From owner-btk-mca@hgmp.mrc.ac.uk Fri Jun 9 15:29:30 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 07B9517B24; Fri, 9 Jun 2000 15:29:29 +0100 (BST) Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 6024) id 2E48E17B06; Fri, 9 Jun 2000 15:29:27 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by mercury.hgmp.mrc.ac.uk (Postfix) with ESMTP id B4A31415D4 for ; Fri, 9 Jun 2000 15:29:26 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id PAA19665 for btk-mca-list@hgmp.mrc.ac.uk; Fri, 9 Jun 2000 15:29:25 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Leonard Pattenden X-Newsgroups: bionet.metabolic-reg Subject: Re: Drug design for HIV-1 PR. Date: Sat, 10 Jun 2000 00:29:21 +1000 Organization: University of Queensland Lines: 183 Message-ID: References: <393D6667.B5C164C6@biokin.com> <3940E7A3.59CB46E0@biokin.com> Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Trace: bunyip.cc.uq.edu.au 960560962 4781 130.102.2.14 (9 Jun 2000 14:29:22 GMT) X-Complaints-To: news@uq.edu.au X-Sender: ddlpatte@dingo.cc.uq.edu.au In-Reply-To: <3940E7A3.59CB46E0@biokin.com> To: btk-mca@hgmp.mrc.ac.uk Sender: owner-btk-mca@hgmp.mrc.ac.uk Precedence: bulk On Fri, 9 Jun 2000, Petr Kuzmic wrote: > 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. I actually have solved two structures of HIV-1 PR with a D25N mutant:substrate-bound a wild type:two cleaved product halves complex. The substrate used is a synthetic cyclic peptide. The data was collected under cryostatic conditions and has defracted to 1.6 angstroms. An overlay of these structures and a parent hydroxyl-ethyl-amine inhibitor show there is no-wat this class of inhibitors mimics the transition state, nor do they bind in the same manner to substrates/products as they are more "puckered" about P1, P1'. The structures suggest the mechanism of catalysis is subtle and not spectacular, with the enzyme locking the N-terminal half to effect efficient electron transfer. Indeed the N-terminal product is in an almost identical position. From my observations I would say there isn't any inhibitors to HIV-1 PR that would mimic the transition state, though I'd be happy to compare any structure that is suggested can. I would confidently say it would also be the case for the larger monomeric aspartyl proteases. > 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. Interesting, I'll check it out. From the Henderson paper and more highlighted in Methods in Enzymology (1994) v241 pp 127-156 "Use of Steady State Kinetic methods to Elucidate the Kinetic and Chemical Mechanisms of Retroviral Proteases." By Meek et al. It is plausible for HIV-1 PR there is a hierarchical classification system of the substrates into several types (class 1, class 2, class 3 and mixed). This system is largely ignored, but is extremely useful. I believe the reason it is ignored is because it is described in an SIV paper, so isn't easily found by a simple lit search on HIV-1 PR and is in a virology journal - not the normal reading matter of enzymologists. In this paper, the authors conclude SIV and HIV have similar cleavage site specificities (most are homologous) and define a sequential ordering of substrate cleavage. The classes are as follows and some show interesting trends which you my not normally consider as a trend; Class 1: Tyr/Phe~Pro (P1~P1' and possibly an aromatic in P1') with hydrophobic residues in P2,P2' (Asp in P2 for non-HIV and Asn for HIV) and Gly/polar groups in P3. These also resemble HTLV-I and FIV consensus sequences except P2 has a hydrophobic preference (HIV also likes a hydrophobic residue in P2). Class 2: All contain Arg in P4 and the sequence Phe-Leu (for all retroviral aspartyl proteases generally in P1'-P2', but can be changed for HIV). The general type being ArgP4..Leu~Phe-LeuP1-P2' (p51-p15). Interesting with respect to this class of enzymes is they like P3 to be polar and P2 to be hydrophobic. Indeed immunodeficiency proteases cleave either side of a hydrophobic residue based purely on the neighbours (co-operative binding). Class3: All contain Gln/Glu at P2' and prefer hydrophobics at P2, P1, P1'. Mixed: the mixed sequence for HIV-1 PR is querky. It's sequence P5-P5' is, Arg-Pro-Gly-Asn-Phe~Leu-Gln-Ser-Arg-Pro, so it contains the class 2 determinants (Phe-Leu and Arg 4 as 4th amino acid from Phe), but it has a shifted cleavage sequence from class 2 because of that Class 3 Gln in P2' complicating things (there is also an Asn at P2 which stabilises hydrophobics into P1 so cleavage follows a class 3 cleavage sequence. Ie, the class 3 factor and stabilising Asn causes an alteration in the cleavage pattern and co-operative shifting. Now Tozser et al 1991 (FEBS 279, 356-360) examined P4 (Ser) substitution in a classic class 1 HIV-1 PR sequence and found bulky long chains and branched chains were bad (hence Arg importance as a Class 2), and smaller, more Ser-like and helix breaking (extended or beta preference) amino acids here stabilise the hydrophobic and Class 1 and mixed cleavage type (I believe Asn in P2 is exactly the same). This happens by increasing the Kcat and/or decreasing the Km. For interests sake, the numbers of each class for HIV-1 PR is thus class 1 (3), class 2 (1 - and rare) class 3 (2) mixed (2). Class 3 appear to be the "preferred" class and hence best for inhibitor design. Meek et al., takes all the classes into account and examines the product inhibition of the different classes concluding that the kinetic mechanism of HIV-1 PR is random, with products forming both binary and ternary enzyme product(s) depending purely on the substrate used. For strong substrates of HIV, once cleaved, one generally leaves and is an uncompetitive inhibitor, whereas the other is held more tightly but is a non-competitive inhibitor. Other scenarios exist (a non-competitive inhibitor (product) that is released and a competitive inhibitor that is held), and it is possible to make a non-native substrate where both inhibitors are competitive which leads to a random release - though they are within experimental error and an ordering may still exist. Indeed if you put the two competitive product inhibitors together you can reform a complete substrate (as has been done), but non/uncompetitive inhibitors such as those with a Pro residue just can't reform (think of the turn-like nature of the Pro and how hard it is to reform a bond to that amino acid in terms of the position of a cleaved peptide that has left and then tries to re-enter. The energy barrier is too high as the substrate binding pocket essentially relaxes/collapses). So what is a good inhibitor going to possess in terms of substrate halves? Substrates with poor Kcat/Km values are generally good product inhibitors, whereas good substrates with high Kcat/Km values show less product inhibition. Why? Because a good product inhibitor is more pre-ordered to form the end product of catalysis and so mimics the transition state poorly and hence the energy transition is higher on binding and through catalysis, but is then nicely relaxed and happy once cleaved. If you think in terms of what half of your inhibitor is doing what and instead of trying to mimic the transition state per se, you consign yourself to purely building a good substrate or a tight binding product (or a combination?), and follow the rules (by the way, combining the rules works *really* well too - I have a peptide inhibitor that is more potent than non-peptides mixing the classes). > > 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. That is exactly what I mean. Especially in my hands. It is impossible to predict selection via an assay system. I believe it is the nature of the assay or the protease, which co-embraces the ligand. Unfortunately the assay I have inherited is apparently flawed It is at the wrong pH and salt concentration. This is significant as the pH optimum can vary by as much as 2 units depending on the particular substrate and can vary from 4-6, though it can operate from 3-7.5. All my assays are conducted at pH 6.5 to be more cytosol-like.I believe cytosolic pH is not relevant to HIV-1 PR activation and activity. The ionic strength has a great effect on the catalytic efficiency (Kcat/Km) and varies with ionic strength Km can change from milli--->micro in a transition from I= 0.1-2M, with Kcat increasing with I. Our assay uses I=0.1. However, the concentration inside a virion is likely to have a larger value of I and lower pH value. To highlight the failings of the assay, think of the optimum crystallisation conditions which often have a semblance to the assay conditions. The norm is to grow using 2-5mL of protein-substrate (pH 5.5 acetate buffer) over a reservoir containing 100mM acetate buffer (pH 5.5) and 35-60% saturated ammonium sulfate as the precipitant. If you look at the structures of these mutant proteases, you will find they are only subtley different, almost non-recognisable. I wonder if the nature of the solution (hydrophilic) and the nature of the inhibitors (hydrophobic) does not create an energetic incentive to form a complex despite the disfavoured Kcat/Km? Indeed, I'm wondering if it would be worth trying an assay with the polyprotein precursors gag and gag-pol without and in the presence of an inhibitor. I wonder how useful examining the protease totally in the context of it's native environment would be? Len... .