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Floppiness Is Not Your Friend: Who Knew?

There’s a trick that every medicinal chemist learns very early, and continues to apply every time its feasible: take two parts of your compound, and tie them together into a ring.
The reason that works so well may not be immediately obvious if you’re not a medicinal chemist, so let me expand on them a bit. The first thing to know is that this method tends to work either really well or not at all – it’s a “death or glory” move. And that gives you a clue as to what’s going on. The idea is that the rotatable bonds in your molecule are, under normal conditions, doing just that: rotating. Any molecule the size of a normal drug has all kinds of possible shapes and rotational isomers, and room temperature is an energetic enough environment to populate a lot of them.
But there’s only one of them that’s the best for fitting into your drug target, most likely. So what are the odds? As your molecule approaches its binding pocket, there’s a complicated energetic dance going on. Different parts of your drug candidate will start interacting with the target (usually a protein), and that starts to tie down all that floppy rotation. The question is, does the gain resulting from these interactions cancel out the energetic price that has to be paid for them? Is there a pathway that leads to a favorable tight-binding situation, or is your molecule going to approach, flop around a bit, and dance away?
Several things are at work during that shall-we-dance period. The different conformations of your compound vary in energy, depending on how much its parts are starting to bang into each other, and how much you’re asking the bonds to twist around. The closer that desired drug-binding shape is to the shape your molecule wants to be in anyway, the better off you are, from that perspective. So tying back the molecule and making a ring in the structure does one thing immediately: it cuts down on the range of conformations it can take, in the same way that tying a rope between your ankles cuts down on your ability to dance. You’ve handcuffed your molecule, which would probably be cruel if they were sentient, but then, a lot of organic chemistry would be pretty unspeakable if molecules had feelings.
That’s why this method tends to be either a big winner or a big loser. If the preferred binding mode of your compound is close to the shape it takes when you tie it down, then you’ve suddenly zeroed in on just the thing you want, and the binding affinity is going to take a big leap. But if it’s not, well, you’ve now probably made it impossible for the thing to adopt the conformation it needs, and the binding affinity is going to take a big leap over a cliff.
There’s another effect to reducing the flexibility of your compound, and that has to do with entropy. All that favorable-interaction business is one component of the energy involved, namely the enthalpy, but entropy is the other. Loosely speaking, the more disordered a system, the higher its entropy. A floppy molecule, when it binds to a drug target, has to settle down into a much tighter fit, and entropically, that’s unfavorable. Energetically, you’re paying to do that. But if your molecule is already much less flexible, there’s not much of a toll as it fits into the pocket. If loss-of-floppiness is a bad thing, then don’t start out with so much of it.
So, how much do I and my medicinal chemistry colleagues think about this stuff, day to day? A fair amount, but there are parts of it that we probably don’t pay enough attention to. Entropy gets less respect from us than it deserves, I think. It’s easy to imagine molecules bumping into each other, sticking and unsticking, but the more nebulous change-in-disorder part of the equation is just as important. And it doesn’t just apply to our drug molecules – proteins get less disordered as they bind those molecules (or more disordered, in some cases), and those entropic changes can mean a lot, too.
I also mentioned molecules finding a pathway to binding, and that’s something that we don’t think about as much, either. We probably make things all the time that would be potent binders, if they just could get past some energetic hump and wedge themselves into place. But there are no crowbars available; our drug candidates have to be able to work their way in on their own. The can’t-get-there-from-here cases come back from the assays as inactive. The tendency is to imagine these in the binding site already, and to try to think of what could be going wrong in there – but it may be that they’d be fine, but that their structures won’t allow them to come in for a landing.
Picturing this accurately is very hard indeed. We have enough trouble with good representations of static pictures of our molecules bound to their targets, so making a movie of the process is a whole different story. Each frame is on a femtosecond scale – molecules flip around rather quickly – and every frame would have to be computed accurately (drug structure, protein structure, and the energetics of the whole system) for the resulting video clip to make sense. It’s been done, but not all that often, and we’re not good at it.

13 comments on “Floppiness Is Not Your Friend: Who Knew?”

  1. milkshake says:

    Sometimes I like to whisper to my molecules: “My little honeydew, in this flask no-one will hear you scream”
    By the way you will notice that majority of natural compounds with a strong bioactivity happen to be cyclic

  2. Ty says:

    Interesting thought, the ‘can’t get there from here’ case. I think that’s part of the kinetics of binding. In corollary, you might need to think about the ‘can’t-get-out-of-here’ cases, too, which is thought to be the more important factor (off-rate) for biological activities (not just enzyme inhibition). In that sense, energetics – making molecules happy upon binding, no matter what paths they may have taken (kinetics) – may be good enough a factor to consider in most cases (happy binding -> slow dissociation -> better potency). The femtosecond feature film of binding, even if it’s possible, may not do much good in terms of finding a better molecule. Of course seeing such things appeals greatly to human curiosity and I bet someday somebody will (claim to) be able to release it.

  3. qetzal says:

    Then, of course, there are the entropic changes that come when solvent molecules or ions are displaced by drug binding….

  4. “…but then, a lot of organic chemistry would be pretty unspeakable if molecules had feelings.”
    That line is a classic!
    Regarding entropy, remember that it is a thermodynamic concept, hence the concepts of “order” and “disorder” correctly apply to energy, not positions. (That’s why the units for S are J/K.) Certainly “position/conformer analysis” can be used, but it is not the final analysis.

  5. Sili says:

    “Floppiness Is Not Your Friend: Who Knew?”
    Well, Pfizer (and my spamfolder) for a start.
    It sounds like the trouble with my labwork was a lack of mad, sadistical laughter: “No, I expect you to break, mr Bond.”

  6. Dlib says:

    The movie is possible, but pharma ain’t interested. I checked ( or maybe my sales pitch isn’t working on the right people ). The scientists I’ve talked to seem interested but the people who make the real decisions to allocate resources don’t seem to care much about this topic — to the detriment of their companies IMHO.
    The floppiness has real implications as it relates to things like drug resistance. by minimizing entropy loss ( rigidification / pre-shaping ) you may initially improve your binding constant , but the molecule is more susceptible to slight geometric alterations of the binding pocket ( HIV protease first gen ). Sadly most of you guys use techniques that can’t help you optimize for the different parts of the driving forces involved ( entropy and energy ) rather you use an indirect measure of both together.
    BTW, the movie ( and a lot more info too ) is available on the millisecond to second time scale ( important time scales for large motions ) through the frequency dispersion of the heat capacity function.

  7. well the whole scenario is so different.

  8. S says:

    This reminds of a few seminars I attended, where folks went trigger-happy with cyclizing the supposed floppy parts of their modestly active compounds. This without even realizing that all those double bonds and aromatic systems in their molecule made the structure rigid-enough. It must be a more pre-meditated approach than just forming a ring.

  9. CMC Guy says:

    Some of this is reminiscent of the recent Noisy Numbers post in that we as Chemists mostly tend to view/deal with things very precisely (i.e. Structural flexibility) however as things move to isolated targets (enzymes), models (cell assays) and then ultimately whole systems (animals and people) where variability and complexity become so much greater there is a mismatch between the capabilities we started with. Seems one can both over-design or under-design molecules but frequently in the end serendipity will play a role in leading to a viable drug and then typically still won’t fully understand how it actually works or may cause certain side effects.

  10. Boghog says:

    Reducing the entropy and enthalpy penalties are clearly two potential benefits.
    Another potential benefit as proposed by Daniel Rich is that rigidity can prevent hydrophobic collapse and thereby increase binding affinity.
    A flexible core will allow hydrophobic substituients to associate in aqueous solution which reduces the contribution of hydrophobic effect to binding. Conversely forcing these substituients apart in solution so that they are maximally solvated will increase hydrophobic driving force for binding.

  11. Anonymous BMS Researcher says:

    A cool way to demonstrate entropy changes in action is a trick with a rubber band that I learned in graduate school.
    Hold it against your lips and stretch it quickly: you will feel its temperature rise. Now keep it stretched and move it away from your lips. Wait a few minutes to let it re-equilibrate to room temperature, then bring it back against your lips and rapidly release the tension while holding it against your lips. It will feel cold, demonstrating the thermodynamic nature of rubber elasticity.
    A metal spring will not do this, because the elasticity of metal is due to bonds being strained. But rubber is an elastomer, lots of long molecules in a disordered state: stretching it puts the molecules into a more ordered = lower entropy state.
    For an even more bizarre demonstration of the thermodynamics of rubber, see this item from the Feynmann lectures on Physics.

  12. Anonymous BMS Researcher says:

    Ooops, forgot to paste in the URL:

  13. JK says:

    An essay from earlier this year by Roald Hoffmann and Henning Hopf begins:
    “From the time we first got an inkling of the geometries and metrics of molecules, the literature of organic chemistry has contained characterizations of molecules as unstable, strained, distorted, sterically hindered, bent, and battered.[1] Such molecules are hardly seen as dull; on the contrary, they are perceived as worthwhile synthetic goals, and their synthesis, or evidence of their fleeting existence, has been acclaimed.
    What is going on here? Why this obsession with abnormal molecules? Is this molecular science sadistic at its core?”
    Angewandte Chemie International Edition, Volume 47, Issue 24, Pages 4474-4481. Published Online: 16 Apr 2008

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