From owner-btk-mca@hgmp.mrc.ac.uk Sat Jun 10 06:11:13 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 6125317A78; Sat, 10 Jun 2000 06:11:12 +0100 (BST) Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 6024) id 2063917A73; Sat, 10 Jun 2000 06:11:10 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by mercury.hgmp.mrc.ac.uk (Postfix) with ESMTP id 82779415D5 for ; Sat, 10 Jun 2000 06:11:10 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id GAA09051 for btk-mca-list@hgmp.mrc.ac.uk; Sat, 10 Jun 2000 06:11:09 +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 15:11:04 +1000 Organization: University of Queensland Lines: 157 Message-ID: References: <393D6667.B5C164C6@biokin.com> <3940E7A3.59CB46E0@biokin.com> <394126A0.B500AEC1@biokin.com> Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Trace: bunyip.cc.uq.edu.au 960613866 13230 130.102.2.14 (10 Jun 2000 05:11:06 GMT) X-Complaints-To: news@uq.edu.au X-Sender: ddlpatte@dingo.cc.uq.edu.au In-Reply-To: <394126A0.B500AEC1@biokin.com> To: btk-mca@hgmp.mrc.ac.uk Sender: owner-btk-mca@hgmp.mrc.ac.uk Precedence: bulk G'day Petr; On Fri, 9 Jun 2000, Petr Kuzmic wrote: > is somewhat complicated by the fact that nobody has ever /seen/ the > transision state for aspartic protease catalysis. Of course, people > often represent it as some kind of "tetrahedral intermediate", adding a > nucleophile to the peptidic carbonyl, but that's really just one part of > the structure, isn't it? How do we know what /other/ changes in the > substrate structure might occur simultaneously with the nucleophilic > attack? I'm in the "enzyme stretches the scissile amide bond" camp, but I don't think there will be a gross "flash" change at the TS. To go from the substrate to the transition state (if we assume the TS is accurately mimicked by the inhibitors) would take a lot of energy and would mean radical changes about the active site, but the whole thing looks much more subtle than that. The final resting place in the product carbonyl is very close to where the nucleophile must originate from. I don't believe it is necessary to bend the substrate too radically as the difference to the inhibitor complex can vary by as much as 1 angstrom - not spectacular. Rather, a substrate in an extended conformation is "pre-ordered" for catalysis, so it is merely a case of presenting the substrate towards the catalytic water for catalysis to occur. The substrate rigidity is already ideal for electron transfer via acid-base catalysis. Further, I have read in many papers that people believe the flaps close tighter on an inhibitor compared to a substrate. From my structures I conclude that the interactions about the flap regions with the conserved water and ligand carbonyls, as well as the related H-bonding from G48/148 to ligand amides shows HIV-1 PR alters interactions and structure about the flap region and through the conserved water to satisfy all H-bonding interactions. This is particularly exemplified in the case of the inhibitor which binds very differently to both the substrate and product ligands, but still maintains good H-bonding through this region. The differences in all complexes within the remainder of the flap region is quite subtle and may facilitate the presentation of I50/I150 to the water molecule and thereby the ligands. The greatest differences about this region being G149 CA atoms that are separated by 1.08 (substrate - product). Since G149 is between the H-bond donor I50 and the H-bond acceptor G148, it is not surprising there is a large shift in this residue between the structures. I interpret the difference of interaction between a substrate, product and inhibitor around the region of the flaps and including the conserved water molecule and ligand carbonyls as a more optimal interaction of the water molecule with the substrates compared to the inhibitor. When all interactions are taken into account in this region, there is no evidence that the flaps close tighter on an inhibitor compared to a substrate ligand. Perhaps the protease doesn't know it's an inhibitor and it's supposed to treat it special ;-). I think a great deal of the problem with hydroxyl ethyl amine inhibitors is the lengthening of the inhibitor by a carbon with respect to the substrates. Indeed, the hydroxyl (gem-diol "mimic") lies only rudely between the two separate states of catalysis. So perhaps we have assumed too much about the transition state and the way it changes with respect to substrates? I am also curious as to the nature of an active site about the time of transition. Do you think the active site could be gaseous or approach a gaseous state? Room temperature data of my substrate complex showed the substrate carbonyl "flipped" to face either of the mutated active site residues, concomitant with this flipping there was rotations of the Asns to satisfy all H-bonding (this is why I went to cryo conditions). It was all very odd that this flipping (occupancy = 40%) can occur in the crystal. > You see, our substrates and inhibitors had a very high positive > electrical charge (several lysines and arginines at pH around 4), so > were able to modulate the association rate constant k1 (k1*) simply by > changind the /ionic strength/ of the buffer. Even though pepsin is an > approximately spherical macromolecule, as a first approximation we used > the Debye-Huckel theory to analyze the data and obtained an approximate > distance between the interacting ions (about 20 angstroms) as well as > the average surface charge of pepsin (isoelectric point about 1.5 !!) > that is "seen" by the charged ligands. Importantly, these /nonspecific/ > interactions physically occur nowhere near the active site, and yet by > modulating the ionic strength (and thus k1, k1*) were able to change the > Km and kcat/Km (for substrates) or Ki (for inhibitors) by more than > three orders of magnitude! Importantly, the kcat stayed constant. > There was no "salting-in" effect because the overall ionic strength was > quite low. Incredible! Perhaps when we think Ionic strength is modulating the enzyme in assays of HIV-1 PR by "salting in" the inhibitor it can also be modulating the enzyme directly by a non-specific interaction of surface residues quite analogous to pepsin? How very interesting. I searched the structure for a long-range charge attractant like a carboxy-anion hole or something and found nothing except the highly conserved Arg 8/108, which is a whole other story in itself, I've made some compounds to "take out" these Arg's but it's a real can of worms. Perhaps the aspartyl proteases doesn't need a charge attractant? Perhaps, as for pepsin, if the pI is around 1.5 for the surface, perhaps this alone is enough to attract charged residues? The more I think about it the more I feel HIV-1 PR is subject to strict control. There is the cysteines which are subject to selective glutathionylation - one enhances, the other inactivates catalysis, there is the charged residues at p6 which act as an inhibitor at cytosolic pH, there is the preference for acidic pH and salt as in a budding virion, even the N-terminal proline target's it for destruction outside a virion. I think MCA studies should be applied to HIV-1 PR to more fully understand the dynamic role of this enzyme and enhance drug design. Any suggestions as to how? Anybody? > Have you tried the fluorescence HIV-1 protease assay we developed? That > might fit your needs quite well. Here is the reference: > > http://www.biokin.com/papers/biblio.html#Pera9542 I saw the reference previously and was going to check it out. Indeed a Medline search using your name churns out some interesting papers. I have a flourometric assay using Abz-NF-6* developed by Toth et al. I might try to use the plate reader and apply some of your conditions for a nice high throughput (and automated) assay. As it is I've been doing the whole thing by hand - but it gives you an intimacy with the protein and inhibitor I like, which allows troubleshooting (like when the hydrophobic inhibitors develop an affinity for the plastic/glassware). > Interestingly, the assay was developed by an /undergraduate/ student > (Ms. Anne Peranteau) working more or less independently with a gentle > nudging on my part. Nobody uses the assay except Jon Erickson's lab (as > far as I know). Funny: one might think that publishing a paper in > Analytical Biochemistry could cause at least some laboratories to try a > sensitive new assay. Wrong. I guess I forgot that science results seem > to travel most effectively by a word of mouth, and even then among > members of the same mutually supportive tribe. My favored quantum > chemistry textbook, by R. Zahradnik, has this motto printed on the book > jacket: "Everybody writes. Nobody reads." Seemed facetious at that > time, but not so much anymore. [;)] I read a lot of papers, but only in the last six months has it become expedient to look at another assay. I also want to try some Microcal, like Todd et al have been pumping out. I am thinking of playing with Arg's to help bioavailability - something you've done by the sounds of it, did you try anything vs cells? I think people don't read because the area is so huge (and growing constantly), but also there is a lack of unity (assay?), so comparisons are difficult. I have a stack of papers I have gotten but not read - so many good intentions on the way to the library.... My work is complicated because I'm also working with rev and the rre. I've just been able to bind the rre to a streptavidin biosensor chip and now trying to get an assay going. I guess when the work is on it is also hard to keep on top (and then there's family time). Well, they're my excuses anyhow.... I want to get my hands on the common drugs to try in my assay for direct comparison, do you happen to have any Saquinavir/ritonavir/Indinavir or VX-478 lying around? Len... .