Transient Incomplete Separation Facilitates Finding Accurate Equilibrium Dissociation Constant of Protein–Small Molecule Complex

Abstract

Current practical methods for finding the equilibrium dissociation constant, K d ,o fp rotein-small molecule complexes have inherent sources of inaccuracy.I ntroduced here is "accurate constant via transient incomplete separation" (ACTIS), which appears to be free of inherent sources of inaccuracy.C onceptually,as hort plug of the pre-equilibrated protein-small molecule mixture is pressure-propagated in ac apillary,c ausing fast transient incomplete separation of the complex from the unbound small molecule.Asuperposition of signals from these two components is measured near the capillary exit and used to calculate af raction of unbound small molecule,which,inturn, is used to calculate K d . Herein the validity of ACTIS is proven theoretically,i ts accuracy is verified by computer simulation, and its practical use is demonstrated. ACTIS has the potential to become ar eference-standardm ethod for determining K d values of protein-small molecule complexes.Reversible binding of proteins (P) to small-molecule ligands (L) plays an important role in the regulation of cellular processes. [1] In addition, most therapeutic targets are proteins, [2] and drugs are developed to form stable PL complexes with them:Complex stability is characterized by the equilibrium dissociation constant K d ,which is defined as:

Assessing Accuracy of an Analytical Method In Silico: Application to “Accurate Constant via Transient Incomplete Separation” (ACTIS)

“…In particular, we explain basic principles of K d determination and TIS of L from PL; the corresponding two sections are a close reiteration of our previously published explanation. 1 General Procedure of Kd Determination. In general, finding K d requires determination of a fraction R of unbound L in the equilibrium mixture of L and P with the initial concentrations [L] 0 and [P] 0 , respectively:…”

“…ACTIS is a separation-based method for finding K d of complexes (PL) between proteins (P) and small-molecule ligands (L): 1 on off P L PL k k    (1) where K d is defined via equilibrium concentrations of P, L, and PL:…”

“…ACTIS is a uniquely deterministic method as it relies on molecular diffusion in a pressure-driven flow which can be described by a system of partial differential equations with fully-defined initial and boundary conditions. [1][2][3] As such, it is perfectly suited for computational assessment of its accuracy.…”

“…A physical ACTIS instrument has a minimum fluidic system with a pump, an injection loop, a separation capillary, and a multiport valve (Figure 2A). [1][2][3] The valve serves as two connectors: one from a pump tube to the injection loop and the other one from the injection loop to the separation capillary. Accordingly, the instrument has five essential fluidic components which can be presented as a series of coaxial pipes of different radii ( Figure 2B).…”

Assessing Accuracy of an Analytical Method in silico: Application to “Accurate Constant via Transient Incomplete Separation” (ACTIS)

“…In particular, we explain basic principles of K d determination and TIS of L from PL; the corresponding two sections are a close reiteration of our previously published explanation. 1 General Procedure of Kd Determination. In general, finding K d requires determination of a fraction R of unbound L in the equilibrium mixture of L and P with the initial concentrations [L] 0 and [P] 0 , respectively: R = [L] eq / [L] 0 ( 4) Finding R, in turn, requires a signal S that is a superposition of signals from L and PL: From eq 5, R can be experimentally determined via measuring three signals, S L , S PL , and S, and using the following expression:…”