Subject: Why is trimethoprim selective for bacterial DHFR??

From: "E.E.Moret" <[email-masked]>
Date: Thu, 6 Apr 95 22:18:17 METDST

Dear colleagues,

The following question is of a rather detailed nature. Still, in view of
the omnipresence of DHFR research in the modelling literature of the last
decade, I think many of you can help us out.

Trimethoprim (TMP) is a valuable antibacterial compound used 
in pharmacotherapy. Its value is based on its 10000 times higher affinity
for bacterial over human Dihydrofolate Reductase (DHFR). 
It is a classical example in medicinal chemistry and drug design.

In ligand modelling, the DHFR and methotrexate (MTX) complex (for example
4dfr.pdb) has been the de facto standard example for testing new programs
like Ludi, Leapfrog, Apex-3D, MCSS, Grow and others.
The X-ray structure of TMP with vertebrate or bacterial DHFR has never 
been deposited at the PDB. 

The selectivity of TMP for bacterial DHFR is believed to be based on a 
somewhat different binding mode, caused by decreased accessibility of 
the hydrophobic pocket, as compared to vertebrate DHFR. Although it is possible
to generate a complex with energy minimisation and docking algorithms, the
giant effect of these subtle changes remains mysterious to me. 
Although our docking method seems to work fine, given the precise
prediction of the crystal structure positions of MTX and DHF, the interaction
energies for TMP in human and bacterial DHFR are roughly the same. 
Some workers have pointed to changes in a histidines protonation state 
and its effect on the co-factor NADPH. Other workers have pointed out 
the importance of buried water molecules. Some workers have pointed at 
the protonation state of an aspartate or glutamate residue. 
A mutative loss of a valine in human DHFR compared to E.Coli DHFR seems to 
explain the loss of one hydrogen bond. The crystallographers describe the 
hydrophobic pocket in vertebrate DHFR as being too wide. A cooperative
binding effect of NADPH has been experimentally determined.

I still do not grasp what interactions are missing in vertebrate DHFR. 
What prevents TMP from adopting the conformation found in bacterial
DHFR? What are the extra interactions in bacterial DHFR which account for
the 10000-fold difference, roughly equal to 23 kJ/mole or 2 to 3 salt bridges?
(No flames on my estimates please; the difference is HUGE and complete
therapy is based on this)

Among you, there should be numerous workers who have studied this complex. 
What do you think is the main determinant for this selectivity?

With best regards,
Ed Moret
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E.E. Moret	(@more@)			    [email-masked]
Computational Medicinal Chemistry/Department of Pharmaceutical Chemistry
Faculty of Pharmacy/Utrecht University/the Netherlands
Telephone	(31-30)536979/536958        Facsimile      (31-30)516674
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