Simulation-Guided Engineering Enables a Functional Switch in Selinadiene Synthase toward Hydroxylation.

Engineering sesquiterpene synthases to form predefined alternative products is a major challenge due to their diversity in cyclization mechanisms and our limited understanding of how amino acid changes affect the steering of these mechanisms. Here, we use a combination of atomistic simulation and site-directed mutagenesis to engineer a selina-4(15),7(11)-diene synthase (SdS) such that its final reactive carbocation is quenched by trapped active site water, resulting in the formation of a complex hydroxylated sesquiterpene (selin-7(11)-en-4-ol). Initially, the SdS G305E variant produced 20% selin-7(11)-en-4-ol. As suggested by modeling of the enzyme-carbocation complex, selin-7(11)-en-4-ol production could be further improved by varying the pH, resulting in selin-7(11)-en-4-ol becoming the major product (48%) at pH 6.0. We incorporated the SdS G305E variant along with genes from the mevalonate pathway into bacterial BL21(DE3) cells and demonstrated the production of selin-7(11)-en-4-ol at a scale of 10 mg/L in batch fermentation. These results highlight opportunities for the simulation-guided engineering of terpene synthases to produce predefined complex hydroxylated sesquiterpenes.

Mechanism of covalent binding of ibrutinib to Bruton's tyrosine kinase revealed by QM/MM calculations.

Ibrutinib is the first covalent inhibitor of Bruton's tyrosine kinase (BTK) to be used in the treatment of B-cell cancers. Understanding the mechanism of covalent inhibition will aid in the design of safer and more selective covalent inhibitors that target BTK. The mechanism of covalent inhibition in BTK has been uncertain because there is no appropriate residue nearby that can act as a base to deprotonate the cysteine thiol prior to covalent bond formation. We investigate several mechanisms of covalent modification of C481 in BTK by ibrutinib using combined quantum mechanics/molecular mechanics (QM/MM) molecular dynamics reaction simulations. The lowest energy pathway involves direct proton transfer from C481 to the acrylamide warhead in ibrutinib, followed by covalent bond formation to form an enol intermediate. There is a subsequent rate-limiting keto-enol tautomerisation step (ΔG ‡ = 10.5 kcal mol-1) to reach the inactivated BTK/ibrutinib complex. Our results represent the first mechanistic study of BTK inactivation by ibrutinib to consider multiple mechanistic pathways. These findings should aid in the design of covalent drugs that target BTK and other similar targets.

Limitations of Ligand-Only Approaches for Predicting the Reactivity of Covalent Inhibitors.

Covalent inhibition has undergone a resurgence and is an important modern-day drug design and chemical biology approach. To avoid off-target interactions and to fine-tune reactivity, the ability to accurately predict reactivity is vitally important for the design and development of safer and more effective covalent drugs. Several ligand-only metrics have been proposed that promise quick and simple ways of determining covalent reactivity. In particular, we examine proton affinity and reaction energies calculated with the density functional B3LYP-D3/6-311+G**//B3LYP-D3/6-31G* method to assess the reactivity of a series of α,ß-unsaturated carbonyl compounds that form covalent adducts with cysteine. We demonstrate that while these metrics correlate well with experiment for a diverse range of small reactive molecules these approaches fail for predicting the reactivity of drug-like compounds. We conclude that ligand-only metrics such as proton affinity and reaction energies do not capture determinants of reactivity in situ and fail to account for important factors such as conformation, solvation, and intermolecular interactions.

Probing the excited state relaxation dynamics of pyrimidine nucleosides in chloroform solution.

Ultrafast transient electronic and vibrational absorption spectroscopy (TEAS and TVAS) of 2'-deoxy-cytidine (dC) and 2'-deoxy-thymidine (dT) dissolved in chloroform examines their excited-state dynamics and the recovery of ground electronic state molecules following absorption of ultraviolet light. The chloroform serves as a weakly interacting solvent, allowing comparisons to be drawn with prior experimental studies of the photodynamics of these nucleosides in the gas phase and in polar solvents such as water. The pyrimidine base nucleosides have some propensity to dimerize in aprotic solvents, but the monomer photochemistry can be resolved clearly and is the focus of this study. UV absorption at a wavelength of 260 nm excites a 1ππ* ← S0 transition, but prompt crossing of a significant fraction (50% in dC, 17% in dT) of the 1ππ* population into a nearby 1nπ* state is too fast for the experiments to resolve. The remaining flux on the 1ππ* state leaves the vertical Franck-Condon region and encounters a conical intersection with the ground electronic state of ethylenic twist character. In dC, the 1ππ* state decays to the ground state with a time constant of 1.1 ± 0.1 ps. The lifetime of the 1nπ* state is much longer in the canonical forms of both molecules: recovery of the ground state population from these states occurs with time constants of 18.6 ± 1.1 ps in amino-oxo dC and ∼114 ps in dT, indicating potential energy barriers to the 1nπ*/S0 conical intersections. The small fraction of the imino-oxo tautomer of dC present in solution has a longer-lived 1nπ* state with a lifetime for ground state recovery of 193 ± 55 ps. No evidence is found for photo-induced tautomerization of amino-oxo dC to the imino-oxo form, or for population of low lying triplet states of this nucleoside. In contrast, ∼8% of the UV-excited dT molecules access the long-lived T1 (3ππ*) state through the 1nπ* state. The primary influence of the solvent appears to be the degree to which it destabilizes the states of 1nπ* character, with consequences for the lifetimes of these states as well as the triplet state yields.