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Drug Assays

Park Your Drug Right Here For a While

Here’s a ten-year look back at the whole concept of residence times in drug discovery, published by Bob Copeland of Epizyme in Nature Reviews Drug Discovery. He’s the one to write it, since he’s one of the key developers of the whole concept, which certainly seems to have made a home for itself in the way that we think about our compounds’ activity.

What it all comes down to is looking at on-rates and off-rates for drugs binding to their targets. (The off-rates vary much more widely than the on-rates, most of the time). The renewed attention to this kinetic behavior fit perfectly with the wider adoption of surface plasmon resonance (SPR) assays, which give you detailed information on both steps for everything you put into them (well, everything that actually works, but you know what I mean). Maximizing your compounds’ residence times, which in practice usually means reducing off-rates, can be a powerful way to think about optimizing drug candidates. That has you improving both potency and pharmacokinetics at the same time, rather than thinking about them as separate problems.

The advent of SPR and other newer biophysical methods has also made it more feasible to get the details on just what actually happens when a compound binds. The more potent a ligand is, the more likely it is to work its way in through a rather complex series of steps – what’s traditionally been called the “induced fit” model, where both the compound and the binding site are adjusting to each other. This is why the old intro-to-biochemistry “lock and key” metaphor can only take you so far. If both lock and key were both mode of some sort of fairly stiff jelly and came to grips with each other in a series of shifts and rearrangements, that would be a more accurate mental picture. The further and more thoroughly a compound works itself into a binding site, the more steps there probably are that have to be reversed to get back out – potency and residence time are thus usually correlated. Maybe we should switch over from locks and keys to trucks backing into tricky loading docks.

You can get into some interesting situations if the residence time is longer than the pharmacokinetic half-life, such as having a drug that continues to work after it should already be gone from the system. In some cases, the limits on its activity are imposed by the half-life of its protein target – how long does a given protein molecule last in the cell before being turned over? These thoughts often come up when discussing covalent compounds (which bind irreversibly to their targets), but it’s important not to put these in some sort of separate mental category. They’re just the far end of the residence-time continuum, out there on the asymptote. You can see these things with noncovalent compounds, too – in the end, what’s important is the residence time itself, not necessarily whether or not a bond has been formed in order to increase it. Here’s a paper on BTK inhibitors that addresses just this point.

That reference (and many other recent ones) are to be found in the NRDD article, making it an excellent opportunity to get caught up on work in the field. Definitely recommended!

19 comments on “Park Your Drug Right Here For a While”

  1. It’s a very useful concept which I have found handy myself, but here’s a contrary viewpoint:

    http://www.ncbi.nlm.nih.gov/pubmed/23500610

    1. ab says:

      It’s not really a contrary viewpoint, it’s just saying that if your plasma clearance is slower than your dissociation rate, then it’s not ligand dissociation kinetics that’s driving duration of action, it’s plasma clearance (this is the situation most of us are used to dealing with). The situation that Copeland refers to is when your plasma clearance is fast relative to your binding off-rate kinetics. In that case, further decreasing your off rate will further improve your duration of action (up to the other limit, which is enzyme turnover in vivo).

      1. Peter Kenny says:

        Slow on-rate means that target is less-engaged when plasma levels start to fall than is case for fast-on rate.

  2. LeeH says:

    Derek – your induced fit model is not consistent with common medchem practice of trying to reduce rotatable bonds and forming rings. If increasing the complexity of how a molecule slithers into a binding site were truly a path to a more potent drug, you should be adding rotatable bonds, no? Why pursue rigid compounds?

    For two compounds with the same binding constant and biodistribution properties, but one having twice the Kon and Koff, I have two possible explanations for the slow Koff compound being better. First, it resides in the binding site proportionately longer after the compound clears from the area just outside the target. Second, the probability of a a molecular event (e.g. putting an enzyme into an inactive state, or signalling for a receptor) increases more than linearly with residence time.

    And while I’ve heard testimonial evidence of the advantages of slow Koff compounds, I have yet to see a really definitive analysis based on a large set of compounds.

    1. ab says:

      Reducing rotatable bonds improves potency by increasing the on rate. Remember that ‘potency’ gets better with a faster on rate or with a slower off rate (Kd = koff/kon). By adding constraints such as a ring, you improve your on rate since the ligand is always in the proper orientation for binding, rather than sampling that binding orientation only a fraction of the time. Adding a ring usually doesn’t change your off rate. So your Kd gets better.

      On the other hand adding rotatable bonds has the potential to decrease both your on rate and off rate and leave ‘potency’ unchanged, for the reasons discussed above. It is further complicated by the fact that on rate is proportional to ligand concentration. So if you are operating in a ‘slow on’ domain, you run the risk of never getting a high enough ligand concentration to drive that binding event, and even though the off rate is slow it doesn’t matter because there isn’t enough target engagement to drive the pharmacology.

      There are a lot of moving parts when thinking about binding kinetics and how that may impact in vivo efficacy and duratin of action. At the very least it’s worth understanding what space you’re operating in, in terms of binding kinetics.

      1. LeeH says:

        That was my point, that the pre-organization (entropic) model and the induced fit model seem to run contrary to each in terms of compound design.

        Also, we’re mixing effects here. The issue is not how to improve the binding constant. No one is disputing that a higher Kd is better. The issue is, for two compounds with equal Kd’s, does the one with a slower Koff have an advantage.

        1. sgcox says:

          Kd : lower, not higher 🙂

          1. LeeH says:

            Lower. Right.

        2. Some idiot says:

          An interesting discussion… But yes, it is important to recognise the different contributions to the effects we are talking about here. On the one side, of course reducing the entropic cost makes a difference. However, too much rigidity would then have a cost on the “induced fit” side. I would argue that the level of flexibility/rotation required to satisfy the induced fit part would be relatively low (consider aryl bonds in an otherwise rigid system). Enough of these would give even a relatively rigid molecule the chance to dance its way in, assuming that these marginally rotatable bonds were in the right places…

          It would be extremely interesting to make these sorts of measurements, although given conflicting effects from the same substitution patterns, trying to decent control experiments would come under the “very tough” category.

    2. Quintin says:

      Of course it might seem counterintuitive that more rigid molecules tend to perform better as drugs if it has more trouble fitting in its binding site. Here I should stress what I’ve been taught in biochemistry: proteins are dynamic. We might like looking at perfectly rigid X-ray crystallography determined structures of our favourite proteins, but they rarely are that rigid and in fact this is part of how they function. So if proteins are so flexible, it would seem flexibility of your drug molecule would not be a limitung factor and so you might focus on getting it to bind more strongly where you do eventually want it to by locking in the necessary conformation.

  3. watcher says:

    With great respect to Bob’s book and articles on slow binding, he certainly was not the first to write of such events (see John Morrison “Trends in Biochemical Sciences” Volume 7, Issue 3, p102–105, March 1982 along with slow tight binders (Morrison and Walsh “Advances in enzymology and related areas of molecular biology” 61:201-301 · January 1988.

    And while it is a useful concept of which to remain aware, there are certainly several downsides.
    The slow binding can involve movement / rearrangement of the protein target and/or the binding molecule itself to clamp down into a tighter complex. Unfortunately, even with today’s modelling methods, there is yet no way to predict such behavior accurately de novo, and hence no way for synthetic chemists to design molecules that will be tight binding; to find such molecules tends to be luck, a fluke. Also, many people who think they have an advantage with a tight binder are disappointed that there is little to no greater effect in vivo due to a relatively slow blood or total tissue clearance. On the upside, it should be pointed out, though, that tight binding can be advantageous for greater target selectivity.

    The concept of tight binders (and their relatives slow tight binders) certainly have a place in the pharmaceutical R&D tool-box, but is not going to be useful all the time, or even most of the time. It’s just one more concept that must be kept in mind and understood, amongst so many others.

  4. alchemist says:

    I am not sure that long residence time compounds only have advantages when the plasma clearance is faster then dissociation.
    At least it is not (always) for GPCRs. I have been twice in a situation where a long residence compound was found to have an additional advantage: the activity of the compounds (in both cases highly protein bound) did not move at all when plasma was added.
    It was quite cool to see a compound with an IC50 on 10 nM in a basic cell assay remain at 25 nM in a whole blood assay, despite a PPB of >99%…
    My assumption is that, with an extracellular target at least, if you have a slow Koff you will not be at equilibrium and the binding to your target will be advantaged.

  5. Peter Kenny says:

    Slow binding kinetics can be considered to be equivalent to slow distribution. Under what circumstances would you design a compound to have slow distribution?

  6. john adams says:

    A fun fact: The detailed mechanism of finasteride inhibition of steroid 5-alpha reductase (see ref 9 in Copeland review; http://pubs.acs.org/doi/pdf/10.1021/ja953069t) was elaborated only AFTER it was noted in early Clinical studies that it inhibited the enzyme activity in man far longer than the PK would dictate. That observation triggered a reexamination of the inhibition kinetics which in term led to the studies of the MOA. In short, finasteride acts as an alternative substrate in which NADPH adds hydride to the “wrong” carbon, setting up a carbanion that attacks NADP to form a binary substrate analog of extraordinary potency (Ki 31 days). Beautiful stuff.

    1. john adams says:

      Correction: Ki 31 days.

      1. john adams says:

        Sorry. Bug in the system. The inhibition constant is less than 100 femtomolar and the half life for dissociation is greater than 31 days.

  7. Investor says:

    Wow not sure I want to take any scientific advice from Copeland, a Founder of Epizyme who dumped almost all of his shares in Epizyme at a high price then watched the shares slide. Scoundrel

    1. eugene says:

      Why would the scientific views become tainted by investment decisions? Besides, from the story it sounds like you would want to take investment advice from him as well as scientific advice. If you don’t know more information than the majority of the people about the things you’re trading in the stock market, then you’re just the sucker of those who make the money, no?

  8. Anon says:

    How GSK could have let Copeland exit the company is beyond belief. He was a great mentor to the younger (and older) enzymologists and med. chemists. Huge loss but glad to see he had the chance to take his skills to a biotech and be a leader in an exciting area of cancer research. Best of luck to Epizyme.

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