Transition States, analogues, and drug development

Abstract

Enzymes achieve their transition states by dynamic conformational searches on the femtosecond to picosecond time scale. Mimics of reactants at enzymatic transition states bind tightly to enzymes by stabilizing the conformation optimized through evolution for transition state formation. Instead of forming the transient transition state geometry, transition state analogues convert the short-lived transition state to a stable thermodynamic state. Enzymatic transition states are understood by combining kinetic isotope effects and computational chemistry. Analogues of the transition state can bind millions of times more tightly than substrates and show promise for drug development for several targets.

Fig. 1

Dynamic motion in natural abundance and heavy PNPs. The green arrows show chemistry-promoting bond vibrational frequency in natural abundance (left) and heavy (right) PNPs. The natural abundance enzyme has higher frequency bond vibrations (longer light green arrows) that those in heavy PNP (shorter dark green arrows). Lower frequency bond vibrations extrapolate to fewer stochastic searches for the transition state per unit time. Bond vibrations orthogonal to the reaction coordinate and shown in red and do are non-productive bond vibrations that do not contribute to barrier crossing. The transition state is characterized by a fully-formed ribocation with minimal bonding to the oxygen of the attacking phosphate nucleophile, an event following the transition state formation. From reference 17 with permission of the publisher.

Fig. 2

Transition state structure for HIV protease in comparison to a clinical inhibitor, indinavir. (A) The structure of bound indinavir in HIV-1 protease (PDB 2AVO, left panel). The transition state structure for the enzyme determined from intrinsic kinetic isotope effects and quantum chemistry are shown in the right panel. In B, the molecular electrostatic potential surface for indinavir is shown and compared to the transition state structure determined for HIV-1 protease (right). Natural bond order charges are indicated in parentheses (blue = electron deficient; red = electron rich). In panel C, the hydrogen bond patterns from the catalytic Asp groups are shown. From reference 20 with permission of the publisher.

Fig. 3

Comparing complexes in mammalian PNPs. The crystallographic structures of the Michaelis complex (left) with inosine and sulfate is compared to Immucillin-H and phosphate (middle) for bovine PNP. Distances (in blue, Å) are between heavy atoms. The transition state structure (right) is taken from transition path sampling computations for barrier crossing with human PNP using guanosine as substrate. Only the inosine atoms and relevant protein groups are shown for comparison with the other complexes. Green arrows show chemistry-promoting bond motions. Note that the leaving group interactions to Asn243 are similar between the transition state analogue and the transition state. In this excursion to form the transition state, the N of His257 is in motion and moves from 3.6 to 2.7 Å in the 20 fsec period including the transition state.

Fig. 4

Transition states for four N-ribosyltransferases and transition state analogues. Bond lengths shown for transition states are in Å. Values shown for the inhibitors are dissociation constants.

Fig. 5

Transition state analogue and structures in complex with ribosome inactivating proteins. (Upper panel) The structure of cyclic G(9-DA)GA 2′-OMe, a transition state analogue mimic for ricin A-chain and saporin L-3. Note the placement of 9-DA between two guanosine residues to mimic the GAG sequence specificity of ribosome inactivating proteins. 9-DA is a transition state mimic of 2′-deoxyadenosine. Atomic numbering for 9-DA follows that for purine nucleosides. (Lower panel) Catalytic site detail of cyclic G(9-DA)GA 2′-OMe bound to (A) ricin A-chain and (B) saporin L-3. In both catalytic sites the 9-DA is involved in π-stacking interactions between two tyrosines. Placing two cationic Arg residues near the adenine position serves to withdraw bonding electrons into the leaving group and a Glu serves as a general base to ionize the water nucleophile. From reference 75 with permission of the publisher.

Fig. 6

Inhibition of H. pylori MTAN blocks growth of bacterial cultures. (A) The effect of BuT-DADMe-ImmH (BuT-Dadme-ImmuA; Fig. 4) is shown to have an IC₉₀ of less than 8 ng/mL. (B) When compared to the same amounts of amoxicillin, metronidazole or tetracycling, BuT-DADMe-ImmH is more effective. From reference 90 with permission of the publisher.