Izbornik
 
Catalytic selectivity toward fluoro-compounds
 

 

 

Trajanje / Duration: 5.8.2019. – 28.9.2019.

 

Financiranje / Funding: Fond „Jedinstvo uz pomoć znanja“ / Unity Through Knowledge Fund

TOTAL funding 31200 kn (4220 €)

 

VODITELJ PROJEKTA / PROJECT LEADER: doc. dr. sc. Aleksandra Maršavelski

SUVODITELJ PROJEKTA / PROJECT CO-LEADER: Professor Lynn Kamerlin, PhD

SURADNIK / PROJECT COLLABORATOR: Prof. dr. sc. Ita Gruić Sovulj

 

INSTITUCIJE KOJE SUDJELUJU U PROJEKTU / INSTITUTIONS

Prirodoslovno-matematički fakultet, Sveučilište u Zagrebu / Faculty of Science, University of Zagreb i/and Department of Chemistry – Biomedicinska Centrum (BMC), Uppsala University, Uppsala

 

NAZIV PROJEKTA / PROJECT TITLE

Katalitička selektivnost prema fluoriranim spojevima / Catalytic selectivity toward fluorinated compounds 

 

SAŽETAK / ABSTRACT

 

Fluorirane molekule su važni bioaktivni spojevi koji imaju široku primjenu. Najvažniji fluorirani lijekovi uključuju antidepresiv fluoksetin (Prozac), antipsihotik haloperidol (Haldol) i lijek za snižavanje kolesterola atorvastatin (Lipitor). Specifično fluoriranje pokazalo se kao važna strategija za dobivanje metabolički stabilnih lijekova, međutim ostale implikacije koje fluor donosi tek trebaju biti razjašnjene. Stoga postoji snažan interes za razumijevanje utjecaja fluoro supstituenata na selektivnost i reaktivnost ovih spojeva. Fluoroacetil-CoA tioesteraza iz bakterije Streptomyces cattleya koja proizvodi organofluorirane spojeve izvrstan je primjer prirodno evoluiranog enzima koji pokazuje visoki stupanj diskriminacije između fluoroacetil-CoA i acetil-CoA. Iako su eksperimentalna istraživanja pokazala da se selektivnost prema fluoroacetil-CoA u odnosu na acetil-CoA u iznosu od šest redova veličine može pripisati specifičnom katalitičkom mehanizmu i entropijski vođenim prepoznavanjem koje favorizira fluorirani umjesto nefluorirani spoj, mnogi aspekti ove selektivnosti ostali su nerazjašnjeni. Cilj ovog projekta je, dakle, pojasniti mehanističke razlike u hidrolizi fluoroacetil-CoA i acetil-CoA fluoroacetil-CoA tioesterazom primjenom metode EVB (engl. empirical valence bond simulations). Očekivani ishodi uključuju detaljan uvid u reakcijske mehanizme hidrolize oba spoja, mapiranjem svih međuprodukata i prijelaznih stanja duž reakcijskog puta, što je nužno za razumijevanje katalitičkih učinaka spojeva koji sadrže fluor. Projekt će omogućiti uspostavu dugoročne suradnje s partenerskim laboratorijem koji ima impresivno iskustvo u proučavanju reaktivnosti enzima. Partnerski laboratorij će, osim ekspertize, omogućiti pristup računalnim resursima neophodnim za opsežne simulacije, a koji nisu dostupni u Hrvatskoj. Sve navedeno doprinijet će kvaliteti i međunarodnoj vidljivosti dobivenih istraživačkih rezultata.

 

Fluorinated molecules are important bioactive compounds used for a broad range of applications. The most important fluorinated pharmaceuticals include the antidepressant fluoxetine (Prozac), antipsychotic haloperidol (Haldol) and the cholesterol-lowering drug atorvastatin (Lipitor). Site-specific fluorination has proved to be an important strategy for drug metabolic stability. However, other implications that fluorine brings are yet to be understood. Therefore, there is a strong interest in gaining detailed insights into how fluorine substituent affects the selectivity and reactivity of these compounds. Fluoroacetyl-CoA thioesterase from the organofluorine-producing bacteria Streptomyces cattleya is an excellent example of a naturally evolved enzyme that demonstrates high discrimination between fluoroacetyl-CoA and acetyl-CoA. Although experimental studies have demonstrated that a 106-fold selectivity against fluoroacetyl-CoA can be attributed to a different catalytic mechanism and entropically driven recognition that favors fluorinated over nonfluorinated compound, many aspects of this selectivity are left unexplained. The aim of this project is, therefore, to elucidate mechanistic differences in the hydrolysis of fluoroacetyl-CoA and acetyl-CoA by fluoroacetyl-CoA thioesterase by applying empirical valence bond simulations. The expected outcome includes detailed insight into the reaction mechanisms of hydrolysis of both compounds, by mapping all intermediates and transitions states along the reaction pathways, which is essential to understanding the catalytic effects of fluorine-containing compounds. The project will allow the establishment of a long-term collaboration with the host laboratory, which has impressive experience in studying enzyme’s reactivity. The host laboratory will, besides expertise, provide access to extensive computing power for simulations, not available in Croatia. Altogether, this will contribute to the quality and international visibility of obtained research results.

 

PROJEKTNI TIM / PROJECT TEAM

Prirodoslovno-matematički fakultet, Sveučilište u Zagrebu: Aleksandra Maršavelski, Ita Gruić Sovulj

Department of Chemistry (BMC), Uppsala University: Lynn Kamerlin

 

MILESTONES REACHED

M1. The referent reaction for the hydrolysis of fluoroacetyl-CoA and

acetyl-CoA needed for calibration of the EVB potential is defined. All calculations in this step are performed by using wB97XD/6-31++G(d,p). 

M2. Topology and parameter files for the referent reaction and reaction in the enzyme is prepared.

M3. Prepared systems are relaxed and the resulting structure are subjected to ten parallel MD runs by randomizing the atomic velocities and running short relaxations.

M4. EVB calculations in the gas phase are done for the first step of the proposed reaction mechanism. The two tunable EVB parameters are obtained. These two parameters are then used for the same reaction in water and protein.

M5. Analysis of EVB runs is done. The extraction of the free energy profile plotted against the energy gap as the reaction coordinate is done. Transition state (TS) structures are located.

 

The goal of the project is realized through the following objectives: a) Reference reaction for hydrolysis of both fluoroacetyl-CoA and acetyl-CoA compounds is defined. b) The two tunable EVB parameters (coupling term and relative shift) involved in the expression of EVB energy function are obtained by fitting to the energetics of the reference reaction. c) Topologies for both systems are prepared.  d) Relaxation, heating up and MD simulations are done. e) FEP file is prepared. f) EVB simulations are done. g) EVB runs are analyzed (extraction of the free energy profiles) h) Group contribution analysis will be done in Zagreb.

 

These realized objectives refer to the reaction catalyzed by Glu of fluoroacetyl-CoA thioesterase. However, since this enzyme shows catalytic promiscuity, reaction through different mechanism initiated by another nucleophile, is continued to be studied.

 

 


Tražilica
 

 

 

Sva prava pridržana. Kemijski odsjek Prirodoslovno-matematičkog fakulteta
Sveučilište u Zagrebu