Drug–tubulin interactions interrogated by transient absorption spectroscopy

Abstract

Colchicine (COL) is a bioactive molecule with antitumor properties. When COL binds to tubulin (TU), it inhibits microtubule assembly dynamics. We have investigated COL-TU interactions using laser flash photolysis (LFP) technique and performing fully flexible molecular dynamics simulations. Excitation of COL at 355 nm in aqueous medium did not lead to any transient absorption spectrum. By contrast, in the presence of TU a transient peaking at l max ca. 420 nm was registered and assigned as triplet excited COL complexed with TU ( 3 COL*@TU). In aerated medium, the lifetime was s ca.160 ms and the quantum yield was 0.138. Likewise, when the bicyclic COL analog MTC was submitted to LFP in the presence of TU, 3 MTC@TU* was detected with a lifetime of ca. 62 ms and a quantum yield of 0.296, Aqueous solutions of MTC did not produce any signal in the microsecond timescale. The triplet energy of MTC was obtained by means of emission measurements and found to be ca. 200 kJ mol À1 , a value that matches with that previously reported for COL (188 kJ mol À1 ).Molecular dynamic simulations, both with the ground and triplet excited state, reveal a strong interaction between COL and TU to give stabilized complexes with restricted mobility inside the protein binding site. These results demonstrate that LFP is a useful methodology to study the binding of COL derivatives to TU and open a new way to evaluate the interactions of nonfluorescent anticancer drugs with this protein.

Hydrodynamic Steering in Protein Association Revisited: Surprisingly Minuscule Effects of Considerable Torques

“…In the above equation k B is the Boltzmann constant, T is the temperature, μ ( q⃗ ) is the spatially dependent mobility of the ligand molecule, and denotes either the potential affecting the ligand or the potential energy of the system in configuration given with q⃗ ;∇ q⃗ denotes the gradient with respect to the components of q⃗ . Introducing the survival probability function, S ( q⃗ o , t ), i.e., the probability that the ligand molecule located initially at q⃗ o has not yet reacted at time t : where P ( q⃗ , t | q⃗ o ,0) is the conditional probability that if the ligand molecule was at q⃗ o at time 0 then it will be at q⃗ at time t , resulting from the solution of the Smoluchowski eq , and the integration is over the whole volume V of the domain enclosed between δω and δΩ , one may evaluate the time τ­( q⃗ o ) of the receptor–ligand association (the mean first-passage time ,, ) as We assume that for t < 0 the studied system is nonreactive, i.e., both δω and δΩ are reflective, and only an activation (or triggering) at t = 0 makes the association between the ligand and the receptor possible, such a situation is usually encountered in experiments involving pump–probe techniques. − Before the activation, the probability of finding the ligand at q⃗ o follows the equilibrium distribution P eq ( q⃗ o ) and thus the average time for association, τ, (or the global first-passage time) can be evaluated by integrating out the dependence of the mean first-passage time (eq ) on the initial position of the ligand: Again, the integration is over the whole volume V of the domain enclosed between δω and δΩ .…”

Effects of Spatially Dependent Mobilities on the Kinetics of the Diffusion-Controlled Association Derived from the First-Passage-Time Approach

“…We attempt to solve the above equation with the following boundary conditions and for the δ-function initial condition The desired solution is the conditional probability, P ( Q⃗ , t | Q⃗ o ,0), that if the ligand molecule was at Q⃗ o at time 0 then it will be at Q⃗ at time t . The probability that the ligand molecule located initially at Q⃗ o has not yet reacted at time t is denoted S ( Q⃗ o , t ) ( S ( Q⃗ o , t ) is the survival probability function) and is given by As −δ t S ( Q⃗ o , t ) d t is the probability that the ligand reaches δω in the time interval ( t , t + d t ), the average time τ­( Q⃗ o ) required for the receptor–ligand association to take place (the mean first-passage time ,, ) can be evaluated as We may now consider a situation in which δω is reflective for t < 0 and at t = 0 the studied system needs to be activated (or triggered) for a reaction to occur (as, e.g., in the case of experiments involving various pump–probe techniques − ). Before the activation, the probability of finding the ligand at the position Q⃗ o ∈ Ω\ω follows the equilibrium distribution P eq ( Q⃗ o ).…”