Conformational methotrexate

We saw in Chapter 3 that bisubstrate reactions can conform to a number of different reaction mechanisms. We saw further that the apparent value of a substrate Km (KT) can vary with the degree of saturation of the other substrate of the reaction, in different ways depending on the mechanistic details. Hence the determination of balanced conditions for screening of an enzyme that catalyzes a bisubstrate reaction will require a prior knowledge of reaction mechanism. This places a necessary, but often overlooked, burden on the scientist to determine the reaction mechanism of the enzyme before finalizing assay conditions for HTS purposes. The importance of this mechanistic information cannot be overstated. We have already seen, in the examples of methotrexate inhibition of dihydrofolate, mycophenolic acid inhibiton of IMP dehydrogenase, and epristeride inhibition of steroid 5a-reductase (Chapter 3), how the [5]/A p ratio can influence one s ability to identify uncompetitive inhibitors of bisubstrate reactions. We have also seen that our ability to discover uncompetitive inhibitors of such reactions must be balanced with our ability to discover competitive inhibitors as well. 

The binding site for trimetrexate is well defined and was compared with the binding sites in related complexes formed with methotrexate and trimethoprim. No major conform -... 

Evidence for, and Proposed Structure of, a New Folded Conformation of Methotrexate. ... 

Fig. 13.21. Histograms of dihedral angles for fragments which are representative for the nine flexible bonds in the inhibitor methotrexate data taken from small molecule crystal structures (for data with trigonal planar and pyramidal geometry at N have been considered). For each fragment the local symmetry has been taken into account. Some of the angles exhibit clear conformational preferences, others show a broad distribution. The torsion angles for the conformation of methotrexate, adopted at the active site of DHFR (3DFR, 4DFR) from two species E. coli, 1. casei) and in small molecule crystal structures (DOJZADOl, QUIASPIO, FABVUZ (two independent molecules)) are indicated (lines to the right, protein data boldfaced)...

Fig. 3 Conformational changes of methotrexate 7. Reprinted from Ref. [16]. Copyright 1995, with permission from Elsevier...

The method was tested by attempting to reproduce the bound conformations of two enzyme-inhibitor complexes Streptomyces griseus proteinase B (SGPB) complexed with the ovomucoid inhibitor from turkey (OMTKY3), and dihydrofolate reductase (DHFR) complexed with methotrexate (MTX). The... 

As a test, the fit of methotrexate into dihydrofolate reductase was attempted. More than 90% of the trials converged, indicating that all ligand atoms are inside spheres, and all distance bounds are satisfied within 0.5 A. Each conformation takes less than a second on a typical workstation. Of 100 random trials, 15% are within 2.5 A rmsd of the X-ray structure. Further, the lowest energy fits were closest to the X-ray structure. 

For the (rare) case where the 3-D structure of the receptor/inhibitor complex is known, the conformation of the inhibitor can directly be used to build a 3-D pharmacophore. For methotrexate, it is known that the pteridine ring system is... 

Figure 7. 3-D pharmacophore query based on methotrexate conformation when it is bound to DHFR...

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