by liquid chromatography and ESI-MS. In this study, the interaction between RAD51181−200 and BRCA1846−871 or mutant peptides was investigated using circular dichroism spectroscopy (CD) and fluorescence spectroscopy. The CD results shown that the secondary structure change of RAD51181−200 induced by BRCA1846−871, BRCA1846−871 K862F (F), BRCA1846−871 K862G (G), BRCA1846−871 K862E (E) and BRCA1846−871 K862N (N) was obvious, which the α-helix content of RAD51181−200 decreased from 58.3–36.1%, 58.3–36.2%, 58.3–36.4%, 58.3–36.5% and 58.3–36.0%, respectively. While, the addition of BRCA1846−871 K862P (P) and BRCA1846−871 K862Y (Y) hardly changed the α-helix content of RAD51181−200. From the fluorescence results, the binding constants of F and Y are 1.00 × 105 M−1 and 1.16 × 105 M−1 respectively, which were both larger than that of BRCA1846−871 (9.01 × 102 M−1). The binding constant of RAD51181−200 with E (7.45 × 103 M−1) is slightly higher than that of BRCA1846−871. The binding constants of RAD51181−200 with P (1.24 × 103 M−1) and N (1.26 × 103 M−1) are similar to that of BRCA1846−871. However, the Kb value of G (1.51 × 102 M−1) is less than that of BRCA1846−871. Comparing all spectral data, F is the most significant one interacting with RAD51181−200. These findings could provide direction for peptide processing and application.

Structure-activity relationship studies of Longicalcynin A analogues, as anticancer cyclopeptides

Cancer has emerged as the main cause of the highest rate of mortality in the world. Drugs used in cancer, although, show some beneficial effects on cancerous organs, demonstrate side effects on other normal tissues. On the other hand, anticancer peptides, being effective on target tissues, should be safe and less harmful on healthy organs, since peptides have several advantages, i.e., high activity, specificity, affinity, being less immunogenic and not accumulate in the body. In the present work, analogues of Longicalcynin A, a naturally occurring anticancer cyclopeptide, were synthesized and evaluated their cytotoxicity in order to gain information from structure-activity relationships of the such cyclopeptides which may lead to find novel and safer anticancer peptide compound(s) to be used in clinic. Peptides were prepared by the solid-phase peptide synthesis method using trityl-resin. Peptide cyclization was performed in liquid phase. To study anticancer activity of the peptide analogues of Longicalycinin A, several methods including MTT, flow cytometry analysis and Lysosomal membrane integrity assay were employed using two cell lines HepG2 and HT-29. Fibroblast cells were used to control the safety of the synthesized cyclopeptides on normal cells. Two cyclopeptides 11 and 17 with the sequences of cyclo-(Thr-Val-Pro-Phe-Ala) and cyclo-(Phe-Ser-Pro-Phe-Ala), respectively were cytotoxic against the colon as well as hepatic cancer cells with safety profile against fibroblast cells, probably with the mechanism of apoptosis as lysosomal membrane integrity damaged. These cyclopeptides showed to be more favorable compounds better than Longicalycinin A and good candidates to develop cyclopeptides as anticancer agents.

Rabies Virus 31D Peptide‐[P(VP‐co‐AA)] Conjugates: Synthesis, Characterization and Cytotoxicity Evaluation

AbstractThe prevention of rabies disease, which has been the subject of many pieces of researches since ancient times, has become possible by vaccination. Therefore, new generation vaccine systems should be developed to achieve more effective, accessible and reliable vaccines than conventional vaccines against this disease. In this study, modificated Rabies virus 31D antigenic peptide epitope was synthesized by microwave supported solid phase peptide synthesis method and the synthesized peptide was characterized. Bioconjugates of the antigenic peptide epitope with poly (N‐Vinyl‐2‐pyrrolidone‐co‐acrylic acid) that a biocompatible polymer was synthesized synthetically were synthesized at different ratios in the presence of 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide. Characterization of bioconjugates was performed with Gel Permeation Chromatography and ZetaSizer. Cytotoxicity test was carried out using ECV304 human epithelial cells to determine the biocompatibility of peptide, polymer and bioconjugates. It is thought that the produced biocompatible synthetic peptide‐polymer based rabies vaccine systems will contribute greatly to the vaccination studies.

Synthesis of Antioxidant Peptide SCAP1 (Leu-Ala-Asn-Ala-Lys)

SCAP1 is a pentapeptide isolated from the hydrolysate of oyster (Saccostrea cucullata), which has antioxidant and anticancer properties. This study aims to find a novel strategy to obtain antioxidant peptide SCAP1 (Leu-Ala-Asn-Ala-Lys) through chemical synthesis. This study reports the first synthesis of SCAP1 through the solid phase peptide synthesis (SPPS) method with Fmoc strategy on 2-chlorotrityl chloride resin. A HATU-HOAt coupling reagent was employed in all amide bond formations and 95% TFA in water was employed to release the desired peptide from the solid support. The crude peptide was purified using successive preparative-RP-MPLC and RP-HPLC to obtain the pure peptide. The synthesized peptide was confirmed by mass spectrometry, showing a correct molecular ion peak at m/z 516.3143 (M+H)+, referring to the desired SCAP1. 1H-NMR spectra of the peptide revealed protons signals consistent to the structure of SCAP1. During the synthesis, the coupling between Ala and Asn(Trt) was not straightforward, which led to a low yield of the final product. The use of Trt-based Asn was not a suitable option, as it could cause aggregation during the synthesis, eventually leading to a poor yield of the final product. SCAP1 was obtained as a white solid with a percent yield of 8.28%.

Abstract 1769: CSox-based sensors for continuous, homogeneous and quantitative monitoring of protein kinase and phosphatase activity

Abstract Introduction: Protein kinases are a diverse group of 518 enzymes whose dysregulation lies at the center of many diseases. Currently, 30% of all drug development efforts are focused on protein kinases. Although 41 drugs are approved and &amp;gt;120 in clinical trials, these are predominately ATP-competitive inhibitors. More recently, there has been an expanded focus on kinase inhibitors with different modes of action, where new tools are needed to effectively characterize inhibitor mechanism of action, predict drug potency and to drive decisions earlier in the drug development process. We developed a simple yet powerful method for the generation of peptide sensors that can be used for the continuous, quantitative and homogenous detection of kinase and phosphatase activity with recombinant enzymes and crude lysates to enable target discovery and drug development. Experimental Procedures: We harnessed chelation-enhanced fluorescence by combining next generation sulfonamido-oxine (Sox) chromophore technology with high-throughput solid-phase peptide synthesis methods to identify optimized sequences based on physiological substrates. Enzyme activity is monitored kinetically using fluorescence intensity (Ex/Em 360/485 nm) or in endpoint mode using Europium and time-resolved fluorescence (Ex/Em 360/620 nm). Results: We demonstrate the ability to rapidly identify novel optimized substrates, where performance measures included higher reaction rates, lower Km's, higher signal/background, increased sensitivity and specificity. We identified highly generic substrates (for robust detection of 80 Tyrosine kinases) and highly-selective substrates (for quantitative detection of targeted kinases in crude cell or tissue lysates for profiling, potency assessments and SAR). We have developed sensors to monitor activity of high-profile tyrosine kinases, including the EGFR and clinically-relevant mutants, JAK kinases, Tec-kinases, and, serine/threonine kinases, including CDK1-9, MAPK pathway (MAP4Ks, MAP3Ks, MAPKs &amp; MAPKAPKs), PKR/EIF2AKs and PIM1. In addition, CSox-based phosphopeptide substrates are used to monitor protein phosphatases with specificity for tyrosine (PTP1B, SHP1/2) or serine/threonine (PP2A, PP2C, PHLPP). Conclusions: The generation of robust activity-based sensors, even where peptide assays previously weren’t available, opens new areas for effective drug discovery. The Sox-based kinetic assay format is ideal for elucidating drug mechanism of action, potency, and enzyme regulation. The PhosphoSens-Red endpoint format is ideal for HTS, SAR and profiling. Together, these formats can be applied across the entire target discovery and drug development workflow, providing a quantum improvement in performance and productivity needed to address the challenges and opportunities of next generation protein kinase and phosphatase inhibitors. Citation Format: Erik M. Schaefer, Susan Cornell-Kennon, Bill Lu. CSox-based sensors for continuous, homogeneous and quantitative monitoring of protein kinase and phosphatase activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1769.