Community symposium on molecular basis of ME/CFS at Stanford Discussion Thread

[Murph]

The possible problem, though, is that the Michaelis-Menten chart that Phair presented appears to indicate they may be giving IDO2 a much greater affinity (~200 μM) for tryptophan than what the literature is saying (i.e., ~7000 to 9000 μM).

Like you, I could find little in the literature that suggested humans ever actually use IDO2. (More precisely, I found nothing, but I didn't read all the literature!) Now, it is highly evolutionarily conserved, but perhaps there is another reason for that.

So the mystery of whether IDO2 mutations matter persists.

I am not yet convinced by the SNP frequency in the severely ill study. 55% (sample) vs 42% (population) frequency in a common mutation with a sample of 20? While other genes affecting the same enzyme show a lower frequency than the population?

Bonferroni: LOL.
Bayes:

(being snarky here but I do acknowledge that Phair is no slouch!)

Anyway, in my experience he loves an email about his theories, and I expect he has got many far stupider questions than yours, @nandixon. (Although he may be suffering more inbox pressure now than pre-symposium!) Why not drop him a line and let us know if he can explain why that flux graph looks as it does? You can find his email in this post by Cort. (I've been told bots roam this site harvesting emails so I'm not posting it here.)

 

[nandixon]

...I could find little in the literature that suggested humans ever actually use IDO2.

There are a number of papers/studies on IDO2’s potential immunomodulatory and regulatory capabilities. For example, try this search string.

 

[raghav]

Is it possible to find out with what copaxone reacts in the unhealthy serum and (cation or anion, whichever the case may be) and separate out that ion and identify what it is ? I am making it sound too simple.

 

[Jenny TipsforME]

they may be giving IDO2 a much greater affinity (~200 μM) for tryptophan than what the literature is saying (i.e., ~7000 to 9000 μM). If that's true, then there’s likely no metabolic trap, per se, because even a perfectly working IDO2 enzyme isn't likely to help enough because its ability to catabolize tryptophan is so poor at physiological levels. But like I said previously, maybe I'm misinterpreting something.

That does sound like a problem. Is it perhaps more to do with how IDO1 responds to 8 weeks of a stressor? I think without this trigger we don’t expect the IDO2 variants to cause any noticeable problems.

[/url]



The computer model indicates a clear difference between a 10 day stressor and an 8 week stressor. Perhaps difference between a cold and EBV infections. EBV/glandular fever is the classic ME trigger.

Is this relevant?

Abstract
Indoleamine 2,3-dioxygenase (IDO1) catalyzes the first step in tryptophan breakdown along the kynurenine pathway. Therapeutic inhibition of IDO1 is receiving much attention due to its proposed role in the pathogenesis of several diseases including cancer, hypotension and neurodegenerative disorders. A related enzyme, IDO2 has recently been described. We report the first purification and kinetic characterization of human IDO2 using a facile l-tryptophan consumption assay amenable to high throughput screening. We found that the Km of human IDO2 for l-tryptophan is much higher than that of IDO1. We also describe the identification and characterization of a new IDO1 inhibitor compound, Amg-1, by high throughput screening, and compare the inhibition profiles of IDO1 and IDO2 with Amg-1 and previously described compounds. Our data indicate that human IDO1 and IDO2 have different kinetic parameters and different inhibition profiles. Docking of Amg-1 and related analogs to the known structure of IDO1 and to homology-modeled IDO2 suggests possible rationales for the different inhibition profiles of IDO1 and IDO2.

Highlights
► IDO is an enzyme involved in cancer, hypotension and neurodegenerative disease. ► We report the first purification and kinetic characterization of human IDO2. ► Human IDO1 and IDO2 have different kinetic parameters and inhibition profiles. ► We describe the discovery of a new IDO1-selective inhibitor compound. ► IDO1 and IDO2 may have different functions and can be selectively targeted

https://www.sciencedirect.com/science/article/pii/S1570963911002184?via=ihub

A lot of the terminology is new to me. What does Km mean? Does high Km here mean less tryptophan is catabolised?
[edit don’t worry @nandixon I found your Km definition]

 

[Jenny TipsforME]

little in the literature that suggested humans ever actually use IDO2

I’ve come across papers indicating a distinctive role for IDO2, not sure if I can find again

 

[Jenny TipsforME]

@Murph @nandixon

Abstract
IDO2 is implicated in tryptophan catabolism and immunity but its physiological functions are not well established. Here we report the characterization of mice genetically deficient in IDO2, which develop normally but exhibit defects in IDO-mediated T-cell regulation and inflammatory responses. Construction of this strain was prompted in part by our discovery that IDO2 function is attenuated in macrophages from Ido1 −/− mice due to altered message splicing, generating a functional mosaic with implications for interpreting findings in Ido1 –/– mice. No apparent defects were observed in Ido2 –/– mice in embryonic development or hematopoietic differentiation, with wild-type profiles documented for kynurenine in blood serum and for immune cells in spleen, lymph nodes, peritoneum, thymus and bone marrow of naive mice. In contrast, upon immune stimulation we determined that IDO1-dependent T regulatory cell generation was defective in Ido2 −/− mice, supporting Ido1–Ido2 genetic interaction and establishing a functional role for Ido2 in immune modulation. Pathophysiologically, both Ido1 −/− and Ido2 −/− mice displayed reduced skin contact hypersensitivity responses, but mechanistic distinctions were apparent, with only Ido2 deficiency associated with a suppression of immune regulatory cytokines that included GM-CSF, G-CSF, IFN-γ, TNF-α, IL-6 and MCP-1/CCL2. Different contributions to inflammation were likewise indicated by the finding that Ido2 −/− mice did not phenocopy Ido1 −/− mice in the reduced susceptibility of the latter to inflammatory skin cancer. Taken together, our results offer an initial glimpse into immune modulation by IDO2, revealing its genetic interaction with IDO1 and distinguishing its non-redundant contributions to inflammation.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4432394/
Bold mine

 

[Jenny TipsforME]

Also

Indoleamine 2,3-dioxygenase-2 (IDO2) is one of three enzymes (alongside tryptophan 2,3-dioxygenase and indoleamine 2,3-dioxygenase (IDO1)) that catalyse dioxygenation of l-tryptophan as the first step in the kynurenine pathway. Despite the reported expression of IDO2 in tumours, some fundamental characteristics of the enzyme, such as substrate specificity and inhibition selectivity, are still to be clearly defined. In this study, we report the kinetic and inhibition characteristics of recombinant human IDO2. Choosing from a series of likely IDO2 substrates, we screened 54 tryptophan derivatives and tryptophan-like molecules, and characterised the 8 with which the enzyme was most active. Specificity of IDO2 for the two isomers of 1-methyltryptophan was also evaluated and the findings compared with those obtained in other studies on IDO2 and IDO1. Interestingly, IDO2 demonstrates behaviour distinct from that of IDO1 in terms of substrate specificity and affinity, such that we have identified tryptophan derivatives that are mutually exclusive as substrates for IDO1 and IDO2. Our results support the idea that the antitumour activity of 1-Me-d-Trp is unlikely to be related with competitive inhibition of IDO2, and also imply that there are subtle differences in active site structure in the two enzymes that may be exploited in the development of specific inhibitors of these enzymes, a route which may prove important in defining their role(s) in cancer.

https://link.springer.com/article/10.1007/s00726-014-1766-3

 

[Jenny TipsforME]

@nandixon it does sound like the IDO2 SNPs do impact tryptophan catabolic activity (somewhere quoted as 90% less effective). Have they also knocked out IDO1 function when they test this?

IDO2 in Human Studies
In human immune physiology, the implication of a genetic linkage between IDO1 and IDO2 is intriguing in light of the broad distribution of two functionally attenuating SNP in the coding region of the IDO2 gene in human populations (3). These SNP variations dramatically reduce or abolish tryptophan catabolic activity, therefore varying the level of this IDO2 function in different individuals, perhaps affecting T-cell-dependent immune control as a result.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4238401/

 

[Jenny TipsforME]

There might be methodology issues

Enzymatic studies demonstrate that IDO2 is significantly less active, particularly human IDO2, though it remains to be seen if the proper substrate and/or enzymatic assay conditions have been correctly identified, as IDO2 activity toward l-Trp and various Trp derivatives is both highly pH and buffer dependent.5–7 Unlike IDO1, which efficiently catabolizes l-Trp, there are several Trp derivatives, particularly 5-methoxytryptophan, that are more efficiently catabolized by IDO2 than Trp itself.5 Underscoring the murky relationship between IDO2 and Trp catabolism, deletion of IDO2 in mouse models does not alter serum kynurenine levels, even under chronic inflammatory conditions
...There is, however, some evidence that IDO2 can affect kynurenine levels in some contexts.6,7,17

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5119657/

(And we are thinking of a specific context rather than everyone who has IDO2 SNPs) Reference 17 from that quote is

Accordingly, kynurenine concentration decreased after silencing either enzyme as compared to the control CTR siRNA (Figure 6F), thus demonstrating the active function of both IDO1 and IDO2 in this setting... IDO2 expression confers to (Human blood dendritic cells) DC tolerogenic features, such as the capacity of starving kynurenine and of generating Tregs.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3905741/

confusing :thumbdown: my brain may implode but I’d like to understand

Does anyone have references about the IDO1/tryptophan response to 8 week+ stressors? I think that’s the key element we haven’t addressed enough in discussion. If the trap exists it has to be sprung.

 

[jaybee00]

@melihtas

What is the cost and availability of Copaxone and generics in Turkey?

Thanks!

 

[melihtas]

@melihtas

What is the cost and availability of Copaxone and generics in Turkey?

Thanks!

COPAXONE 20 MG/ML 28 Syringes = 1727 TL = 282 USD = 246 EUR
Generic 20 MG/ML 28 Syringes = 1048 TL = 171 USD = 149 EUR

You cannot buy it online and nobody can ship it to you from Turkey. Customs doesn't allow any drugs in or out via post. The only way to get it out of Turkey is to bring it in your luggage.

 

[Sidereal]

It doesn't sound as though the IDO2 knockout mouse experiences crippling fatigue.

 

[Jenny TipsforME]

It doesn't sound as though the IDO2 knockout mouse experiences crippling fatigue.

But did they experience 8 weeks of a continuous stressor as a trigger? That is the key element with this hypothesis re the difference between healthies walking around with IDO2 variants and people stuck in a trap.

In a sense it’s almost like the IDO2 variants don’t matter (so many people have them). Except that it seems like you maybe don’t get ME if you don’t have them. ‘Typical IDO2 is protective against ME’ might be a better way around to describe it? IDO2 variants on their own don’t result in ME but if you don’t have the protective ones you might be in trouble when you encounter a strong trigger such as mono.

 

[jaybee00]

@melihtas thanks.

Are the 40 mg syringes available also? The non-generic copaxone is Teva brand?

Thanks again

 

[melihtas]

@melihtas thanks.

Are the 40 mg syringes available also? The non-generic copaxone is Teva brand?

Thanks again

COPAXONE 40 MG/ML 12 Syringes = 1951 TL = 319 USD = 278 EUR

The generic brand is GLIA from a Turkish company. They have only 20ML.

 

[jaybee00]

Thanks.....that’s strange though because In the USA 12 syringes of 40mg is cheaper than 30 of 20

 

[sb4]

@Jenny TipsforME Fascinating stuff. Great work. Keep it up.

 

[Sidereal]

But did they experience 8 weeks of a continuous stressor as a trigger?

Given that rodents have a far shorter lifespan than humans you probably wouldn't need anywhere near to 8 weeks of continued stress exposure to trigger the metabolic trap, if such a trap exists. I would like to see these researchers develop an animal model of disease.

 

[Lisa108]

'Targeting the Broadly Pathogenic Kynurenine Pathway'
Sandeep Mittal (ed.), Springer (2015), 430 pp.

Anyone read this already? Just skipped a few pages, so not sure if a recommendation.

If your library doesn't have a copy, let's just say a certain russian lib has one...

P.S. DR. DAVIS'S WARNING (just a reminder to all the potential smugglers on here...)

 

[wigglethemouse]

'Targeting the Broadly Pathogenic Kynurenine Pathway'
Sandeep Mittal (ed.), Springer (2015), 430 pp.

Anyone read this already? Just skipped a few pages, so not sure if a recommendation.

What an excellent resource. It links all the main studies together in one place and covers the vast kynurenine pathway in many bodily systems. Some good info in there, especially if you happen to be a mouse or a rat! I hope Robert Phair has a copy!