Volume of Hsp90 Protein–Ligand Binding Determined by Fluorescent Pressure Shift Assay, Densitometry, and NMR

Abstract

Human heat shock protein 90 (Hsp90) is a key player in the homeostasis of the proteome and plays a role in numerous diseases, such as cancer. For the design of Hsp90 ATPase activity inhibitors, it is important to understand the relationship between an inhibitor structure and its inhibition potential. The volume of inhibitor binding is one of the most important such parameters that are rarely being studied. Here, the volumes of binding of several ligands to recombinant Hsp90 were obtained by three independent experimental techniques: fluorescent pressure shift assay, vibrating tube densitometry, and high-pressure NMR. Within the error range, all techniques provided similar volumetric parameters for the investigated protein-ligand systems. Protein-ligand binding volumes were negative, suggesting that the protein-ligand complex, together with its hydration shell, occupies less volume than the separate constituents with their hydration shells. Binding volumes of tightly binding, subnanomolar ligands were significantly more negative than those of weakly binding, millimolar ligands. The volumes of binding could be useful for designing inhibitors with desired recognition properties and further development as drugs.

Thermodynamic, kinetic, and structural parameterization of human carbonic anhydrase interactions toward enhanced inhibitor design

“…There are several techniques that are capable of determining the volume of protein–ligand binding: the fluorescent pressure shift assay (PressureFluor, FPSA) (Toleikis et al ., 2011, 2016; Skvarnavičius et al ., 2017), vibrating tube densitometry(Barbosa et al ., 2003; Son et al ., 2012; Toleikis et al ., 2016), and the high-pressure NMR (Wilton et al ., 2009; Roche et al ., 2012; Toleikis et al ., 2016). They can determine the change of system volume and compressibility caused by the bound ligand.…”

Interaction of the new inhibitor paxlovid (PF-07321332) and ivermectin with the monomer of the main protease SARS-CoV-2: A volumetric study based on molecular dynamics, elastic networks, classical thermodynamics and SPT

“…In this sense, multiple authors have suggested continuing studies of ivermectin and its interactions with the various targets of SARS-CoV-2 in order to use it as a model to guide efforts towards the development of new compounds and treatment strategies ( Tang and Dill, 1998 , Tang and Kaneko, 2020 , Toleikis et al, 2016 ) as has already been done ( Rabie, 2021 ). In fact, ivermectin is currently being investigated in the UK as part of the Platform Randomized Trial of Treatments in the Community for Epidemic and Pandemic Illnesses (PRINCIPLE), the world's largest clinical trial of possible COVID-19 treatments for recovery at home and in other non-hospital settings.…”

Protein–Ligand Binding Volume Determined from a Single 2D NMR Spectrum with Increasing Pressure

“…In this study, we used two compounds that have a primary sulfonamide group in their structures (Figure 2) and exhibit sub-millimolar binding affinities for CA I and CA II. 40,51 Twodimensional 1 H− 15 N HSQC NMR spectra showed that both 29,32 W denotes water molecules that the protein (P) and ligand (L) release into bulk water after their partial dehydration upon binding. isoforms of carbonic anhydrase are in the slow exchange (in NMR time-scale) between two protein statesthe ligandbound and ligand-freefor compounds 1 and 2.…”

“…These results were compatible with previous findings showing correlations between protein−ligand binding volumes and affinities. 29 To the best of our knowledge, no other values of ΔV b for the family of carbonic anhydrases were reported in the literature. Other globular protein and ligand systems showed rather similar values of ΔV b.…”

“…High pressure is a key that unlocks various volume-related properties of proteins and reveals their functions. Changes in the protein volume upon unfolding have been extensively investigated. ,− High pressure also allowed one to probe structural properties of protein subdomains, ,− observe folding pathways and switches between protein conformations, − and investigate volume changes that arise upon binding small molecules. − As a quantity of the change in the protein volume upon ligand binding, we use a parameter Δ V b , which is called the protein–ligand binding volume and is defined as the difference between the molar volumes of the bound (hydrated protein–ligand complex plus excess water molecules) and unbound (hydrated protein and hydrated ligand) states. Figure illustrates a simplified definition of the Δ V b .…”