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A Condensate-Modifying Compound, Put to the Test

I’ve written several times here about phase-separated condensates in cells, but now comes a rarity: a paper with some evidence for a therapeutic application. Everyone in the field has been thinking along such lines, naturally, but this is the first small-molecule screen that I’ve seen that tries to tie modifying condensate behavior in that way to a disease state.

In this case, it’s the handling of stress granules in the context of ALS. Stress granules have been the focus of a lot of work in the condensate field, which is natural enough. They’d been recognized for many years in cell biology as structures that appeared and disappeared in response to heat stress, hypoxia, and so on, and recently their liquid-liquid phase-separated nature has been the focus. Like many condensates, stress granules seem to be a mixture of proteins and nuclei acids, with various RNA-binding proteins prominent in their composition. The connection to ALS is via mutations in some RNA-binding proteins (such as FUS and TDP-43_ that have shown up in ALS patients, and it’s believed that these mutants can either allow for more stress granule formation or cause them to be abnormally persistent. Protein deposition is, of course, a hallmark of the disease. It’s been observed in many condensate systems (both in vitro and in vivo) that some phases that start out as liquid droplets can “harden” over time, becoming more gel-like and harder to reverse, and even progressing to insoluble aggregates. It’s quite tempting, then, to see aberrant condensate formation (or aberrant re-formation of a single phase) as related to such disease processes.

This new paper is a multicenter effort with Tony Hyman of Dresden as corresponding author, and it’s worth noting that he’s on the founding team at Dewpoint Therapeutics, who are trying to turn discoveries in condensate biology into actual therapeutics. Now, there are compounds known that seem to modulate condensate behavior, but they’re not too drug-like. 1,6-hexanediol shows up the literature quite often as a general “condensate dissolver”, even to the point of treating living cells, but that’s not a feasible path in a whole animal from a pharmacokinetic and toxicological standpoint. You also would probably not want to just go in and dissolve every condensate in the cell simultaneously! In this case, the team screening 1600 compounds (mostly known bioactives and drugs) to see which ones affected FUS condensate behavior, but did not act as “universal solvents”.

Lipoamide and lipoic acid both came out of the screen as such compounds, testing them in HeLa cells that were engineered with a fluorescent FUS protein at roughly endogenous levels. (That’s important – sometimes to see these condensates people have had to crank up expression, which likely wouldn’t be appropriate for a screening effort). Under normal conditions, FUS is mostly in the nucleus, sometimes as part of yet another membraneless structure, the paraspeckles. But under stress, it exports back to the cytoplasm and helps to form stress granules. In this case, the stress was in the form of arsenate treatment, which messes up oxidative metabolism in general and is known to reliably bring on the stress-granule response. Lipoic acid was able to stop stress granule formation, but did not seem to affect the FUS-driven paraspeckles or other known condensate structures (such as the nucleolus itself).

The follow-up assays are quite interesting. Lipoic acid was shown to inhibit stress granule formation in living C. elegans roundworms, and to ameliorate motor defects in Drosophila flies that have been engineered to express human FUS protein instead of their own homolog. In fact, these flies had an even bigger problem; they were made to express mutant human FUS (P525L or R521C mutations), which are known to cause more severe motility problems. P525L is an already-characterized mutation in humans, and if you express it in iPSC-derived motor neurons, you get defects that are consistent with the clinical phenotype of the disease (among other things, they have a much greater tendency to form stress granules). Lipoic acid treatment prevented the axonal die-back seen in the untreated cells, and also restored axonal transport to wild-type levels, both of which were associated with a corresponding lack of stress granule pathology.

To the best of my knowledge, this is the first study that ties together condensates, small molecule treatment, and a disease state in this manner. Could lipoic acid itself be a treatment for ALS? The paper proposes that idea, but I’ll leave it up to the clinicians. You need pretty high concentrations (tens to hundreds of micromolar), and I’m just not sure if that’s clinically feasible. On the other hand, lipoic acid itself is pretty benign, and I would think that this falls into the “What do you have to lose” category. But you’d also think that you’d want to treat people as early in the disease state as possible, before more irreversible damage has had a chance to pile up – I don’t think lipoic acid (or any such compounds) is going to reverse that, but rather keep further damage from occurring. Drug discovery in this space is going to be quite an experience:

The necessary properties of a compound affecting protein phase separation are likely different to those of a canonical drug binding a well-defined structured protein site. Condensates are formed by protein liquid-liquid phase separation involving many transient interactions , unlike strong enzyme-substrate or protein-protein interactions typically targeted by drugs. Small molecules can interfere with phase separation and alter the properties of phase boundaries (surfactants). For instance, ATP has been identified as a hydrotrope which helps keep proteins soluble. The identification of lipoamide and lipoic acid suggests that further small molecules could be identified that target phase separated compartments.

And the hunt is on for them, especially now that this paper has come out. We’re going to be hearing a lot more about this sort of thing, so we’d better all get used to it. . .



21 comments on “A Condensate-Modifying Compound, Put to the Test”

  1. Oxidized says:

    I wonder how the disulfide in lipoic acid contributes to its bioactivity

    1. John Wayne says:

      Disulfides make everything better

  2. MrXYZ says:

    Do people with ALS have reduced amounts of lipoic acid?

  3. Barry says:

    When the goal is modulating phase-separated droplets, the notion a small molecule selectively binding to a biological target is out the window. Shoichet would flag most (all?) of these. The compounds mentioned are prone to aggregate in aqueous media, of course. But maybe that’s part and parcel of the effect?

  4. Kelvin says:

    So mild non-specific detergents as a drug???

    1. C_B says:

      I knew the Tide Pods kids were onto something…

      1. mfernflower says:

        Miltefosine would like to have a chat

  5. JB says:

    Novartis/Avexis just got caught manipulating data that was used to get approval for the most expensive drug in the world. And here I thought this was a problem with data fraud was due to those pesky academics only, and that industry were much better purveyors of scientific truth.

    Sorry, couldn’t help myself on this unrelated topic.

    1. anon says:

      $$$$$$$$$$$$$$$$$$$$$$$$ has always been a heck of a motivator throughout human history

    2. Diver Dude says:

      Preclinical rather than clinical but it’s not a good look.

  6. charlesj says:

    In a sad footnote, the corresponding author is the husband of the late Suzanne Eaton, who was murdered last month in Crete while attending a scientific conference.

  7. Greg Maguire says:

    Most cases of ALS are sporadic, not hereditary and probably not genetic. Instead, an exposome mediated induction of protein misfolding and prion-like spreading of the dysfunctional proteins may be causal. In a model of this process, stem cell released molecules (exosomes) have been shown to reduce stress granule formation using a “systems therapeutic” for “physiological renormalization.”

  8. one man CRO says:

    i had a very interesting time developing lipoic acid for an ophthalmic application (presbyopia). oxidative damage in the form disulfide linkages causes the lens to stiffen. that results in loss of near visual acuity and thus the need for reading glasses. lipoic acid is reduced in the eye to dihydrolipoate. the reduced species can then be reoxidized in the lens by mediating disulfide reduction. lens plasticity is restored. problem is getting lipoic acid to the lens. the acid has little corneal penetration; a problem formulation could not solve. tried numerous ester/amide prodrugs. none worked. then found a real cool way to sneak the stuff in. make the choline ester of lipoic acid. the charged quaternary ammonium allows for easy corneal penetration. once in the anterior chamber of the eye, butylcholine esterases (which are all over the place) will cleave the ester into choline and lipoic acid. pretty interesting and the chemistry to synthesize such a simple molecule was actually a surprising challenge. took more than a few labs over a year to make GMP material for trials.

    it was one of those exciting cases where the clinical trials were stopped early because the thing was working so well.

    1. Barry says:

      Sure, Lipoic acid as a redox shuttle makes sense. But I’d bet the SAR around it is really flat; the enantiomer would serve just as well. That would make it very much an outlier among small-molecule drugs.

      1. Mol biologist says:

        IHMO it is An Awful Idea to dissolve condensate or SRg until you have understood exactly what are they doing to protect the cell from stress.
        Biology rules: It reminds a story of using cathepsin inhibitors and raise stroke incidents in patients.

  9. Joe psycho says:

    That is scary to know that big pharma companies hide and prevent effective and seemingly very safe drug candidates from further development and helping people (and still profiting from it) please tell me more about other potential purposefully hidden helpful drug candidates especially asthma treatments(email is fake)

    1. Derek Lowe says:

      Ah, you misunderstood. That trial was stopped early because it worked so well, and the compound was then approved by the FDA. If a compound is clearly working very well in trials, and it’s generated enough data to be approvable even though the trial hasn’t completed, the advisory board can recommend that it would be unethical to continue gathering more data instead of going ahead and submitting the data to the FDA.

      1. Joe psycho says:

        I’m sorry for wasting your time , but I misunderstood due to the ambiguous wording at the very end and corruption in the pharmaceutical industry is a concern of mine.(again email address is fake)

  10. Paul Zhang says:

    How much research has been done on condensate involvement in other neurodegenerative diseases e.g. Alzheimer’s? Seems like it might be quite interesting.

  11. PSW says:

    They used arsenic as the stress inducer. The reason arsenic is so toxic to respiratory metabolism is that it forms irreversible complexes with the lipoic acid component of key enzyme complexes such as pyruvate dehydrogenase and ketoglutarate dehydrogenase. If one adds extra free lipoic acid or lipoamide, one would expect that it would bind to the arsenic, thus reducing the effect on the dehydrogenases. A chelating agent for arsenic, in effect. So it doesn’t surprise me that lipoic acid reduced the stress effect.

    Does lipoic acid have any effect on stress not induced by arsenic poisoning? Am I missing something here?

    1. Preprint says:

      Quote from the preprint: “Lipoamide action in cells is non-enzymatic and non-antioxidant
      The stressor, arsenate, will react with the thiol groups of lipoamide. Therefore, to exclude the possibility that lipoamide is acting only by removing the stressor we tested whether it can prevent stress granule formation triggered by other non-arsenate stresses: Mitochondrial electron transport chain inhibition (rotenone), heat stress (42°C), hyperosmotic stress (sorbitol, a non-metabolisable sugar), glycolysis inhibition (6-deoxyglucose in the absence of glucose) or serum starvation. 10 μM lipoamide reduced stress granule formation in HeLa cells with mitochondrial, hyperosmotic or arsenate stress but not heat or glycolysis stress (Figure 3A). Lipoamide is therefore not only reacting with arsenate but reduces stress granule formation under several cellular stresses.”

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