Unit:
Department of PharmacyLibrary of the School of Science
Author:
Fantel Anna-Maria
Dissertation committee:
ΕΜΜΑΝΟΥΗΛ ΜΙΚΡΟΣ, ΚΑΘΗΓΗΤΗΣ, ΤΜΗΜΑ ΦΑΡΜΑΚΕΥΤΙΚΗΣ, ΕΚΠΑ
ΑΝΔΡΕΑΣ ΠΑΠΑΠΕΤΡΟΠΟΥΛΟΣ, ΚΑΘΗΓΗΤΗΣ, ΤΜΗΜΑ ΦΑΡΜΑΚΕΥΤΙΚΗΣ, ΕΚΠΑ
ΠΑΝΑΓΙΩΤΗΣ ΜΑΡΑΚΟΣ, ΚΑΘΗΓΗΤΗΣ, ΤΜΗΜΑ ΦΑΡΜΑΚΕΥΤΙΚΗΣ, ΕΚΠΑ
ΠΟΥΛΗ ΝΙΚΟΛΑΪΣ, ΚΑΘΗΓΗΤΡΙΑ– ΔΙΕΥΘΥΝΤΡΙΑ, ΤΜΗΜΑ ΦΑΡΜΑΚΕΥΤΙΚΗΣ, ΕΚΠΑ
ΚΩΝΣΤΑΝΤΙΝΟΣ Ε. ΒΟΡΓΙΑΣ, ΚΑΘΗΓΗΤΗΣ, ΤΜΗΜΑ ΒΙΟΛΟΓΙΑΣ, ΕΚΠΑ
ΚΟΛΟΚΟΥΡΗΣ ΑΝΤΩΝΗΣ , ΚΑΘΗΓΗΤΗΣ, ΤΜΗΜΑ ΦΑΡΜΑΚΕΥΤΙΚΗΣ ΕΚΠΑ
ΒΑΣΙΛΕΙΟΣ ΜΥΡΙΑΝΘΟΠΟΥΛΟΣ , ΕΠΙΚΟΥΡΟΣ ΚΑΘΗΓΗΤΗΣ, ΤΜΗΜΑ ΦΑΡΜΑΚΕΥΤΙΚΗΣ, ΕΚΠΑ
Original Title:
Ορθολογικός σχεδιασμός και ανάπτυξη αναστολέων των ενζύμων παραγωγής υδροθείου.
Translated title:
Rational design and development of H2S producing enzymes inhibitors.
Summary:
Hydrogen sulfide H2S is a gasotransmitter considered as an important biological and pharmacological mediator. In mammalian cells, endogenous H2S is synthesized by four enzymes Cystathionine β-synthase CBS, Cystathionine γ-lyase CSE, cysteine aminotransferase CAT, and 3-mercaptopyruvate sulfurtranferase 3-MST. CBS is responsible for 20-75% of total hydrogen sulfide production and up to 95% of production in the brain. CBS enzyme is located in the central nervous system as well as in other tissues (cardiovascular, gastrointestinal) and it is a pyridoxal phosphate(PLP)-depended enzyme that contains heme as a cofactor. The full-length human CBS is a functional homotetramer of 63kDa or 551 residues. The enzyme exhibits a modular organization and complex regulation. The N-terminal domain of CBS encompasses the heme binding-region followed by a central catalytic core. The C-terminal region is the allosteric domain of CBS that binds S-adenosylmethionine (AdoMet) and consists of 140 residues (CBS motifs). Human colorectal cancer cells contain high levels of CBS and recent studies show that CBS is also implicated in the pathogenesis of ovarian and breast cancer. Currently, there are two widely recognized non-selective inhibitors of CBS, aminooxy acetic acid (AOAA) and hydroxylamine (HA). Thus, selective inhibitors of hCBS are in demand for both further investigations of H2S signaling and the development of novel treatments of H2S-related disease states.
This study presents an integrated approach for developing potent and selective CBS inhibitors by using a combination of in silico and in vitro techniques. To facilitate in vitro screening experiments, full-length human CBS was expressed and purified as a recombinant fusion protein containing GSH-S-transferase (GST) tag, poly-histidine (HIS) tag and no tag. CBS tetramer has a strong tendency to aggregate and susceptibility to proteolysis, thus the purification system of the enzyme has been persistently studied. Different strategies were organized based on a variety of standard chromatographic methods (affinity chromatography, ion chromatography, gel filtration, hydrophobic interaction chromatography) to succeed enzyme purity <90%. Also, CBS- regulatory domain known as CBS-Motif has been expressed and purified. In order to discover compounds that bind selectively to CBS a stepwise screening protocol was implemented.
An in vitro high throughput screening was performed using methylene blue assay, a colorimetric assay used for hydrogen sulfide detection. Enzymatic activity of the fusion protein was measured by the ability to produce hydrogen sulfide in presence of PLP, L-cysteine, Homocysteine. More than 800 synthetic compounds were tested against CBS as potential inhibitors, all molecules were selected from the NKUA compound library (in house collection ~2K compounds). IC50 values of the most potent modulators were calculated by dose-response curves. The pyrazolopyridine derivative 1116 with a promising CBS inhibitory potential was discovered and the compound activity was readily comparable to the most potent CBS inhibitor currently known, aminoacetic acid AOAA. Notably, the IC50 of known inhibitor AOAA is 8.5uM comparable to 9.6uM of the pyrazolopyridine compound 1116. The synthetic scaffold of pyrazolopyridine was selected for further analysis due to its originality with respect to CBS putative activity. Compound 1116 is an organic synthesis product forming a unique structure. There is a central pyrazolo[3,4-c]pyridine core substituted with three important functional groups, one methyl piperazine group placed in the opposite direction of a methoxy group and a second methyl piperazine group connected to the core. In order to exclude any possibility that the pyrazolopyridine derivative may bind the enzyme regulatory domain due to its high structural similarity with cofactor S-adenosylmethionine, differential scanning fluorimetry experiments were performed. Dose response curves were generated and the corresponding IC50s established for 8 existing and 8 newly synthesized pyrazolopyridine analogs, providing preliminary structure-activity relationships. In silico experiments were performed using crystal structure of full length hCBS and crystal structure with absence of CBS regulatory domain (PDB: 4PCU, 1JBQ). All pyrazolopyridine derivatives were docked in the active site of CBS using the IFD induced-fit docking protocol and ligands were prepared in three different states of protonation. Followed by determination of MM-GBSA binding free energy of the lead compound 1116 in the active site of CBS. Subsequently, a hypothesis regarding the exact binding mode of the inhibitor 1116 was devised on the basis of the available structure-activity relationships (SAR), the aniline methylpiperazine side substitute seems to be the most important feature of the compound.
Main subject category:
Science
Other subject categories:
Health Sciences
Keywords:
Cystathionine beta synthase CBS, PLP- Dependent Enzymes, Hydrogen Sulfide, High Throughput Screening, Methylene Blue Assay MBA, Differential Scanning Fluorimetry, CBS Inhibitors, Computational Chemistry, Induced Fit Docking Calculation
Number of references:
147
