Proteomics in drug development – optimizati on of sample preparation for the two-dimensional gel-electrophoresis

Uvod: Dvodimenzionalna gel-elektroforeza (2-DE) metoda je analize kompleksnih proteinskih smjesa izdvojenih iz stanica, tkiva, tjelesnih tekućina ili drugih bioloških uzoraka, koja objedinjuje dva različita elektroforetska postupka: izoelektrično fokusiranje i SDS-poliakrilamidnu gel-elektroforezu. Metode: U ovom radu ispitan je utjecaj četi riju različiti h pufera na proteomske profile dviju staničnih linija karcinoma kolona, SW 620 i HCT 116 uzgojenih klasičnim metodama uzgoja stanica in vitro uz pomoć metode 2-DE, u pH rasponu 3 – 10 NL, te rasponu molekulskih težina od 14 do 100 kDa. Rezultati : Dobiveni rezultati pokazuju kako se pojedini puferi međusobno razlikuju po prirodi proteina koji su specifi čno otopljeni, što je djelomično ovisilo i o staničnoj liniji. Diskusija: Topljivost proteina jedan je od najvećih problema pri korištenju 2-DE, što naročito dolazi do izražaja kod hidrofobnih membranskih proteina, proteina jezgre i proteina izrazito sklonih agregaciji. Jedan od načina poboljšanja topljivosti proteina je i opti mizacija pufera za izdvajanje proteina, pri čemu se u pufer dodaju zwitt er-ionski amfi fi lni surfaktanti poput CHAPS, SB 3-10 (kaprilil sulfobetain) i ASB-14, te uporabom ti ouree u kombinaciji s ureom. Gelovi dobiveni uz pomoć pufera koji sadrži CHAPS imali su najbolju rezoluciju i na njima je bilo moguće detekti rati najveći broj jedinstvenih proteinskih vrsta obiju ispitanih staničnih linija. Zaključak: Postupkom opti mizacije pripreme uzorka za analizu mehanizama učinka novosinteti ziranog protutumorskog spoja na razini proteoma uz pomoć 2-DE pokazano je kako je pufer sastava 7M urea + 2M ti ourea + 4% (w/v) CHAPS opti malan pufer za izolaciju i razdvajanje smjese proteina dobivene iz tumorskih staničnih linija kolona HCT-116 i SW 620.Introducti on: The two-dimensional gel-electrophoresis (2-DE) is a method of choice for complex protein mixture separati on from cells, ti ssue, body fl uids or other biological samples which comprises two diverse electrophoreti c principles: the isoelectric focusing and the SDS-polyacrilamide gel electrophoresis. Methods: In this paper we tested the effects of four diff erent 2-DE buff ers on the protein profi les obtained by 2-DE in the pH range 3-10 NL, and the molecular weight range 14-100 kDa. The proteins were isolated from two colon cancer cell lines SW 620 i HCT 116 grown in vitro by using standard cell propagation procedures. Results: The obtained results revealed diff erent 2-DE buff er properti es for each buff er tested as the protein profi les showed a diff erent patt ern both as the consequence of the buff er type and the type of cell line. Discussion: One of the major problems in 2-DE is protein solubility which is parti cularly emphasized when dealing with hydrophobic membrane proteins, the nuclear proteins as well as with proteins prone to form aggregates. One of the possible soluti ons to this problem is the opti mizati on of the 2-DE buff er by additi on of zwitt er-ionic amphyphilic surfactants like CHAPS, SB 3-10 (sulphobetain) and ASB-14, as well as by additi on of thiourea and urea. The gels obtained with the buff er containing CHAPS showed the best resoluti on properti es and had the best unique protein species score for both tested cell lines in comparison to all other gels. Conclusion: Through the 2-DE buffer optimization in the sample preparati on step for proteomic analysis of newly synthesized anti tumor drug mechanisms of acti on, it has been shown that the buff er containing 7M urea + 2M thiourea + 4% (w/v) CHAPS was the most eff ecti ve for isolati on and separati on of proteins from HCT-116 and SW 620 tumour cell lines

Određivanje aminokiselinske sekvencije farmaceutskih peptida MALDI-TOF tandemnom spektrometrijom masa

Although the peptide ion mechanisms of pre-dissociation, dissociation and post-dissociation have been well-studied over the past fifteen years, their practical application still has to be implemented into modern mass spectrometry-driven proteomics and bioanalysis. Unambiguous peptide sequence determination by mass spectrometry relies on the idea that only one continuous series of ions in mass spectrum can assure full sequence determination and meets the requirements of peptide analysis quality. This set of rules defined for the peptide analysis by tandem mass spectrometry would generally improve an overall reliability and data accuracy. Based on the process mechanisms of gas-phase peptide bond dissociation, a relatively small and large model peptides are unambiguously analyzed (bivalirudin and exenatide) showing that derivatization concepts of the C- or N-terminus derivatization (SPITC and Lys-tag) can be avoided.Iako su mehanizmi preddisocijacije, disocijacije i postdisocijacije iona izučavani dugi niz godina, postoji još cijeli niz mehanizama raspada iona koji se mogu korisno upotrijebiti u modernoj spektrometriji masa, proteomici i bioanalizama. Nedvojbena aminokiselinska analiza spektrometrijom masa počiva na ideji da samo jedan i kontinuirani slijed iona u tandemnom spektru masa može potpuno potvrditi cjelovitu aminokiselinsku sekvenciju i na taj način zadovoljiti zahtjeve nedvojbene analize peptida. Niz pravila kojima se definira cjelovita i nedvojbena analiza peptida tandemnom spektrometrijom masa u konačnici može unaprijediti iščitavanje rezultata analize i pouzdanost dobivenih podataka. Na teorijskim zasadama disocijacije iona u plinskoj fazi u ovom je radu prikazana cjelovita analiza peptida. Na modelnim farmaceutskim peptidima bivalirudinu i eksenatidu pokazano je kako se i bez raširene upotrebe derivatizacijskih tehnika (SPITC i Lys-tag) može ostvariti nedvojbena analiza, iako se modifikacije cisteina akrilacijom, metilacijom ili karboksimetilacijom pri tome ne mogu izbjeći

Novosti u mozaiku metastaziranja zloćudnih tumora

Despite the abundance of attention that cancer has attracted, it continues to constitute one of the deadliest scourges of the modern era. Tumour heterogeneity greatly contributes to the ineffectiveness of current therapies and hampers the study and treatment of cancer. There are two models accounting for tumour heterogeneity and propagation, namely clonal evolution model and cancer stem cell model. In particular, cancer stem theory has attracted much attention lately, as these cells with self-renewal and differentiation abilities are responsible for the initiation of tumour development, growth, and its ability to metastasize and reoccur, and provide a reasonable explanation for poor prognosis for patients in advanced stages of solid tumours. Advances in technologies such as proteomics open new avenues in metastasis research by specifically revealing complex protein networks involved in tumour progression, which should facilitate early diagnosis and provide the basis for designing more effective treatment strategies.Novosti u mozaiku metastaziranja zloćudnih tumora Unatoč napretku u istraživanju i proučavanju zloćudnih tumora, ta je bolest i dalje velik izazov modernoj medicini. Biološka raznolikost, ali i klasično poimanje mehanizama metastaziranja tumora, razlog su neučinkovitosti postojećih načina liječenja. Dva su modela kojima se objašnjava ta raznolikost – model klonske evolucije te model matičnih stanica novotvorina. Ovaj drugi u posljednje je vrijeme privukao pozornost jer su matične stanice novotvorina, zbog svojih sposobnosti samostalnog obnavljanja i diferenciranja, odgovorne za nastanak i razvoj tumora te njihovu sposobnost metastaziranja i pojave recidiva. Metode globalnih analiza poput proteomike otvaraju nove mogućnosti u istraživanju procesa metastaziranja, jer omogućuju identifikaciju složenih mreža proteina uključenih u progresiju novotvorina, što može pridonijeti ranoj dijagnostici i omogućiti razvoj učinkovitijih lijekova protiv metastaza

Novel Bis- and Mono-Pyrrolo[2,3-d]pyrimidine and Purine Derivatives: Synthesis, Computational Analysis and Antiproliferative Evaluation.

Novel symmetrical bis-pyrrolo[2,3-d]pyrimidines and bis-purines and their monomers were synthesized and evaluated for their antiproliferative activity in human lung adenocarcinoma (A549), cervical carcinoma (HeLa), ductal pancreatic adenocarcinoma (CFPAC-1) and metastatic colorectal adenocarcinoma (SW620) cells. The use of ultrasound irradiation as alternative energy input in Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) shortened the reaction time, increased the reaction efficiency and led to the formation of exclusively symmetric bis-heterocycles. DFT calculations showed that triazole formation is exceedingly exergonic and confirmed that the presence of Cu(I) ions is required to overcome high kinetic requirements and allow the reaction to proceed. The influence of various linkers and 6-substituted purine and regioisomeric 7-deazapurine on their cytostatic activity was revealed. Among all the evaluated compounds, the 4-chloropyrrolo[2,3-d]pyrimidine monomer 5f with 4,4'-bis(oxymethylene)biphenyl had the most pronounced, although not selective, growth-inhibitory effect on pancreatic adenocarcinoma (CFPAC-1) cells (IC50 = 0.79 µM). Annexin V assay results revealed that its strong growth inhibitory activity against CFPAC-1 cells could be associated with induction of apoptosis and primary necrosis. Further structural optimization of bis-chloropyrrolo[2,3-d]pyrimidine with aromatic linker is required to develop novel efficient and non-toxic agent against pancreatic cancer

Novel Bis- and Mono-Pyrrolo[2,3-d]pyrimidine and Purine Derivatives: Synthesis, Computational Analysis and Antiproliferative Evaluation

Novel symmetrical bis-pyrrolo[2, 3-d]pyrimidines and bis-purines and their monomers were synthesized and evaluated for their antiproliferative activity in human lung adenocarcinoma (A549), cervical carcinoma (HeLa), ductal pancreatic adenocarcinoma (CFPAC-1) and metastatic colorectal adenocarcinoma (SW620) cells. The use of ultrasound irradiation as alternative energy input in Cu(I)-catalyzed azide- alkyne cycloaddition (CuAAC) shortened the reaction time, increased the reaction efficiency and led to the formation of exclusively symmetric bis-heterocycles. DFT calculations showed that triazole formation is exceedingly exergonic and confirmed that the presence of Cu(I) ions is required to overcome high kinetic requirements and allow the reaction to proceed. The influence of various linkers and 6-substituted purine and regioisomeric 7-deazapurine on their cytostatic activity was revealed. Among all the evaluated compounds, the 4-chloropyrrolo[2, 3-d]pyrimidine monomer 5f with 4, 4′-bis(oxymethylene)biphenyl had the most pronounced, although not selective, growth-inhibitory effect on pancreatic adenocarcinoma (CFPAC-1) cells (IC50 = 0.79 µM). Annexin V assay results revealed that its strong growth inhibitory activity against CFPAC-1 cells could be associated with induction of apoptosis and primary necrosis. Further structural optimization of bis-chloropyrrolo[2, 3-d]pyrimidine with aromatic linker is required to develop novel efficient and non-toxic agent against pancreatic cancer

Amidine- and Amidoxime-Substituted Heterocycles: Synthesis, Antiproliferative Evaluations and DNA Binding

The novel 1, 2, 3-triazolyl-appended N- and O- heterocycles containing amidine 4–11 andamidoxime 12–22 moiety were prepared and evaluated for their antiproliferative activitiesin vitro. Among the series of amidine-substituted heterocycles, aromatic diamidine 5 and coumarine amidine 11 had the most potent growth-inhibitory effect on cervical carcinoma (HeLa), hepatocellular carci- noma (HepG2) and colorectal adenocarcinoma (SW620), with IC50 values in the nM range. Although compound 5 was toxic to non-tumor HFF cells, compound 11 showed certain selectivity. From the amidoxime series, quinoline amidoximes 18 and 20 showed antiproliferative effects on lung adeno-carcinoma (A549), HeLa and SW620 cells emphasizing compound 20 that exhibited no cytostatic effect on normal HFF fibroblasts. Results of CD titrations and thermal melting experiments indicated that compounds 5 and 10 most likely bind inside the minor groove of AT-DNA and intercalate into AU-RNA. Compounds 6, 9 and 11 bind to AT-DNA with mixed binding mode, most probably minor groove binding accompanied with aggregate binding along the DNA backbone

Sphingosine 1-Phosphate Signaling and Metabolism in Chemoprevention and Chemoresistance in Colon Cancer

Colorectal carcinoma (CRC) is the leading cause of cancer-related deaths worldwide. Despite advances in prevention and treatment modalities for CRC, rapidly developing resistance to chemotherapy limits its effectiveness. For that reason, it is important to better understand the mechanisms that undergird the process of chemoresistance to enable design of novel anticancer agents specifically targeting malignant properties of cancer cells. Over recent decades, bioactive sphingolipid species have come under the spotlight for their recognized role in cancer development and progression, and the evidence has surfaced to support their role as regulators of anti-cancer drug resistance. Colon cancer is characterized by a shift in sphingolipid balance that favors the production and accumulation of oncogenic species such as sphingosine 1-phosphate (S1P). S1P is known to govern the processes that facilitate cancer cell growth and progression including proliferation, survival, migration, invasion and inflammation. In this review paper, we will give a comprehensive overview of current literature findings on the molecular mechanisms by which S1P turnover, transport and signaling via receptor-dependent and independent pathways shape colon cancer cell behavior and influence treatment outcome in colon cancer. Combining available modulators of S1P metabolism and signaling with standard chemotherapy drugs could provide a rational approach to achieve enhanced therapeutic response, diminish chemoresistance development and improve the survival outcome in CRC patients

Određivanje aminokiselinske sekvencije farmaceutskih peptida MALDI-TOF tandemnom spektrometrijom masa

Although the peptide ion mechanisms of pre-dissociation, dissociation and post-dissociation have been well-studied over the past fifteen years, their practical application still has to be implemented into modern mass spectrometry-driven proteomics and bioanalysis. Unambiguous peptide sequence determination by mass spectrometry relies on the idea that only one continuous series of ions in mass spectrum can assure full sequence determination and meets the requirements of peptide analysis quality. This set of rules defined for the peptide analysis by tandem mass spectrometry would generally improve an overall reliability and data accuracy. Based on the process mechanisms of gas-phase peptide bond dissociation, a relatively small and large model peptides are unambiguously analyzed (bivalirudin and exenatide) showing that derivatization concepts of the C- or N-terminus derivatization (SPITC and Lys-tag) can be avoided.Iako su mehanizmi preddisocijacije, disocijacije i postdisocijacije iona izučavani dugi niz godina, postoji još cijeli niz mehanizama raspada iona koji se mogu korisno upotrijebiti u modernoj spektrometriji masa, proteomici i bioanalizama. Nedvojbena aminokiselinska analiza spektrometrijom masa počiva na ideji da samo jedan i kontinuirani slijed iona u tandemnom spektru masa može potpuno potvrditi cjelovitu aminokiselinsku sekvenciju i na taj način zadovoljiti zahtjeve nedvojbene analize peptida. Niz pravila kojima se definira cjelovita i nedvojbena analiza peptida tandemnom spektrometrijom masa u konačnici može unaprijediti iščitavanje rezultata analize i pouzdanost dobivenih podataka. Na teorijskim zasadama disocijacije iona u plinskoj fazi u ovom je radu prikazana cjelovita analiza peptida. Na modelnim farmaceutskim peptidima bivalirudinu i eksenatidu pokazano je kako se i bez raširene upotrebe derivatizacijskih tehnika (SPITC i Lys-tag) može ostvariti nedvojbena analiza, iako se modifikacije cisteina akrilacijom, metilacijom ili karboksimetilacijom pri tome ne mogu izbjeći

Small Molecules Targeting Biological Clock; A Novel Prospective for Anti-Cancer Drugs

The circadian rhythms are an intrinsic timekeeping system that regulates numerous physiological, biochemical, and behavioral processes at intervals of approximately 24 h. By regulating such processes, the circadian rhythm allows organisms to anticipate and adapt to continuously changing environmental conditions. A growing body of evidence shows that disruptions to the circadian rhythm can lead to various disorders, including cancer. Recently, crucial knowledge has arisen regarding the essential features that underlie the overt circadian rhythm and its influence on physiological outputs. This knowledge suggests that specific small molecules can be utilized to control the circadian rhythm. It has been discovered that these small molecules can regulate circadian-clock-related disorders such as metabolic, cardiovascular, inflammatory, as well as cancer. This review examines the potential use of small molecules for developing new drugs, with emphasis placed on recent progress that has been made regarding the identification of small-molecule clock modulators and their potential use in treating cancer