Sinteza in biokemijsko vrednotenje novih zaviralcev mikobakterijskega encima InhA s tetrahidropiranskim skeletom

Brčina, Špela (author), Pajk, Stane (mentor), Hrast Rambaher, Martina (co-mentor)

Tuberkuloza je zelo razširjena bolezen, ocenjuje se, da je z njo okuženih že četrtina prebivalstva, letno pa zaradi okužbe umre več kot milijon ljudi. Najbolj uporabni zdravili pri zdravljenju tuberkuloze sta rifampicin in izoniazid. Poleg njiju sta v prvi liniji obrambe še pirazinamid in etambutol. Zdravljenje aktivne oblike je precej dolgotrajno in v najboljšem primeru traja 6 mesecev, kadar pride do rezistence pa se še podaljša. Izoniazid je predzdravilo, ki za svoje delovanje potrebuje predhodno celično aktivacijo in sicer oksidacijo s KatG, kar mu omogoča zaviralno delovanje na encim InhA. InhA je ključen encim v biosintezi mikolnih kislin, zaradi katerih lahko mikobakterijska celica preživi v ekstremnih razmerah, saj tvorijo voskasto plast, ki obdaja celico. Izoniazid z zaviranjem InhA prepreči njihovo sintezo, kar vodi v lizo celice. A vendar je vedno večja pojavnost točkovnih mutacij v KatG, zaradi katerih se onemogoči aktivacija izoniazida, kar vodi v rezistentne oblike tuberkuloze. Zato bi bili novi direktni zaviralci encima InhA zelo dobrodošli. V okviru magistrske naloge smo se lotili sinteze novih potencialnih direktnih zaviralcev InhA s tetrahidropiranskim skeletom, kjer smo na fenilni obroč in metilenski distančnik vnesli manjše spremembe. S tem smo želeli raziskati aktivno mesto encima InhA. Sintetizirali smo 5 končnih spojin, katerim smo na Univerzi v Ljubljani, Fakulteti za farmacijo, določili sposobnost zaviranja encima InhA (IC50). Najboljša spojina (16) je zavirala encim InhA v nanomolarnem območju (IC50 = 329 nM) in se je po aktivnost približala že znanim zaviralcem. Poleg tega so kolegi na Veterinarski fakulteti Univerze v Ljubljani določili tudi protibakterijsko delovanje (MIK) na Staphylococcus aureus in Escherichia coli, ker imata podobno encimsko pot za sintezo maščobnih kislin kot Mycobacterium Tuberculosis, bakterijo odgovorno za okužbo s tuberkulozo. Žal nobena spojina ni zavirala ne ene ne druge bakterije pri koncentraciji 128 µg/mL ali nižje, kar pa ne izključuje možnosti, da ne zavira bakterije M. tuberculosis.

• Language: Slovenian
• Keywords: tuberkuloza, direktni zaviralci InhA, tetrahidropiran, Mycobacterium tuberculosis
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2024
• PID: 20.500.12556/RUL-154237
• Publication date in RUL: 03.02.2024

Secondary language

• Language: English
• Title: Synthesis and biochemical evaluation of new inhibitors of the mycobacterial enzyme InhA with a tetrahydropyran scaffold

Abstract

Tuberculosis is a widespread disease, it is estimated that a quarter of the population is infected with it, and more than a million people die from the infection every year. The most useful drugs in the treatment of tuberculosis are rifampicin and isoniazid. In addition to them, pyrazinamide and ethambutol are also in the first line of defence. The treatment of the active form is rather long-term and in the best-case scenario it lasts 6 months, and when resistance occurs it is even longer. Isoniazid is a prodrug that requires prior cellular activation for its action, i.e. oxidation by KatG, which enables it to inhibit the InhA enzyme. InhA is a key enzyme in the biosynthesis of mycolic acids, which enable the mycobacterial cell to survive in extreme conditions by forming a waxy layer that surrounds the cell. By inhibiting InhA, isoniazid prevents their synthesis, which leads to cell lysis. However, there is an increasing incidence of point mutations in KatG, which disable the activation of isoniazid, which leads to resistant forms of tuberculosis and thus to the search for new, direct inhibitors of the InhA enzyme. As part of this master's thesis, we started the synthesis of new potential direct inhibitors of InhA with a tetrahydropyran skeleton, where we made minor changes to the phenyl ring and the methylene linker. With this, we wanted to investigate the active site of the enzyme InhA. We synthesized 5 final compounds, whose ability to inhibit InhA (IC50) was determined at the University of Ljubljana, Faculty of Pharmacy. The compound 16 inhibited InhA enzyme in nanomolar range (IC50 = 329 nM) and that value came closer to the IC50s of already known inhibitors. In addition, colleagues at the Veterinary Faculty of the University of Ljubljana also determined antibacterial activity (MIC) on S. aureus and E. coli, because they have similar fatty acid synthesis to Mycobacterium Tuberculosis, the bacteria responsible for the infection. Unfortunately, neither compound inhibited either bacteria at a concentration of 128 µg/mL, or lower, which does not rule out the possibility that it does not inhibit M.tuberculosis.

• Keywords: tuberculosis, direct InhA inhibitors, tetrahydropyran, Mycobacterium Tuberculosis