Sinteza in vrednotenje protirakavega delovanja 4,5,6,7-tetrahidrobenzotiazolnih zaviralcev C-končne domene proteina toplotnega šoka 90

Oberč, Rok (author), Tomašič, Tihomir (mentor), Dernovšek, Jaka (comentor)

Proteini toplotnega šoka so šaperoni, ki sodelujejo pri zvijanju novo nastalih proteinov v nativno konformacijo. Poleg tega ščitijo celične proteine pred poškodbo in agregacijo, ko je organizem pod stresom. Do zaščite pride tudi pri proteinih v rakavih celicah, v katerih ima eno ključnih vlog Hsp90, saj stabilizira mutirane proteine, ki se nakopičijo v celici in so vpleteni v patofiziologijo raka. Zato je Hsp90 postal zanimiva tarča za razvoj novih protirakavih učinkovin. Sprva so raziskovalci razvili spojine z ATP-kompetitivnim zaviralnim delovanjem na N-končno domeno (NKD) Hsp90. A zaradi odziva toplotnega šoka, ki ga sprožajo nekateri zaviralci NKD Hsp90, tovrstne spojine povečini niso primerne za klinično rabo. Razvoj se je zato preusmeril k zaviralcem alosteričnega vezavnega mesta na C-končni domeni (CKD) Hsp90, ki odziva toplotnega šoka ne inducirajo. Pri eksperimentalnem delu smo sintetizirali štirinajst zaviralcev CKD Hsp90. Za osnovni skelet smo uporabili 4,5,6,7-tetrahidrobenzotiazolni obroč, na katerega smo s tvorbo amidne vezi na mestih 2 in 6 pripeli različne substituente. Knjižnica A vsebuje spojine, pri katerih smo na mestu 6 pripeli 3,4-diklorobenzenski obroč, medtem ko smo na mesto 2 uvajali različne substituente z bazičnim centrom. Pri spojinah iz knjižnice B smo spremembe uvajali v aromatskem delu na mestu 6 osnovnega skeleta, na mestu 2 pa smo uvedli β-alanin. Namen naloge je bil ugotoviti odnos med strukturo in delovanjem (SAR) zaviralcev C-končne domene Hsp90, da bi lahko v prihodnje sintetizirali protitumorne učinkovine s še močnejšim zaviralnim delovanjem. Biološko testiranje smo izvajali na človeški celični liniji raka dojke MCF-7 in rezultate podali v obliki vrednosti IC50 antiproliferativnega delovanja. Najmočnejše zaviralno delovanje iz knjižnice A je izkazal trans izomer 5f (1-(1S,3S)–3-aminociklobutil)etan-1-on) z vrednostjo IC50 = 0,37 ± 0,03 µM, kar kaže na to, kako pomembna je usmerjenost aminske skupine v aktivnem mestu. Prav zaradi drugačne usmerjenosti amina njegov cis izomer 5e (1-(1R,3S)–3-aminociklobutil)etan-1-on) verjetno težje tvori ionske vezi z glutamatom (Glu489A) v CKD, kar posledično zmanjša jakost delovanja spojine. Iz knjižnice B je najmočnejše zaviralno delovanje imela spojina 8d z IC50 = 1,07 ± 0,15 µM in ima tako 2-krat močnejše delovanje od referenčne spojine TMM-7b. Spojina 8d ima na mestu 6 naftalenski obroč, ki se sodeč po vrednosti IC50 bolje prilega hidrofobnemu žepu CKD Hsp90 kot 3,4-diklorbenzenski obroč spojine TMM-7b, medtem ko je uvedba ostalih aromatskih obročev neugodno vplivala na antiproliferativno delovanje spojin. Z novimi ugotovitvami pri raziskovalnem delu smo odkrili nekaj informacij o SAR 4,5,6,7-tetrahidrobenzotiazolnih zaviralcev Hsp90, kar bi lahko v prihodnje vodilo do spojin z močnejšim delovanjem.

• Language: Slovenian
• Keywords: Hsp90, rak dojke, zaviralci C-končne domene, 4, 5, 6, 7-tetrahidrobenzotiazol, SAR
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-151777
• Publication date in RUL: 20.10.2023

Secondary language

• Language: English
• Title: Synthesis and evaluation of the anticancer activity of 4,5,6,7-tetrahydrobenzothiazole-based inhibitors of the C-terminal domain of heat shock protein 90

Abstract

Heat shock proteins (Hsp) are molecular chaperones involved in the folding of newly synthesized proteins into their native conformation. Hsp protect cellular proteins from damage and aggregation when the organism is under stress. Protection also occurs in cancer cells, where Hsp90 plays a key role by stabilizing mutant proteins that accumulate in the cell and lead to cancer. As a result, Hsp90 has become a target for new chemotherapeutic agents. Initially, researchers synthesized compounds with inhibitory activity against the Hsp90 N-terminal domain (NTD), which serves as an ATP-binding site, but all triggered a heat shock response (HSR), which reduced their anticancer activity. Further research led to the discovery of compounds that inhibit the allosteric site at the Hsp90 C-terminal domain (CTD) without inducing HSR. Our experimental work was based on the synthesis of fourteen inhibitors of the Hsp90 CTD. We used a 4,5,6,7-tetrahydrobenzothiazole ring as a scaffold to which substituents were attached by forming an amide bond at positions 2 and 6. Library A contains compounds with a 3,4-dichlorobenzene ring at position 6, while various substituents were introduced at position 2. In library B, modifications were made at position 6 of the scaffold, while a β-alanine substituent was introduced at position 2. The aim of the study was to establish the structure-activity relationship (SAR) of Hsp90 CTD inhibitors in order to synthesize more potent anti-tumor compounds in the future. Biological assays were performed on the human breast cancer cell line MCF-7, and the results were presented as IC50 values for antiproliferative activity. The most potent inhibition from library A was exhibited by trans-isomer 5f (1-(1S,3S)–3-aminocyclobutiyl)ethan-1-one) with IC50 = 0.37 ± 0.03 µM, highlighting the importance of the orientation of the amino group in the binding site. Due to the different orientation of its amino group, cis-isomer 5e (1-(1R,3S)–3-aminocyclobutyl)ethan-1-one) forms weaker ionic bonds with glutamate (Glu489A) in the CTD, reducing the activity of the compound. Compound 8d from library B showed the strongest inhibitory activity with IC50 = 1.07 ± 0.15 µM and was twice as active as the reference compound TMM-7b. Compound 8d has a naphthalene ring at position 6 that fits better into the hydrophobic pocket of Hsp90 CTD than the 3,4-dichlorobenzene ring of compound TMM-7b. The introduction of other aromatic rings significantly decreased the activity of the compounds. We have discovered new information about the SAR of 4,5,6,7-tetrahydrobenzothiazole inhibitors of Hsp90, which could be used to design even better inhibitors of Hsp90 CTD.

• Keywords: Hsp90, breast cancer, C-terminal domain inhibitors, 4, 5, 6, 7-tetrahydrobenzothiazole, SAR.