Evaluation of Log P, pKa and Log D Predictions from the SAMPL7 Blind Challenge

<div>The Statistical Assessment of Modeling of Proteins and Ligands (SAMPL) challenges focuses the computational modeling community on areas in need of improvement for rational drug design. The SAMPL7 physical property challenge dealt with prediction of octanol-water partition coefficients and pKa for 22 compounds. </div><div>The dataset was composed of a series of N-acylsulfonamides and related bioisosteres.</div><div>17 research groups participated in the logP challenge, submitting 33 blind submissions total. For the pKa challenge, 7 different groups participated, submitting 9 blind submissions in total. Overall, the accuracy of octanol-water logP predictions in the SAMPL7 challenge was lower than octanol-water logP predictions in SAMPL6, likely due to a more diverse dataset. Compared to the SAMPL6 pKa challenge, accuracy remains unchanged in SAMPL7.</div><div>Interestingly, here, though macroscopic pKa values were often predicted with reasonable accuracy, there was dramatically more disagreement among participants as to which microscopic transitions produced these values (with methods often disagreeing even as to the sign of the free energy change associated with certain transitions), indicating far more work needs to be done on pKa prediction methods.</div>

Evaluation of Log P, pKa and Log D Predictions from the SAMPL7 Blind Challenge

<div>The Statistical Assessment of Modeling of Proteins and Ligands (SAMPL) challenges focuses the computational modeling community on areas in need of improvement for rational drug design. The SAMPL7 physical property challenge dealt with prediction of octanol-water partition coefficients and pKa for 22 compounds. </div><div>The dataset was composed of a series of N-acylsulfonamides and related bioisosteres.</div><div>17 research groups participated in the logP challenge, submitting 33 blind submissions total. For the pKa challenge, 7 different groups participated, submitting 9 blind submissions in total. Overall, the accuracy of octanol-water logP predictions in the SAMPL7 challenge was lower than octanol-water logP predictions in SAMPL6, likely due to a more diverse dataset. Compared to the SAMPL6 pKa challenge, accuracy remains unchanged in SAMPL7.</div><div>Interestingly, here, though macroscopic pKa values were often predicted with reasonable accuracy, there was dramatically more disagreement among participants as to which microscopic transitions produced these values (with methods often disagreeing even as to the sign of the free energy change associated with certain transitions), indicating far more work needs to be done on pKa prediction methods.</div>

Characterization of retinaldehyde dehydrogenase 3

RALDH3 (retinal dehydrogenase 3) was characterized by kinetic and binding studies, protein engineering, homology modelling, ligand docking and electrostatic-potential calculations. The major recognition determinant of an RALDH3 substrate was shown to be an eight-carbon chain bonded to the aldehyde group whose kinetic influence (kcat/Km at pH 8.5) decreases when shortened or lengthened. Surprisingly, the β-ionone ring of all-trans-retinal is not a major recognition site. The dissociation constants (Kd) of the complexes of RALDH3 with octanal, NAD+ and NADH were determined by intrinsic tryptophan fluorescence. The similarity of the Kd values for the complexes with NAD+ and with octanal suggests a random kinetic mechanism for RALDH3, in contrast with the ordered sequential mechanism often associated with aldehyde dehydrogenase enzymes. Inhibition of RALDH3 by tri-iodothyronine binding in competition with NAD+, predicted by the modelling, was established kinetically and by immunoprecipitation. Mechanistic implications of the kinetically influential ionizations with macroscopic pKa values of 5.0 and 7.5 revealed by the pH-dependence of kcat are discussed. Analogies with data for non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans, together with the present modelled structure of the thioacyl RALDH3, suggest (a) that kcat characterizes deacylation of this intermediate for specific substrates and (b) the assignment of the pKa of the major ionization (approximating to 7.5) to the perturbed carboxy group of Glu280 whose conjugate base is envisaged as supplying general base catalysis to attack of a water molecule. The macroscopic pKa of the minor ionization (5.0) is considered to approximate to that of the carboxy group of Glu488.

Reductive half-reaction of the H172Q mutant of trimethylamine dehydrogenase: evidence against a carbanion mechanism and assignment of kinetically influential ionizations in the enzyme–substrate complex

The reactions of wild-type trimethylamine dehydrogenase (TMADH) and of a His-172 Gln (H172Q) mutant were studied by rapid-mixing stopped-flow spectroscopy over the pH range 6.0-10.5, to address the potential role of His-172 in abstracting a proton from the substrate in a ‘carbanion’ mechanism for C-H bond cleavage. The pH-dependence of the limiting rate for flavin reduction (klim) was studied as a function of pH for the wild-type enzyme with perdeuterated trimethylamine as substrate. The use of perdeuterated trimethylamine facilitated the unequivocal identification of two kinetically influential ionizations in the enzyme-substrate complex, with macroscopic pKa values of 6.5±0.2 and 8.4±0.1. A plot of klim/Kd revealed a bell-shaped curve and two kinetically influential ionizations with macroscopic pKa values of 9.4±0.1 and 10.5±0.1. Mutagenesis of His-172, a potential active-site base and a component of a novel Tyr-His-Asp triad in the active site of TMADH, revealed that the pKa of 8.4±0.1 for the wild-type enzyme-substrate complex represents ionization of the imidazolium side-chain of His-172. H172Q TMADH retains catalytic competence throughout the pH range investigated. At pH 10.5, and in contrast with the wild-type enzyme, flavin reduction in H172Q TMADH is biphasic. The fast phase is dependent on the trimethylamine concentration and exhibits a kinetic isotope effect of about 3; C-H bond cleavage is thus partially rate-limiting. In contrast, the slow phase does not show hyperbolic dependence on substrate concentration, and the observed rate shows no dependence on isotope, revealing that C-H bond cleavage is not rate-limiting. The analysis of H172Q TMADH, together with data recently acquired for the Y169F mutant of TMADH, reveals that C-H bond breakage is not initiated via abstraction of a proton from the substrate by an active-site base. The transfer of reducing equivalents to flavin via a carbanion mechanism is therefore unlikely.

Supracrystallographic resolution of interactions contributing to enzyme catalysis by use of natural structural variants and reactivity-probe kinetics

1. The influence on the reactivities of the catalytic sites of papain (EC 3.4.22.2) and actinidin (3.4.22.14) of providing for interactions involving the S1-S2 intersubsite regions of the enzymes was evaluated by using as a series of thiol-specific two-hydronic-state reactivity probes: n-propyl 2-pyridyl disulphide (I) (a ‘featureless’ probe), 2-(acetamido)ethyl 2′-pyridyl disulphide (II) (containing a P1-P2 amide bond), 2-(acetoxy)ethyl 2′-pyridyl disulphide (III) [the ester analogue of probe (II)] and 2-carboxyethyl 2′-pyridyl disulphide N-methylamide (IV) [the retroamide analogue of probe (II)]. Syntheses of compounds (I), (III) and (IV) are reported. 2. The reactivities of the two enzymes towards the four reactivity probes (I)-(IV) and also that of papain towards 2-(N'-acetyl-L-phenylalanylamino)ethyl 2′-pyridyl disulphide (VII) (containing both a P1-P2 amide bond and an L-phenylalanyl side chain as an occupant for the S2 subsite), in up to four hydronic (previously called protonic) states, were evaluated by analysis of pH-dependent stopped-flow kinetic data (for the release of pyridine-2-thione) by using an eight-parameter rate equation [described in the Appendix: Brocklehurst & Brocklehurst (1988) Biochem. J. 256, 556-558] to provide pH-independent rate constants and macroscopic pKa values. The analysis reveals the various ways in which the two enzymes respond very differently to the binding of ligands in the S1-S2 intersubsite regions despite the virtually superimposable crystal structures in these regions of the molecules. 3. Particularly striking differences between the behaviour of papain and that of actinidin are that (a) only papain responds to the presence of a P1-P2 amide bond in the probe such that a rate maximum at pH 6-7 is produced in the pH-k profile in place of the rate minimum, (b) only in the papain reactions does the pKa value of the alkaline limb of the pH-k profile change from 9.5 to approx. 8.2 [the value characteristic of a pH-(kcat./Km) profile] when the probe contains a P1-P2 amide bond, (c) only papain reactivity is affected by two positively co-operative hydronic dissociations with pKI congruent to pKII congruent to 4 and (d) modulation of the reactivity of the common -S(-)-ImH+ catalytic-site ion-pair (Cys-25/His-159 in papain and Cys-25/His-162 in actinidin) by hydronic dissociation with pKa approx. 5 is more marked and occurs more generally in reactions of actinidin than is the case for papain reactions.(ABSTRACT TRUNCATED AT 400 WORDS)