Kabozantinib ile İnsan Serum Albumini Arasındaki Bağlanma Etkileşimine İlişkin İlk Spektroskopik Bulgular

Abstract

Bu çalışmada, tirozin kinaz inhibitörü kabozantinib (CAB) ile insan serum albumini (HSA) arasındaki etkileşim ilk kez floresans spektroskopisi kullanılarak araştırılmış; bağlanma mekanizması, bağlanma kuvveti ve olası konformasyonel etkiler detaylı olarak incelenmiştir. CAB ve HSA arasındaki ilişki ağırlıklı olarak floresan spektroskopik analiz yoluyla araştırıldı. Bu etkileşimi değerlendirmek için, artan CAB konsantrasyonlarının sabit bir HSA çözeltisi konsantrasyonuna titre edilmesiyle bir dizi söndürme ölçümü sistematik olarak gerçekleştirildi. Söndürme mekanizmasını belirlemek ve termodinamik parametreleri hesaplamak üzere sıcaklık bağımlı ölçümler de yapılmıştır. Söndürme davranışı ve bağlanma sabitleri, Stern–Volmer analizi ve çift logaritmik eğri uydurma yöntemleri ile değerlendirilmiştir. Artan CAB konsantrasyonları karşısında HSA'nın doğal floresansının kademeli olarak azaldığı gözlemlenmiş ve bu durum CAB–HSA kompleksinin oluşumunu işaret etmiştir. Sıcaklık arttıkça Stern–Volmer söndürme sabitlerinde (KSV) belirgin bir düşüş kaydedilmiş, bu da etkileşimin statik söndürme mekanizmasına dayandığını göstermiştir. Elde edilen bağlanma sabitleri (Ka) 104 M-1 mertebesinde olup, CAB ile HSA arasında orta kuvvette bir etkileşim olduğunu ve CAB’ın plazmada taşınabilirliğini desteklemektedir. Termodinamik veriler bağlanmanın kendiliğinden gerçekleştiğini göstermiş; hidrofobik etkileşimlerin başlıca katkıyı sağladığı, ayrıca hidrojen bağları ve zayıf van der Waals etkileşimlerinin de rol oynadığı anlaşılmıştır. Ayrıca, emisyon maksimumlarında gözlenen hafif kırmızı kaymalar ve triptofan ile tirozin kalıntıları çevresinde belirgin floresans söndürülmesi, protein yapısında CAB bağlanmasına bağlı lokal konformasyonel değişikliklerin meydana geldiğini ortaya koymuştur.

Keywords

• Kabozantinib
• Kanser
• Floresans Spektroskopisi
• İnsan Serum Albumini
• UV-Görünür Spektroskopisi

The First Molecular-Scale View of Cabozantinib–Human Serum Albumin Binding: Quantitative Spectroscopic Insights

Abstract

This study aims to examine, for the first time, the interaction between the tyrosine kinase inhibitor cabozantinib (CAB) and human serum albumin (HSA), emphasizing the binding mechanism, affinity, and possible structural alterations by fluorescence spectroscopy. The association between CAB and HSA was predominantly explored through fluorescence spectroscopic analysis. To evaluate this interaction, a set of quenching measurements was systematically carried out by titrating increasing concentrations of CAB into a fixed concentration of HSA solution. Temperature-dependent measurements were also conducted to analyze the quenching mechanism and to calculate thermodynamic parameters. Stern-Volmer analysis, as well as double logarithmic fitting, were used to evaluate the quenching behavior and binding affinity. The progressive decrease of HSA’s native fluorescence upon incremental addition of CAB concentrations indicated the formation of the CAB-HSA complex. A noticeable reduction in Stern-Volmer quenching constants (KSV) was recorded as the temperature increased, suggesting a static quenching mechanism. Binding constants (Ka) were found to be in the order of 104 M-1, pointing to a relatively intermediate affinity between CAB and HSA, compatible with its transport in blood plasma. The spontaneity of the binding was supported by thermodynamic data, highlighting hydrophobic interactions as the principal contributor, in combination with hydrogen bonding and weak van der Waals attractions. In addition, no shifts in emission maxima and significant fluorescence quenching around tryptophan and tyrosine residues indicated microenvironmental perturbations, suggesting localized conformational changes in the protein structure upon CAB binding.

Keywords

• Cabozantinib
• Fluorescence spectroscopy
• Human serum albumin
• Interaction studies
• UV-Vis spectroscopy

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