That reply clarified a lot about this proposal. It’s encouraging to see there is an assay in hand, and they have some leads.
However, I’m concerned about the assay. In the sample assay in the google doc with tannin, the “protection” comes from an increased background level, not a decrease in absorbance. This runs the risk of being a false positive. The ELISA is concerning because it has a low dynamic range (the max absorbance is 0.1). And it’s not clear if the ELISA is linear within that range. I would also note that most IC50s were extrapolated, which increases the error in the assay. Properly, the IC50s for most are <4.8 ug/mL. A second assay that is more robust seems needed to validate hits and confirm estimated IC50s.
The MTT assay has low precision, which is concerning. I do not think a ‘cytovitalic’ action can be concluded here. Instead, I would interpret negative numbers to indicate that extracts interfere with the assay. That means you’ll need a different assay to validate cytotoxicity. LDH is a terrible viability assay, so would recommend against that one. You might be able to do calcein release in a plate format, though.
This still looks unfocused to me. There are many things that elevate cytokines, and the contribution of AGEs in the ECM to inflammaging and other phenotypes is questionable. These end up being different questions than ‘how do these plant extracts interfere with AGE accumulation?’ and measure potential downstream effects. Since RAGE is a therapeutic target, checking impacts on RAGE seems higher value to me than some of the others.
This is where I would expect mechanistic studies to start, and then get more physiologic.
Bone-marrow derived macrophages might give you better numbers for scaling up- you can expand them in GM-CSF for 21 days and freeze them down after day 7. Or is the purpose of using the peritoneal macrophages because you elicited them with peptone (which has AGEs in it)? If that’s the case, why not aged thioglycollate medium?
It may be a communication issue, but this sentence indicates to me that the library is not ready to go. The raw materials are available, but the extracts that will be used are not yet ready.
Based on the time calculations, it’s not clear if the screen will go in 384-well plates or not. With a dedicated tech, and careful strategy (screen high concentration on all of them first, and eliminate any that fail to show activity there), you might be able to cut the number of plates you use down.
I’m unconvinced this is a problem. It seems like it would distract from the science. If VitaDAO wants to fund videos, that would be better done as a seperate initiative. If you were submitting to the US National Science Foundation, though, this might be a reasonable Broader Impact.
I think $30k was what was quoted to me by my TTO for the cost of filing just in US and Europe. Additional countries cost more. However, others in VitaDAO have more experience on this end than I, and will know precise amounts. Maybe it was just cost for filing the provisional patent?
Hello, @MAC! Partially getting rid of glycated blood proteins has its benefits for sure. Primarily, circulating AGEs act on their cognate receptors – RAGEs. These receptors belong to the immunoglobulin family and are dedicated to sensing so-called damage-associated molecular patterns (DAMPs). AGE-dependent activation of RAGEs stimulates oxidative stress and expression of pro-inflammatory markers, so discarding glycated blood components is undoubtedly useful. However, removing AGEs from the extracellular matrix is by far a more complex task, hence the present proposal. I hope I answered your question.
@bowtiedshrike Members of the BostonMatrix team would like to express their gratitude for your effort in reviewing their proposal. Currently, the team is looking forward to collecting community feedback and will respond and introduce amendments to the text in bulk.
Separating popularization and research projects has always been a rule of some sort. We’ve tried it. By we I mean all of us as a field. And I wouldn’t say we succeeded much in shifting the mindset of people, in forming the vision of longevity research. And we do need that for the field to grow, to grow fast enough for us to see the results of our work (we are not arguing about that, right?). The old ways of popularization, of promotion, of explanation don’t really work. We haven’t found the right format (among other reasons, it’s actually not that easy topic to cover in one paragraph, we do need to look, to experiment in the life-extension-popularization field too).
So what we propose is the new format: unite the actual research and a movie about it. Make it real. Show people how REAL it is. It’s not somewhere behind close doors, it’s very transparent, and anyone is welcome to join and help. Most people can only guess how the research itself looks like, what are the day-to-day obstacles, routine practices, how does the result and the intermediate result look like and thus, what is the possible pace of research.
I believe VitaDAO was created to build an active community. One tool for it is to publicly post scientific proposals and then publicly discuss them. Exactly what we are doing right now. But here we are: only 6 people voted and 4 people joined the discussion. The very discussion that will affect the protocol of the experiment and its chances to be executed at all. This exact discussion and these discussions in the field as a whole need scaling. And for this we need new formats, new ways of getting people engaged. That’s why the movie would be an important part of research.
I have mixed feelings on popularization in general. I would say popularization needs to be targeted, with a measurable effect, and that researchers tend to be the worst at marketing and sales. Selling extended life to the boomers ruling most countries and controlling governmental purse strings should be closer to selling heroin to junkies than ice to Alaskans.
If the plan is to do marketing, we need a clearly defined marketing campaign and budget. I suspect there are many Bowties that would do a great job developing resources if the pay was right. VitaDAO has also been active with media creation, so may not need outside help.
If the need is more people doing proposal review, I don’t think you’ll get what you want from a fancy video. Most lay people lack the expertise to evaluate the proposals, and will trust experts they otherwise trust. Some would rather read the discussion and use that to inform their decision, even if they don’t have anything to add.
Targeted recruitment for early career faculty or TTO personnel (and $Vita) would help get outside reviews on the proposals. That’s a cold email campaign, though, not a video campaign.
The other big challenge for decentralized organizations is time. Poll the VitaDAO membership and see how much time they are willing to invest in VitaDAO, between reading posts to stay up to speed, to evaluating different things. Each of the project proposals takes an hour or more to read, digest, and evaluate.
With a few exceptions, the most engaged people are already in Working Groups, where these ideas received a round of evaluation already. Most of them do not comment except to clarify, presumably because they don’t want to bias the community’s evaluation.
Though we are in the VitaDAO space right now and I did a reference about our current conversation and campaign, this was just a mere example of what’s happening in the field in general. I actually don’t expect more interest and expertise from within the existing community and don’t expect the movie to work for that cause. We are all stretched pretty thin already. The question is how to grow the field in general.
We’re in desperate need of exponential growth of fundamental research. Meaning governmental funding. Meaning a massive shift in distribution of budgets. Meaning life extension has to win as an idea above any other. Win over the public, win over the people in power. Also, not only should we talk about “life extension is good”—this reasoning doesn’t seem to be working—but also about New Normal. Spending your lifetime trying to conquer death is the new normal and the only reasonable thing to do. Anything else is not cool. How does a life of such person look like? Here, the movie will show you (and hopefully many other movies and other engaging and immersive media will follow). It just seems like a waste not to put on camera the people who are already doing what everyone else is supposed to be doing with their lives (not being researchers necessarily but being part of the movement).
And yes, I absolutely agree that we need a measurement system, a KPI for popularization. And I suggest to work out an index for the life extension field in general: does it grow or not? Does the current fuss serves our goal of life extension? Do our efforts in enlarging the field work? Should we be optimistic about the current state of things or not? Different specialists in the field vary in their estimations, and all of them rely on their intuition. But I don’t want for it to be a competition of “I told you so” (which it comes down to eventually). I need to know properly in advance. I need the metric—our lives are at stake here. The metric could be a combination of different indexes or something fun like The Big Mac Index. A number of startups plus the budget of Buck Institute plus the number of TV shows, which portray radical life extension positively plus a number of politicians standing for the growth of research budgets plus… I’m just randomly naming things here, obviously we can’t just summarize it all. But I hope you get my point.
So my proposition (not within this research proposal but since we’re discussing this) is to work out the index for the field, create content on the new ethics, and see how one effects the other.
Going back to the video content: people tend to believe what they understand. And visa versa. If they don’t understand how life extension research work, they ignore it. If they see it and it looks doable, it inspires them to take part, to sponsor, to start their own research, to learn more. And the video made from within is just more sincere and more accurate. And yes, of course, not the researchers themselves are to be making the production—there are specialists to hire (director, editor, camera people…). It’s just we should be in control of the end message here.
@everyone
The VDP has been updated with details on diabetic cardiomyopathy as a surrogate indication and expanded on the methodology. Since the text has been changed considerably, votes have been reset. Please vote again if you think the the project should be funded with the requested amount.
As had been discussed at longevity dealflow working group meetings and in the designated thread in the WG Discord channel, in order to critically evaluate the project’s feasibility and attractiveness in terms of fundability, the applicant team has been asked to show proof-of-concept (PoC) efficacy of extracts in an in vivo model. Consequently, the team is asking $30k (see Experimental plan and Financing and milestones sections) to conduct a short PoC study in an experimental glycation animal model.
Hello, @bowtiedshrike! Please see below the applicant team’s response to your comments:
Protection might seem to derive from an increased background level. However, the direct correlation between the background value and tannin concentration in non-glycated samples indicates the reliability of the result. There is no such relationship for glycated samples. Perhaps 100 uM of tannin (high concentration) disturbs the spatial organization of the protein, contributing to the nonspecific adhesion of labeled antibodies. We believe that blank subtractions are a reliable way to filter out this artifact.
The discussed ELISA protocol is preliminary and will be validated further. ELISA parameters may be revised to improve data quality.
A repeat study will be performed. Initial screening of extracts is conducted in a concentration range of 48-4.8 ug/ml, after which the investigated concentrations are reduced to 0.48 ug/ml (or lower) for potential lead extracts. The range of investigated concentrations in primary screening can exceed two orders of magnitude.
The low accuracy of the results for the extracts that exhibited a controversial “cytovitalic” effect may be related to the optical density values exceeding the linearity range of the test system. At the same time, for quercetin, which expressed cytotoxicity, the variability was within the acceptable range.
To determine potential cytotoxicity in vitro, we usually use a combined approach that includes an assessment of the effects on cell growth and metabolic function:
assessment of morphological changes (Diff-Quik staining).
assessment of cytoplasmic membrane integrity (LDH test, trypan blue test, we also consider calcein test that you have recommended).
evaluation of metabolic functions of the cell (MTT-test in two modifications: under microscopy control and by spectrophotometry)
The above protocols aim to detect physiological and/or morphological changes that may have occurred as a result of cytotoxic action.
The conclusion about the “cytovitalic” effect is controversial and requires verification. However, it is unlikely that the result is due to interference from the extracts. We remove the medium containing the extract and, in some cases, perform a series of cell washings before introducing the MTT reagent. To verify the result, we are going to perform a modification of the MTT test with microscopy detection. This implies mandatory microscopic control of formazan crystal formation, and only samples with intracellular crystal localization will be selected for further spectrophotometric evaluation, which will exclude the possibility of nonspecific interaction of the extracts under study with tetrazolium salts in the extracellular space.
In case of confirmation, we will consider the result as a certain qualitative sign indicating a possible “cytovitalic” effect of the extract components, which, however, will require further study of the mechanisms of the effect. For some plant polyphenols (ellagitannins, urolithin A), the ability to enhance mitochondrial activity and stimulate mitophagy was previously shown [link]. We cannot rule out a similar effect from some components of our extracts.
Many factors contribute to cytokine levels, but according to our understanding, AGEs, as well as matrix stiffness contribute significantly to the inflammatory phenotype through pattern recognition receptors [link], Hippo pathway [link], and other mechanisms. The view that AGEs and inflammation are linked is supported not only in scientific publications, but also in various projects [link]. Some interleukins are considered as markers of inflammation (e.g. IL-6 [link]), and causality of AGEs for markers of inflammation (e.g. IL-6 and IL-8 [link]) is described. Thus, we plan to evaluate the ability of our agents to suppress the AGE-induced inflammatory cell response (regardless of the detailed mechanism of the effect). One of the mechanisms of such action may be RAGE blockade. We recognize the importance of studying RAGE for understanding the mechanism of action of the agents, and are considering including it in the plan. However, the original exclusion of RAGE was because RAGE is a target whose development may require reevaluation of the financial plan. The question of including RAGE is not a question of whether or not we will include it in the study design, but it is a question of at what point in the research we will include it. However, we note that isolated blockade of RAGE may not be sufficiently effective (e.g., azeliragon clinical trials link).
The idea of reducing matrix stiffness through an antiglycation action is based on the fact that we prevent the glycation of proteins that have a long (but not super long) turnover period. The turnover period of such proteins is sufficient for formation of AGEs and cross-links, but it is not counted in years or decades. Such proteins are usually found in the structures of the pericellular matrix – laminin, type IV collagen, etc. (their turnover periods are as follows [link]). If these proteins are protected from glycation, the AGE load of their new generations will be lower, and therefore the associated stiffness and DAMP-potential less pronounced. The rate of matrix stiffness reduction, like the rate of matrix clearance from AGEs, will be proportional to the turnover period. This approach can be regarded as independent, but due to its partial nature, we consider it as a step in a complex therapy to restore tissues from the effects of glycation and aging. Subsequent steps, the implementation of which is difficult without reducing the AGEs load in ECM, include restoration of the synthetic activity of resident matrix cells, restoration of the stem cell population and their niches, senolysis, and stimulation of the turnover of extra-long-lived proteins. All named steps are considered by us as part of our master strategy [link].
We can offer another marker of matrix stiffness reduction that is promising specifically for plant polyphenols. Plant polyphenols can inhibit hyaluronidase [link], thereby slowing down the breakdown of hyaluronan. Hyaluronidase inhibition kits are available [link]. What do you think about including this marker in our application?
The choice of peptone was based on reliability, simplicity, and in order to save costs. At the same time, if we are able to secure additional funding, we can change the experimental protocols, in particular consider the use of bone marrow-derived macrophages, use other activators and colony-stimulating factors.
At the planning stage of the study, we did not consider the connection between the mechanism of macrophage activation by peptone and the presence of AGE in peptone, but we will consider the potential significance of such a connection in future work. The first step will be to investigate the peptone used for the presence of nontryptophan fluorescence (fluorescent AGEs) in it. Perhaps its role will prove to be significant.
We agree. This is the way we organize our project.
The raw material is ready for the extraction procedure, but the extracts are prepared in sequence as soon as we are ready to take a new batch into the screening. The extraction procedure takes 24 hours, and it takes some time to determine the concentration of the extract. Thus, the extraction procedure is not a time-limiting stage of the project. There is enough raw material in stock to prepare quantities of the extract sufficient for brief in vivo studies for lead candidates. In addition, there is the possibility of determining plant ITS markers for clear species identification if more raw material is needed. These factors contribute to extraction costs.
During the screening, we use 96-well plates, although we can technically increase the number of wells to 384. The plan to examine all extracts for activity over ~5-month period is not based on a predictive calculation, but on real estimates of the throughput of the test system we had performed earlier.
We would appreciate to have your detailed feedback on what costs we might incur and how to put them in an optimal sequence, as patenting is not our strongest side.
On behalf of OpenLongevity and the BostonMatrix Team, we thank you for your questions.
We will continue to collect feedback from the VitaDaO community to improve our project further.
Hello BM team - can you clarify per stage 3 if the extract structures are not amendable to synthesis you propose to move to medical chemistry to explore a traditional small mol approach?
As we discussed live, in general I am not a fan of natural products. However this project becomes interesting if at minimum we gain understanding of MOA which can be used to launch discovery using standard drug like modalities. At best, one of the extract components can be synthesized easily & offers an immediate path forward.
Also, I do not understand the need for a promotional video….
Hello, thank you for the interest to our proposal!
If the first-best compound is not amendable to synthesis, we will select second best, third best etc. We plan to assess 2500 extracts, which increases the chance of finding several leads. However, we agree that it is sensible to add this point into “Risks” section of this application.
Should all lead compounds not be amendable to synthesis, it is possible to use the lead structures as the blueprint for synthesizable drug-like molecules. We can narrow the pool of predicted drug-like molecules by testing them for antiglycation activity using our proprietary in silico methods [published here] and by in silico ADME-Tox screening. This approach will result in a novel synthesizable drug-like component with properties close to the nature-derived lead.
We also note that there is a chance that the lead can be synthesized at scale by biocatalysis. There are known cases of applying this approach to synthesize several flavonoids [1, 2].
We agree that there is a high chance of finding at least one lead. We planned to uncover MoA of lead compounds in glycation reaction (transglycation, carbonyl compound binding, antiradical action, binding of transition metals etc). These trials are planned at Stage 1 (for extracts) and Stage 3 (for isolated compounds) of this application. In addition, we will conduct phenotypical screening to assess the compound’s propensity to suppress glycation-related pathological processes. Moreover, we are interested in further exploring MoA of our lead compounds.
As we proposed in our follow up [link] to live discussion, after identification of the active compound it can be immobilized on resin or magnetic beads and incubated with homogenized tissues. Further, captured molecules will be subjected to mass spectrometry and chromatography. These kinds of experiments are promising and results might be very interesting but it will require additional funding. We would expect to see such targets as kinases of NFkB or MAPK pathways, as these were previously identified to be involved in AGEs-associated pathogenesis, and also we hope to see GLO, PARK7, or AKR enzymes as endogenous glycation suppressors.
The establishment of detailed mechanisms of antiglycation activity represents considerable interest for us, and we are interested in identification of other targets as well. This justifies multiple tests for different types of antiglycation activity that we consider to qualify as a detailed analysis of the mechanism of action.
Thanks a lot for your questions, and have a great day.
Hi! Well, if the movie idea continues to raise questions, we’re not insisting on it within this fundraising campaign and can drop it. But we still like it and still would like to pursue with it (we’ll just have to look for the funding for the movie elsewhere). As to why, to summarize my longer previous answer, I think we have to use any given chance to expand the field in general, and making the movie about the research helps promote it and explains its inside to the general public. Personally, I would love to see the movies about all the ongoing research in the field: to meet the teams, understand their reasoning better, see the designs and the routines of the experiments explained, understand the difficulties, the struggles, how much does and doesn’t rely on human factor and on the accessibility to resources, tools, and logistics, to estimate the chances of success… I really think that all this will help with funding for the field in general. But again, we are not pushing.
Sorry if this has been asked elsewhere but I’d like to see funding the 3rd and possibly 2nd milestones contingent on other funding sources coming in. $300k is becoming more than vitadao will typically invest. Also it is important to show traction with multiple funders.
We would definitely need to find a lot more additional funding for the project to succeed and are looking for it already (in parallel to VitaDAO). It is though quite hard, given the very early stage of the project. We would be OK with contingent funding for a 3rd milestone. Would something like a combination of charity, gitcoin, other crowdfunding, private funding and/or grants suffice or are you looking for something more specific? What sum shall we agree upon?
Seems fair @strygah. @rpill can you update the proposal to make the 3rd milestone contingent on let’s say at least 10X of VitaDAO’s 3rd milestone contribution of $155K.
@timrpeterson
Such a structure poses novel challenges, I think we need to address them.
Originally we discussed IP ownership by VitaDAO (with little co-ownership by the team trough IP-NFT), and the plan was to decide later (after this part of the project) what to do with the IP (ie license out or create a company) and what part of the team and with how much involvement will continue working on the bringing the candidate through the development pipeline.
However, the need to find additional capital would require us either accelerate NewCo formation or offer clear alternatives (IP-NFT participation or otherwise). Non-profit charity-like funding and crowdfunding will still probably work, but grants and especially investors would require very clear IP right ownership or company share ownership. We will be very lucky if we are able to raise 1.5M+ just from non-profit funding, and it seems to maximize the chances of success we would require turning to VC/angel route earlier. But going this route would require proper company, full founders and team involvement, and IP controlled by founders (would it?).
So it begs the question - how to properly structure the VitaDAO-BM team relationships and IP co-ownership given the new contingency requirement? Note also, according to the original plan, we would be able to generate the IP only after 3-rd Milestone and we won’t have any patent applications unless we find $150k in other sources.
Any additional thoughts on how to maximize the chances of other funds being involved? Any restrictions from VitaDAO we should be aware of?
Further to our last query on structuring the project and IP arrangements in light of raising additional funding, we would kindly ask for your advice on whether we could start preliminary communication with potential third-party investors/grantors regarding additional financing of the project (with full disclosure of and attribution priority to VitaDAO’s current granting process).
If you find such communication reasonable, would you consider it possible for us to seek financing for the same scope of research activity as indicated in our experimental plan for VitaDAO (with particular focus on stages 2 and 3) or we should rather concentrate on obtaining financing for the project’s subsequent, spin-off or adjacent research activity.
I am voting against funding it in its current form because the plan is flawed, imo.
The $30k would be stranded if nobody else is willing to support it, and if we decide not to fund the rest, why sink $30k in it?
I’d much rather we decide to fund an amount that makes sense, but make sure there are other funders coming in for the rest, either now, or after successful milestones.
If the phase 3 vote is negative, I’d love to see a new vote with a better funding plan, perhaps iteration on the experimental plan as well, and clarity on who’d spin this out and fundraise further (if VitaDAO, I’d like to know that we can find a potential CEO before funding it).
Thanks for all the wonderful work put into this, especially @rpill and the Boston Matrix / Open Longevity team!
To put this on Snapshot, it needs to be shorter, less than half fits in the max length. @rpill, can we please make a simpler version, that maybe links to more supporting material on IPFS or a google doc in the VitaDAO drive?