Synthesis and Biological Study of 4-Aminopyridine-Peptide Derivatives Designed for the Treatment of Neurodegenerative Disorders.

BACKGROUND: Alzheimer's disease (AD) and Multiple sclerosis (MS) lead to neurodegenerative processes negatively affecting millions of people worldwide. Their treatment is still difficult and practically incomplete. One of the most commonly used drugs against these neurodegenerative diseases is 4-aminopyridine. However, its use is confined by the high toxicity. OBJECTIVES: The aim of this work is to obtain new peptide derivatives of 4-aminopyridine with decreased toxicity compared to 4-aminopyridine. METHODS: Synthesis was conducted in solution using a consecutive condensation approach. The new derivatives were characterized by melting points, NMR, and Mass spectra. Important ADME (absorption, distribution, metabolism, and excretion) properties have been studied in silico using ACD/Percepta v.2020.2.0 software. Acute toxicity was determined in mice according to a Standard protocol. All new derivatives were tested in vitro for cytotoxic activity in a panel of human (HEP-G2, BV-173) and murine (NEURO 2A) tumor cell lines via a standard MTT-based colorimetric method. ß-secretase inhibitory activity was determined by applying the fluorescent method. RESULTS: New derivatives of 4-aminopyridine containing analogues of the ß-secretase inhibitory peptide (Boc-Val-Asn-Leu-Ala-OH) were obtained. The in vivo toxicity of the tested compounds was found to be as high as 1500 mg/kg. Cell toxicity screening against tumor cell lines of different origins showed negligible growth-inhibitory effects of all investigated 4-aminopyridine analogues. CONCLUSION: Synthesis of new peptide derivatives of 4-aminopyridine is reported. Acute toxicity studies revealed a ca. 150 times lower toxicity of the new compounds as compared to 4-aminopyridine that may be ascribed to their peptide fragment.

Synthesis and biological study of new galanthamine-peptide derivatives designed for prevention and treatment of Alzheimer's disease.

The Alzheimer's disease leads to neurodegenerative processes and affecting negatively million people worldwide. The treatment of the disease is still difficult and incomplete in practice. Galanthamine is one of the most commonly used drugs against the illness. The main aim of this work is design and synthesis of new derivatives of galanthamine comprising peptide moiety as well as study of their ß-secretase inhibitory activity and the anti-aggregating effect. All new derivatives of galanthamine containing analogues of Leu-Val-Phe-Phe (Aß17-Aß20) were synthesized in solution using fragment and consecutive condensation approaches. The new derivatives were characterized by melting points, NMR, and HPLC/MS. They were tested in vitro for ß-secretase inhibition activity by means of fluorescent method and were investigated in vitro for anti-aggregation activity on sheep platelet-rich plasma. Although the new compounds do not contain a structural element responsible for the ß-secretase inhibition, five of them show high or good ß-secretase inhibitory activity between 19.98 and 51.19% with IC50 between 1.95 and 5.26 nM. Four of the new molecules were able to inhibit platelet aggregation between 55.0 and 90.0% with IC50 between 0.69 and 1.36 µM. Four of the compounds were able to inhibit platelet aggregation and two of them have high anti-aggregating effects.

Serum 7-alfa-hydroxy-4-cholesten-3-one and fibroblast growth factor-19 as biomarkers diagnosing bile acid malabsorption in microscopic colitis and inflammatory bowel disease.

BACKGROUND: Bile acid malabsorption is common in microscopic colitis, irritable bowel syndrome with diarrhea, and inflammatory bowel disease. We investigated the diagnostic accuracy of 7-alfa-hydroxy-4-cholesten-3-one and compared it with fibroblast growth factor-19 as biomarkers for bile acid malabsorption. METHODS: We enrolled consecutively 109 chronic diarrhea patients with standard laboratory tests, fecal calprotectin, and endoscopy separated into six groups: n = 30 with active inflammatory bowel disease, n = 21 with inflammatory bowel disease in remission reporting >3 bowel movements per day, n = 21 with inflammatory bowel disease after surgery, n = 23 with irritable bowel syndrome with diarrhea, n = 14 with microscopic colitis and 11 healthy subjects (controls). We defined bile acid malabsorption as >3 bowel movements and lower fibroblast growth factor-19 (<60 pg/ml). RESULTS: Median levels of 7-alfa-hydroxy-4-cholesten-3-one in inflammatory bowel disease active were 53.1 ng/ml, inflammatory bowel disease remission were 52.2 ng/ml, inflammatory bowel disease after surgery were 85.7 ng/ml, irritable bowel syndrome with diarrhea were 7.5 ng/ml, microscopic colitis were 69.3 ng/ml, and healthy controls were 3.7 ng/ml. We estimate a 7-alfa-hydroxy-4-cholesten-3-one cutoff of 48.9 ng/ml with 82.6% sensitivity and 84.3% specificity for detecting bile acid malabsorption. Both 7-alfa-hydroxy-4-cholesten-3-one >48.9 ng/ml and fibroblast growth factor-19 (<60 pg/ml) were found in 52% of the patients, compared with those 8% of patients below this 7-alfa-hydroxy-4-cholesten-3-one cutoff (P < 0.001). Serum 7-alfa-hydroxy-4-cholesten-3-one correlated with the number of bowel movements/day (r = -0.709; P < 0.001) and correlated inversely with fibroblast growth factor-19 (r = -0.741; P < 0.001). CONCLUSIONS: Serum 7-alfa-hydroxy-4-cholesten-3-one above 48.9 ng/ml and fibroblast growth factor-19 below 60 pg/ml identify patients with diarrhea likely attributable to bile acid malabsorption with high diagnostic accuracy and they can be used as screening biomarkers for bile acid malabsorption in microscopic colitis and inflammatory bowel disease.

HPLC-UV and LC-MS Analyses of Acylquinic Acids in Geigeria alata (DC) Oliv. & Hiern. and their Contribution to Antioxidant and Antimicrobial Capacity.

INTRODUCTION: Geigeria alata is a traditional plant used in Sudanese folk medicine for treatment of diabetes, cough, epilepsy and intestinal complaints. OBJECTIVE: To analyze phenolic acids in Geigeria alata roots and leaves and to evaluate their antioxidant and antimicrobial activities. METHODOLOGY: Phenolic acids in the aqueous-methanol extracts were identified by LC-MS. Major compounds were isolated using low-pressure liquid chromatography. The quantitative analysis of phenolic acids was performed by a validated HPLC-UV method with limits of detection ranging from 0.04 to 0.57 µg/mL. 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azinobis-(3-ethylbenzothiazine-6-sulphonic acid) (ABTS) and ferric reducing antioxidant power (FRAP) methods were used for antioxidant activity evaluation. In addition, the minimal inhibitory concentration and the minimal bactericidal concentration against a panel of pathogenic bacteria and fungi were determined by the broth microdilution test. RESULTS: For the first time protocatechuic, caffeic, p-coumaroylquinic, caffeoylsinapoylquinic, caffeoylferuloylquinic, three feruloylquinic, six caffeoylquinic acids, and a caffeic acid hexoside were detected in Geigeria alata roots by LC-MS. HPLC-UV analyses showed that 3,5-dicaffeoylquinic acid (25.96 ± 2.08 mg/g dry weight (DW)) was the most abundant phenolic acid in roots, while 4,5-dicaffeoylquinic acid (8.99 ± 0.56 mg/g DW) was the main compound present in leaves. 3,5-Dicaffeoylquinic acid demonstrated stronger radical scavenging activity and reducing power compared with the crude extracts and the positive control 5-caffeoylquinic acid. 3,4,5-Tricaffeoylquinic acid revealed the highest antibacterial potential against the penicillin sensitive and resistant Staphylococcus aureus strains, as well as methicillin-resistant S. aureus. CONCLUSION: The caffeoylquinic acids content of up to 6.22% in Geigeria alata roots establishes this species as a new source rich in these bioactive molecules. Copyright © 2016 John Wiley & Sons, Ltd.

In vivo investigation of homocysteine metabolism to polyamines by high-resolution accurate mass spectrometry and stable isotope labeling.

Polyamines are essential polycations, playing important roles in mammalian physiology. Theoretically, the involvement of homocysteine in polyamine synthesis via S-adenosylmethionine is possible; however, to our knowledge, it has not been established experimentally. Here, we propose an original approach for investigation of homocysteine metabolites in an animal model. The method is based on the combination of isotope-labeled homocysteine supplementation and high-resolution accurate mass spectrometry analysis. Structural identity of the isotope-labeled metabolites was confirmed by accurate mass measurements of molecular and fragment ions and comparison of the retention times and tandem mass spectrometry fragmentation patterns. Isotope-labeled methionine, spermidine, and spermine were detected in all investigated plasma and tissue samples. The induction of moderate hyperhomocysteinemia leads to an alteration in polyamine levels in a different manner. The involvement of homocysteine in polyamine synthesis and modulation of polyamine levels could contribute to a better understanding of the mechanisms connected with homocysteine toxicity.

Comparative analysis of polyamine metabolism in benign and neoplastic keratinocytic proliferations.

INTRODUCTION: Polyamines (putrescine, spermidine, and spermine) are polycationic compounds that play a central role in keratinocytic proliferation, differentiation, and regulation. The objective was to elucidate the polyamine metabolic changes that occur in various benign and neoplastic skin proliferations. METHODS: The study included 58 patients: 31 with the plaque form of psoriasis vulgaris and 27 with non-melanoma skin tumors. The levels of putrescine, spermidine, and spermine were detected in lesional and non-lesional skin samples. RESULTS: Findings were representative (p < 0.05). Psoriatic lesions showed a twofold elevation of all polyamines in lesional skin compared to non-lesional skin. Spermine had the highest concentration, which suggested a leading position of propylamine synthesis in psoriatic pathogenesis. Results on the polyamine metabolism of basal cell carcinoma represented basic characteristics similar to those of psoriasis. Conversely, squamous-cell carcinoma lesions showed the highest concentration of putrescine, suggesting a crucial role of spermidine-spermine acetyltransferase in their pathogenesis. DISCUSSION: Our findings showed different polyamine metabolic changes in lesions from benign and neoplastic keratinocytic proliferations. Basal-cell carcinoma polyamine metabolism revealed a closer relationship to psoriasis than to squamous-cell carcinoma, which might explain its long-term benign course and non-metastatic nature.

Evaluation of recombinant caspase specificity by competitive substrates.

The specificity of 10 recombinant caspases was investigated using a set of competitive substrates. The caspase activity was determined by high-performance liquid chromatography using highly fluorescent peptides containing 2-aminoacridone (AMAC) as reporting group. The sequences of the used substrates were designed according to literature data for being specific for 10 of the caspases. The described approach allows the concentration changes of several substrates to be monitored simultaneously in a single sample. Because the substrates are in competitive conditions, the preferences of particular caspases to given peptide sequences are most clearly demonstrated. In the studied competitive assay conditions, all tested caspases except caspase 2 exhibit activity toward more than one substrate. None of the used peptide sequences was found to be highly specific for a defined caspase. The results obtained indicate that there is well-expressed group specificity among the caspases.

Determination of plasma aminothiols by high performance liquid chromatography after precolumn derivatization with N-(2-acridonyl)maleimide.

Design, synthesis and properties of new derivatization reagent N-(2-acridonyl)-maleimide (MIAC) for thiol groups is presented. The reaction of MIAC with aminothiols is specific, very fast and yield highly fluorescent products. The HPLC method for determination of homocysteine, cysteine and glutathione based on utilization of MIAC is developed. A baseline separation of derivatives is achieved by isocratic elution on reverse phase column within 6 min. The method is linear in the range of 0.5-25 microM for homocysteine and glutathione, and in the range of 0.5-200 microM for cysteine. The limits of detection for homocysteine, cysteine and glutathione are 1.2, 1.4 and 2.0 pmol, respectively, per 20 microl injection. Within and between-run precision expressed as relative standard deviations are in the range of 1.35-4.38% and 0.89-4.13%, respectively.