Identification of Species-Specific Allosteric Binding Sites in Phosphofructokinase for Drug Design Studies
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Date
2020
Authors
Ayyıldız, Merve
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Publisher
Kadir Has Üniversitesi
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Abstract
Glikolitik yolaktaki en kritik enzimlerden biri olan fosfofruktokinaz (PFK), türe özgü ilaç tasarım çalışmalarında kullanılmak üzere hedeflenmiş ve bakteri, parazit ve insan olmak üzere üç türdeki allosterik bağlanma bölgeleri araştırılmıştır. Evrimsel süreçte allosterik bölgeler, proteinlerin aktif bölgesinden daha az korunmuş oldukları için, türe özgü ilaç ilaç tasarım çalışmaları için güçlü hedef bölgeler haline geldiler. Bu doğrultuda, çözücü haritalama, elastik ağ modeli, dizi ve yapı hizalama gibi iyi bilinen yöntemleri birleştiren yeni bir yaklaşım geliştirdik. İlk olarak, tüm bağlanma bölgeleri hesaplamalı çözücü haritalama yöntemiyle araştırıldı. Bu haritalama sonucunda tespit edilen bölgelerin çoğu, reseptörün monomerik alt birimleri arasındaki arayüz bölgelerinde bulundu. Daha sonra, ligand bağlanmasına bağlı olarak proteinin global dinamiklerinde daha fazla değişikliğe sebep olabilecek bölgelerin belirlenmesi için elastik ağ modelini baz alan yüzde frekans kayması hesaplamaları ile enzimler tarandı. Bu bölgelerin bazılarının, metodolojimizin doğruluğunu destekleyerek iy rapor edilmiş aktif ve allosterik bölgelerin yakınında olduğu bulunmuştur. Ayrıca, sekans ve yapısal hizalama yöntemleri ile insan ve bakteri/parazit arasındaki sekans ve yapısal farklılıklar araştırıldı. Yüksek yapısal benzerliğe rağmen, bakteri/parazit ve insan arasındaki özgüllüğü artıran düşük sekans benzerliği gözlenmiştir. Sonuç olarak, hem bakteri hem de parazitte yeni allosterik bölgeler tanımlanmıştır. Son olarak, S. aureus'a karşı kullanılabilecek potansiyel türe özgü ilaç molekülleri için yerleştirme yoluyla sanal tarama gerçekleştirildi. Sırasıyla 1416 ve 2922 ilaç molekülleri içeren ZINC veri tabanından çıkarılan FDA onaylı ve World-not-FDA onaylı alt kümeler, S. aureus PFK'daki potansiyel allosterik bölgelere yerleştirildi. Her iki alt gruptan altı potansiyel ilaç molekülü tanımlandı ve bunlar daha sonra in-vitro çalışmalarda daha fazla değerlendirme için kullanılacaktır.
Phosphofructokinase (PFK), one of the most critical enzymes in glycolytic pathway was targeted for use in species-specific drug design studies and its allosteric binding sites in three species, bacteria, parasite and human, were investigated. As allosteric regions are evolutionarily less conserved than active sites, they became powerful target sites for species-specific drug design studies. In accordance, we developed a novel approach which combines well-known computational methods including solvent-mapping, elastic network modeling and sequence/structural alignments. First, all binding regions were explored by computational solvent mapping. The majority of the regions detected in mapping were located at the interface regions in between monomeric subunits of the receptor. Then, percent frequency shift calculations based on elastic network model were conducted to scan the enzymes in order to determine the areas which are more likely to perturb the global dynamics upon ligand binding. Some of these regions were found to be located near well-reported active and allosteric regions supporting the accuracy of our methodology. Furthermore, sequence and structural differences between human and bacteria/parasite were investigated by sequence and structural alignment methods. Despite high structural similarity, low sequence similarity which increase the specificity between bacteria/parasite and human was observed. As a result, novel allosteric regions in all both bacteria and parasite have been identified. Finally, virtual screening was performed via docking for potential species-specific drug molecules that can be used against S. aureus. FDA approved and World-not-FDA approved subsets extracted from ZINC database which contains 1416 and 2922 drug molecules respectively were docked to potential allosteric sites in S. aureus PFK. Six potential drug molecules were identified from both subsets which will be later used in in- vitro studies for further assessment.
Phosphofructokinase (PFK), one of the most critical enzymes in glycolytic pathway was targeted for use in species-specific drug design studies and its allosteric binding sites in three species, bacteria, parasite and human, were investigated. As allosteric regions are evolutionarily less conserved than active sites, they became powerful target sites for species-specific drug design studies. In accordance, we developed a novel approach which combines well-known computational methods including solvent-mapping, elastic network modeling and sequence/structural alignments. First, all binding regions were explored by computational solvent mapping. The majority of the regions detected in mapping were located at the interface regions in between monomeric subunits of the receptor. Then, percent frequency shift calculations based on elastic network model were conducted to scan the enzymes in order to determine the areas which are more likely to perturb the global dynamics upon ligand binding. Some of these regions were found to be located near well-reported active and allosteric regions supporting the accuracy of our methodology. Furthermore, sequence and structural differences between human and bacteria/parasite were investigated by sequence and structural alignment methods. Despite high structural similarity, low sequence similarity which increase the specificity between bacteria/parasite and human was observed. As a result, novel allosteric regions in all both bacteria and parasite have been identified. Finally, virtual screening was performed via docking for potential species-specific drug molecules that can be used against S. aureus. FDA approved and World-not-FDA approved subsets extracted from ZINC database which contains 1416 and 2922 drug molecules respectively were docked to potential allosteric sites in S. aureus PFK. Six potential drug molecules were identified from both subsets which will be later used in in- vitro studies for further assessment.