Publication: Fosfoinositid 3-kinaz (PI3K) enziminin kovalent olmayan inhibisyon mekanizması üzerine kuvantum kimya hesaplamaları
Abstract
FOSFOİNOSİTİD 3-KİNAZ (PI3K) ENZİMİNİN KOVALENT OLMAYAN İNHİBİSYON MEKANİZMASI ÜZERİNE KUVANTUM KİMYA HESAPLAMALARI Kinaz enzimlerinin özellikle kanser tedavisi için önemli farmakolojik hedefler olduğubilinmektedir. Son zamanlarda lizin kalıntıları ile gerçekleştirilen hedeflenmiş-kovalentinhibisyon çalışmaları ise etkili ilaç aktif madde tasarımları açısından birçok avantajasahiptir. Yeni bir deneysel çalışmada, fosfoinositid 3-kinaz delta (PI3Kδ) enzimini aktifbölgesindeki lizini hedefleyerek seçimli ve kovalent olarak inaktive eden bir dizi yenifenil ester inhibitörü geliştirilmiştir. Ancak, fenil esterlerin tersine, metil ester türevinin kovalent olmayan inhibisyona yol açması dikkat çekmiştir. Etkin ve seçici yeni inhibitörlerin tasarlanabilmesi için inhibitör davranışlarının kimyasal mekanizma ile aydınlatılması gerekmektedir. Bu tezin amacı, kuantum kimyasal mekanizma modellemeleri yaparak, söz konusu metil ester türevinin, PI3Kδ enzimini neden kovalent bağlanarak inhibe etmediğini açıklığa kavuşturmak ve kovalent inhibisyonun sağlanabilmesi için yeni inhibitör adayı önermektir. Bu amaçla, enzim yapısından kesilmiş bir aktif bölge modeli üzerinde PI3Kδ enzimindeki lizin-779 ile metil ester inhibitörü arasındaki ester aminoliz tepkimesine ait çeşitli mekanizma yolları modellenmiştir. Yapı optimizasyonları 2 katmanlı ONIOM yaklaşımı kullanılarak M06-2X/ 6-31+G(d,p):PM6 yöntemleri ile gerçekleştirilmiş ve 3 farklı mekanizmaya ait Gibbs serbest enerji profilleri elde edilmiştir. Lizinin estere kovalent bağlanması sonucu zwiteriyon ara ürünü oluşması tüm mekanizma yollarının ortak ilk adımıdır. Hesaplanan aktivasyon enerjileri, bu araürünün oluşabileceğini ancak oldukça kararsız olduğunu ve kovalent bağın tersinir olarak geriye ayrılabileceğini ortaya koymaktadır. Ancak, tüm mekanizma yollarının son adımları çok yüksek aktivasyon enerji bariyerlerine sahiptir. Bu durum zwiteriyon'un ileri yönlü reaksiyonunun engellenmesine ve kovalent olmayan enzim-inhibitör kompleksine geri dönmesine neden olur. Bu bulgular, deneysel gözlem ile tutarlıdır ve metil ester inhibitörünün kovalent olmayan davranışının nedenini açıklamaktadır. Elde edilen sonuçlara dayanarak enzimi kovalent inhibe edeceği öngörüsü ile inhibitörün tiyoester türevi tasarlandı ve reaksiyon enerji profili elde edildi. Beklenildiği gibi, ayrılan grup tiyolat iyonunun daha kararlı olması ayrılma basamağının enerji engelini azaltarak, tiyoester türevinin kovalent inhibisyona neden olabileceğini göstermiştir. Tez kapsamında mekanizmanın detaylarının aydınlatıldığı bu modellemeler, amaca uygun etkili yeni PI3Kδ inhibitörlerinin tasarlanması için yol gösterici niteliktedir.
QUANTUM CHEMICAL CALCULATIONS ON THE NONCOVALENT INHIBITION MECHANISM OF PHOSPHOINOSITIDE 3-KINASE ENZYME (PI3K) Kinase enzymes are important pharmacological targets especially for cancer treatment. Recently, covalent inhibitors targeting lysine residues have attracted great interest as lysines have many advantages in the design of effective drug active substances. In a recent experimental study, a series of novel phenyl ester inhibitors have been developed that selectively and covalently inactivate the phosphoinositide 3-kinase delta (PI3Kδ) enzyme by targeting a lysine at its active site. However, it was noted that, unlike phenyl esters, the methyl ester derivative caused non-covalent inhibition. The elucidation of inhibitory behavior with the chemical mechanism is necessary for the design of effective and selective new inhibitors. The aim of this thesis is to clarify why the methyl ester derivative does not act as a covalent inhibitor of the PI3Kδ enzyme and to propose a new inhibitor candidate for covalent inhibition. For this purpose, various mechanistic paths of the ester aminolysis reaction between lysine-779 in the PI3Kδ enzyme and the methyl ester inhibitor were modeled on an active site model truncated from the enzyme structure. The geometry optimizations were performed using the 2-layer ONIOM approach with M06-2X/ 6- 31+G(d,p):PM6 method and Gibbs free energy profiles of all mechanisms were obtained. The formation of a zwitterion intermediate as a result of covalent binding of lysine to the ester is the common first step of all pathways. The calculated activation energy barrier reveals that this intermediate can form but is highly unstable and therefore dissociates back to the reactants. However, the final steps of all pathways have very high activation energy barriers. This prevents the forward reaction of the zwitterion and drives it back to the non-covalent enzyme-inhibitor complex. These findings are consistent with the experimental observation and explain the noncovalent behavior of the methyl ester inhibitor. Based on these results, we designed the sulfur analogue of the noncovalent inhibitor (thioester) with the assumption that it would covalently inhibit the enzyme. As expected, the thioester derivative, bearing a more stable leaving group, have displayed an energy profile of a typical covalent inhibitor with a vii reduced energy barrier of the dissociation step. The inhibition mechanisms elucidated within the scope of this thesis can serve as a guide for the design of new effective PI3Kδ inhibitors.
QUANTUM CHEMICAL CALCULATIONS ON THE NONCOVALENT INHIBITION MECHANISM OF PHOSPHOINOSITIDE 3-KINASE ENZYME (PI3K) Kinase enzymes are important pharmacological targets especially for cancer treatment. Recently, covalent inhibitors targeting lysine residues have attracted great interest as lysines have many advantages in the design of effective drug active substances. In a recent experimental study, a series of novel phenyl ester inhibitors have been developed that selectively and covalently inactivate the phosphoinositide 3-kinase delta (PI3Kδ) enzyme by targeting a lysine at its active site. However, it was noted that, unlike phenyl esters, the methyl ester derivative caused non-covalent inhibition. The elucidation of inhibitory behavior with the chemical mechanism is necessary for the design of effective and selective new inhibitors. The aim of this thesis is to clarify why the methyl ester derivative does not act as a covalent inhibitor of the PI3Kδ enzyme and to propose a new inhibitor candidate for covalent inhibition. For this purpose, various mechanistic paths of the ester aminolysis reaction between lysine-779 in the PI3Kδ enzyme and the methyl ester inhibitor were modeled on an active site model truncated from the enzyme structure. The geometry optimizations were performed using the 2-layer ONIOM approach with M06-2X/ 6- 31+G(d,p):PM6 method and Gibbs free energy profiles of all mechanisms were obtained. The formation of a zwitterion intermediate as a result of covalent binding of lysine to the ester is the common first step of all pathways. The calculated activation energy barrier reveals that this intermediate can form but is highly unstable and therefore dissociates back to the reactants. However, the final steps of all pathways have very high activation energy barriers. This prevents the forward reaction of the zwitterion and drives it back to the non-covalent enzyme-inhibitor complex. These findings are consistent with the experimental observation and explain the noncovalent behavior of the methyl ester inhibitor. Based on these results, we designed the sulfur analogue of the noncovalent inhibitor (thioester) with the assumption that it would covalently inhibit the enzyme. As expected, the thioester derivative, bearing a more stable leaving group, have displayed an energy profile of a typical covalent inhibitor with a vii reduced energy barrier of the dissociation step. The inhibition mechanisms elucidated within the scope of this thesis can serve as a guide for the design of new effective PI3Kδ inhibitors.