Leonard Pattenden wrote:
>> On Tue, 6 Jun 2000, Petr Kuzmic wrote:
> [...]
> > Some people
> > were then looking at small peptide substrates for proteases, trying to
> > select those with low Km and chemically modify them into successful
> > inhibitor drugs. And failing, of course.
>> I assume you were specifically thinking of HIV-1 protease, is that correct
> Petr?
Not only HIV-1 protease, but long before that also pepsin as a "model
aspartic protease". Various people spent years hunting for small
inhibitors of /renin/ (as opposed to "rennin") for blood pressure
regulation. Pepsin was used to model the relationship between substrate
reactivity (Km, kcat, kcat/Km) and inhibitor potency (Ki). Oh yes, and
then there was Bartlett's work on thermolysin, penicillopepsin, and
carboxypeptidase A. Bartlett was showing these nice looking pictures
showing correlations between kcat/Km and Ki as an indication that the
inhibitors were "transition state analogs". Oh well.
> Do you feel the reason for the spectacular failure lay in the
> methods by which people arrived at their optimised inhibitor? Too often
> people tried to functionalise from left to right, then threw their arms in
> the air when co-operative effects influenced the binding to such an extent
> they could no longer tell why their inhibitor failed.
I believe that co-operative effects ("subsite interactions") are largely
responsive for the failure of substrate vs. inhibitor correlations, but
perhaps even more importantly, /nonspecific/ interactions might be even
more important. We got our hands on one of those phenomena -- long
range, nonspecific, electrostatic interactions in pepsin catalysis
(http://www.biokin.com/papers/biblio.html#Kuzm9175). Those show
clearly that extrapolating from substrate kinetics (a /dynamic/ event)
to the binding of reversible inhibitors (a /static/ event) is very
tricky.
> [...] I believe the work outlined by
> Henderson et al (Henderson et al., (1988) "Molecular Characterization of
> gag Proteins from Simian Immunodeficiency Virus (SIVmne)" J. Virol
> 62; 2587-2595) was a brilliant start point that should never have been
> rejected.
Interesting, I missed that paper somehow. Will have to look at it.
> Indeed, following these rules you can make a *very* potent
> inhibitor. Perhaps now in light of viral resistance we can return to real
> drug design. Which brings me to a question - why do you think it is that
> assays for HIV-1 PR/inhibitors conducted with mutant enzymes and the
> compounds that select them fail to predict the said selection?
Can you elaborate a little bit what do you mean by "selection"? I must
admit that I've not been following the work on mutant protease kinetics
as much as I should.
Do you mean to say that HIV-Pr inhibitors toward which the virus becomes
resistant are /still/ potent in vitro inhibitors of the protease?? I
could just look up some recent papers from Jon Erickson's lab, but they
are not easily accessible for me right now.
-- Petr
_____________________________________________________________________
Petr Kuzmic, Ph.D. * BioKin, Ltd. * Consulting & Software Development
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