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Journal Club Presentation 11/8/19

Journal Club Presentation

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Nourianz (istradefylline)

10 Comments

  1. The purpose of this paper is to describe the discovery and characterization of μ-opioid receptor PAMs and silent allosteric modulators. Due to various issues with current opioid agonists such as tolerance, constipation, dependence, allodynia, and various others, novel ligands that act in alternative ways can prove beneficial in better treating pain without some of the less desirable side effects. One potential alternative to the common orthosteric opioid agonists is allosteric modulators. Allosteric modulators can be positive or negative depending on whether they enhance or inhibit efficacy or affinity of an orthosteric agonist. There are also silent allosteric modulators which have no direct effect on the active site but will competitively inhibit both PAMS and NAMS. PAMS in particular show potential in reducing some of the downstream compensatory effects associated with building of tolerance and dependence. An HTS campaign was carried out to identify potential opioid receptor ligands and two ligands, BMS-986121 and BMS-986122, were identified. To assess BMS-986121 and BMS-986122’s μ-PAM activity three functional assays were carried out: β-arrestin recruitment, inhibition of adenylyl cyclase activity, and G protein activation. Concentration–response curves for endomorphin I–mediated recruitment of β-arrestin indicated a leftward shift in the curve when the μ-PAM’s were introduced. BMS-986121 and BMS-986122 significantly increased the inhibition of forskolin-stimulated adenylyl cyclase activity produced by an 30 pM concentration of endomorphin-I in CHO-μ cells. BMS-986122 was found to significantly positively influence the affinity of orthosteric agonist DANGO in the G protein activation study. Further study on the μ-PAM’s ability to influence efficacy indicated that BMS-986122 can positively modulate agonist efficacy, measured as an increase in maximal responses to the partial agonists morphine and endomorphin-I. The leftward shift in the potency of DAMGO in the presence of BMS-986122 was confirmed in mouse brain. Two other compounds were found to have SAM-like activity, causing BMS-986122 to only increase DANGO’s potency less than two-fold, down from it’s original seven-fold increase, indicating that these two compounds are likely SAM’s.
    Overall the methodology seems sound and appropriate for the question at hand. While there is still some uncertainty at times regarding things such as whether the two μ-PAM’s also act as NAM’s and PAM’s, the results generated seem to support their overall conclusion that BMS-986121 and BMS-986122 are μ-PAM’s that may have therapeutic potential in pain management with improved side-effect profiles and reduced tolerance and dependence liabilities. One critique I have is why they used different types of cells testing BMS-986121 and BMS-986122. For example, Chinese hamster ovary cell line recombinantly expressing human μ-opioid receptors was used to test μ-PAM’s influence on inhibition of adenylyl cyclase but when carrying out GTPγS-binding studies, C6 glioma cells stably expressing recombinant μ-opioid receptors were used. Why not remain consistent? Also, they explained how they were able to deduce that the two ligands, BMS-986121 and BMS-986122, were likely PAM’s for μ-opioid receptors but they could not rule out that they were not also potentially SAM’s on δ-opioid receptors. I read another paper about measuring ligand efficacy at the mu-opioid receptor using a conformational biosensor and was curious if, for one, they could measure efficacy for allosteric modulators that don’t interact with the active site, and secondly, if they could do this for δ-opioid receptors specifically. Perhaps other methods such as patch clamping and fluoresce membrane potential assays may prove useful in this as well.
    The introduction was brief and well written. It described the purpose of the study, the significance, and the rationale for scientific premise. The summary/conclusion was concise and I felt that it should’ve expanded more on future experiments that need to be carried out to further assess the therapeutic potential of these μ-PAM’s. Overall, the correlated literature and the results from this study indicate potential therapeutic use and, while the work in this study seems strong, there is still a lot of necessary work to be carried out to determine the true potential of μ-PAM’s in pain management.

  2. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.

    This study investigated the use of μ-opioid receptor allosteric ligands like positive allosteric modulators (PAMs) and silent allosteric modulators (SAMs) in an effort to develop novel opioid receptor-based therapeutics to fine-tune pain control while alleviating the side effects of current opioid therapeutics. They proposed this study based on the need to identify better efficacious treatments for pain management due to side effects related to current μ-opioid receptor-based treatments, such as respiratory suppression, constipation, allodynia, tolerance, dependence, as well as abuse potential. In the study, they describe the discovery and characterization of μ-opioid receptor PAMs and silent allosteric modulators, identified from high-throughput screening using a βarrestin–recruitment assay. They conclude that their studies describe the existence of μ-selective PAMs and SAMs, implicating positive allostery as a potential avenue for the discovery of tightly regulated pain therapeutics; and that they may possess therapeutic potential in pain management with improved side-effect profiles and reduced tolerance and dependence liabilities.

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.

    I think the methodological approaches used in this study were pertinent to the proposed research question to be answered. They used necessary techniques to validate their findings and support their analysis, as well as their conclusions. In the study, they utilized high-throughput screening (HTS) using the Path-Hunter enzyme complementation assay technology to identify potential μ-opioid receptor PAMs (μ-PAMs). In which they identified two compounds BMS-986121 and BMS-986122. Next, they used U2OS Path-Hunter cells expressing PK-tagged δ-opioid receptors (U2OS-OPRD1) to assess if both of the potential compounds bound to the μ-opioid receptor as opposed to being associated proteins. They found that neither compounds displayed any significant effect in the absence or the presence of the δ agonist leu-enkephalin. Thus, they concluded that the compounds appeared to be selective for μ- over δ-opioid receptors, which would support a direct interaction with μ receptors. However, they were unable to rule out if the compounds may be SAMs at the δ-opioid receptor as SAMs bind to the allosteric site but do not produce any effect on orthosteric-agonist potency or efficacy. Next, to further assess μ-PAM activity, both compounds were tested in three functional assays, β-arrestin recruitment, inhibition of adenylyl cyclase activity, and G protein activation using [35S]GTPγS binding, as well as in in receptor-binding assays. The only critique I have concerning their methodology is the absence of in-vivo data substantiating their claims. If the proposed advantage/premise for the study is improved side-effect profiles and dependence issues compared to orthosteric opioid receptor agonists, I think I would have been pertinent to show in vivo data in this paper. None-the-less, they do state that their studies only provide proof-of-concept for the development of opioid allosteric modulators, I assume they intend to look into it further.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    I think the paper does a great job at explaining the rational and premise of the study. The introduction does a great job at giving adequate background and stating the problem state. The organization as well as the flow of the article in terms of the methodology and the progression of the results and discussion were thorough. Other than the critique concerning their absence of in-vivo data substantiating their claims, I think their in-vitro data does demonstrate the potential for the development of opioid allosteric modulators that may have therapeutic potential in pain management. However, I am hesitant to agree with their conclusions on the assumptions of improved side-effect profiles and reduced tolerance and dependence liabilities without any in vivo studies. As we’ve seen many times in literature, compound may work in vitro but lose function in vivo or even have more severe side effects pre-clinically as well as in the clinic.

  3. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.

    – Hypothesis: opioid receptor PAMs could produce less tolerance and dependence than exogenous orthosteric agonists
    – BMS-986121 and BMS-986122 were identified as potential mu-opioid receptor PAMs and neither produced significant B-arrestin recruitment on their own, but both significantly augment the B-arrestin-recruitment response produced by endomorphin-I; they are also selective for mu- over delta-opioid receptors
    – Both compounds produced both concentration-dependent and saturable leftward shifts in the presence of endomorphin-I and exhibited some agonist activity that caused inhibition of cAMP accumulation in the absence of any orthosteric agonist
    – BMS-986122 can enhance the affinity of DAMGO for the mu-opioid receptor and positively modulates agonist efficacy
    – Though they tested other analogs of BMS-986122, it was the one that exhibited the greatest PAM activity and that it can be antagonized by BMS-986124 and both BMS-986123; BMS-986124 may be mu-opioid receptor SAMs competitive agonists at the allosteric site to which BMS-986122 binds

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.

    – While I understand that there may have been a word limit, I would have appreciated the methods all being included in the main paper, rather than the supplemental information
    – I appreciated that the researchers included the reproducibility issues they had, rather than exclude that data or try to take their nicest results; however, I would have liked to see them include how they tried to fix the reproducibility issues or ideas on why they thought they might have been having issues

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    – I am curious what potential in vivo experiments could be conducted that show the clinical applications of all the compounds mentioned, since they may prove to have no clinical value at all
    – The authors kept the introduction sections very general and while they were thorough in the significance of opioid receptors, there was little to no mention of how finding these new ligands would be useful clinically or why this research is important for practical applications
    – The discussion at the end also did not give me any more insight than with the introduction of why this research is important to the field as a whole; while it does include a very general overview of the negative effects of opioids again, the authors don’t explain much about the ligands they did discover, besides being a proof of concept

  4. Student Aminatta Tejan-Kamara Instructor Dr. Selley Date 11/07/19
    Please answer the following. Do not exceed 1 page for your answer; you may use outline/bullet points.
    1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.
    • Hypothesis- Authors hypothesized μ opioid receptor allosteric ligands could be a potential therapeutic for pain, the emphasis/focus of the paper was on the characterization and specificity of the allosteric ligands BMS-986122 and BMS-986121.
    • Major Results- Authors found that BMS-986122 and BMS-986121 both enhanced the orthosteric ligands endogenous effects (when agonist was bound to the receptor), however BMS-986122 appeared to have a greater enhancement of potency with respect to the full agonist DAMGO, “[BMS-986122] resulted in DAMGO potency shifting leftwards by sevenfold…BMS-986121 produced a fourfold leftward shift.”
    • Overall conclusion- Authors concluded that the two characterized μ opioid receptor PAMs BMS-986122 and BMS-986121 enhanced agonist mediated beta arrestin recruitment, cAMP decrease/adenylyl cyclase inhibition and positive affinity modulation of DAMGO which strengthens their argument that μ opioid receptor PAMs may have therapeutic use in pain management/control without some of the undesirable side effects.

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.

    To note, authors desired to first characterize the μ opioid receptor PAMs BMS-986122 and BMS-986121 with beta arrestin recruitment assay. From the literature, this assay has been described as accurate and specific. The experimenters’ different conditions of PAM detection mode, agonist detection, for OPRM1 and OPRD1 allowed the experimenters to assess if the allosteric ligands did/did not have agonist properties and how the BMS-986122 and BMS-986121 compounds augmented Beta arrestin recruitment, given that the compounds “significantly” augmented recruitment, this provided the first piece of evidence the compounds may be acting through allosteric modulation in the presence of ligand bound to the opioid receptor. The subsequent assays assessing adenylyl cyclase activity and G protein activation were suitable assays and both demonstrated enhancement of orthosteric ligands (enkephalin) endogenous effects. The DAMGO affinity data was an adequate method to illustrate the distinction between the two PAMs as BMS-986122 enhanced DAMGO’s binding affinity (BMS-986122 increased DAMGO’s potency sevenfold as compared to BMS-986121 fourfold increase). Thus the results appear to be substantiated by the data presented in the paper.
    In terms of critiques, the authors could have further tested the roles of SAMs in their presented studies, they make mention of the SAMs and the potential role but SAMs would provide more information as to whether the presented mu opioid receptor PAMs are in fact working through allosteric modulation of orthosteric ligands.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    Introduction is thorough, authors convey the importance and relevance of opioid receptor agonists, and the dangerous side effects. In doing so, they successfully demonstrate the need for alternative therapeutic pain control/management avenues such as allosteric modulation through PAMs (and NAMs). The results of the beta recruitment assay, adenylyl cyclase activation assay and binding/competing assays support overall hypothesis and provide the evidence and foundation for further investigation into allosteric ligand mediated pain therapeutics.

  5. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.
    The authors hypothesized that there are existing mu-selective positive allosteric modulators (PAMs) and silent allosteric modulators (SAMs) that could be used as potential therapeutics for pain. Mu-opioid receptor PAM ligands were identified using Path-hunter enzyme complementation assays in order to identify two compounds, BMS-986121 and BMS-986122, which were selective for mu-receptors. Using binding assays, it was determined that both ligands inhibited cAMP responses and increased inhibition of adenylyl cyclase activity, B-arrestin recruitment, and stimulation of G protein activation. It was also confirmed that BMS-986122 could positively modulate agonist efficacy which was measured as an increase in maximal response to the partial agonists morphine and endomorphin-I. PAM activity on the mu-receptor was also observed for DAMGO-mediated G protein activation in the mouse brain tissue with endogenous levels of receptor and G protein. Additional analogs of the ligand were tested to determine the structure-activity relationships, and it was found that minor modifications to the structure affected its PAM activity (reduced Emax) but retained relative potencies. It was also found that probe dependence was not significant because BMS-986121 produced similar potentiation of various peptide and small molecule agonist responses. The overall conclusion was that PAMs have the ability to have therapeutic potential in pain management with improved side-effect profiles and reduced tolerance and dependence.

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.
    • Statistical analyses seemed appropriate; a two-way ANOVA with a Bonferroni posttest and nonlinear regression analyses were used.
    • In the binding assay, they mentioned that the low-efficacy agonist activity of BMS-986121 was not reproducible or too low to determine a fit of the concentration response data. They could have explored the reason for this further.
    • It would have beneficial for the methods to be separated from the results for overall clarity of the paper.
    • The authors kept mentioning that results could be affected by activity of SAMs at the delta-opioid receptor which was undetectable. They could have explored this further, but this is an overall limitation of the study that could have skewed the results.
    • Different cell types were used for the various assays, which could be a limitation.
    • Overall, the methods were appropriate and the results are substantiated the data.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.
    They provided a very thorough introduction delving into the specifics of GPCR mechanism of action as well as background on PAMs, NAMs, and SAMs. They also discussed the possible implications of opioid receptor positive allosteric modulators for drug discovery, which was a strength of the paper. However, it would be strengthened by the addition of previous literature or findings in the field. The rationale for the scientific premise was the potential therapeutic value of PAMs of the mu-opioid receptor in treating chronic pain, as direct agonists such as morphine have significant side effects like respiratory depression and tolerance. They stated that PAMs have an advantage of specifically modulating activity of the receptor only when the orthosteric agonist is present and fewer side effect profiles and dependency issues. I’m not sure that the overall conclusion is supported in entirety because there is no significant proof that the PAMs will improve side effects or reduce tolerance in the long term, as they didn’t study this and there were no in vivo studies. The discussion was mostly a reiteration of the advantages of PAMs as well as potential future studies on biased signaling, and I would have liked to hear a deeper delve into the significance of this data.

  6. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.
    H: Opioid receptor PAMs produce less tolerance and dependence than exogenous orthosteric agonists through modulation of β-arrestin recruitment and G-protein signaling mechanisms.
    R: After screening compounds for activity of β-arrestin activity, 2 compounds were shown to modulate recruitment in the presence of an orthosteric opioid agonist, endomorphin-I: BMS-986121 and BMS-986122. Varying concentrations of these compounds were analyzed in agonist and PAM-detection modes in combination with various orthosteric agonists to reveal that the compounds are selective for modulation of the µ-opioid receptor. The compounds were then shown to leftwardly shift the curves and EC50s of endomorphin-1 stimulated β-arrestin recruitment in U2OS-OPRM1 cells with BMS-986122 being slightly less potent although equally as efficacious as BMS-986121. When tested in agonist and PAM-detection modes for inhibition of forskolin-stimulated cAMP accumulation in CHO cells, both compounds showed high PAM activity and some agonist activity. Finally, BMS-986122 was also shown to leftwardly shift the curves of the % stimulation of [35S]GTPγS binding in multiple cell types.
    C: Considering the compounds’ ability to dramatically shift the dose response curves of these assays, the authors concluded that the two compounds are in fact PAMs of the µ-opioid receptor and that the effects of the PAMS could be blocked by the use of a intrinsically inactive compound known as a SAM. They were able to characterize a SAM in-vitro and confirm this effect. The authors then characterized a few more compounds as SAMs in a β-arrestin recruitment assay and in a DAMGO-mediated [35S]GTPγS binding assay. BMS986124 was shown to block the effects of BMS-986122 suggesting it blocks the binding of the orthosteric agonist (DAMGO) to the receptor.
    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.
    M: The authors utilized good in-vitro assays for the characterization of the PAMs, as well as their implications in drug tolerance and dependence. The assays provided results for multiple doses, crucial for dose response curves and the evaluation of PAM-like effects. The use of multiple cell types in the [35S]GTPγS binding assay was a great addition to strengthen their overall conclusions. While many great assays relating to tolerance and dependce were used, the use of tolerance or dependence related in-vivo assays would have further strengthened their conclusions. For example, the ability to leftwardly shift the dose response curves of drug discrimination of opioids would give more indication of the compounds’ ability to serve as a PAM in-vivo or clinically.
    R: Plenty of data analysis was provided for results, including EC50s and confidence intervals. Drug doses for some, but not all assays were included for accurate conclusions (only a single dose was used for examining the effect of BMS-986122 on μ-opioid agonist-stimulated [35S] GTPγS binding in membranes). Overall, the data supported the final results.
    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.
    I: The authors gave good rationale for exploring a novel method of targeting the opioid receptor. They also described the advantages of allosteric modulation. While they did explain that allosteric modulators had been studied in other systems, such as glutamate receptors, there was no rationale provided for the existence of an allosteric site for the µ-opioid receptor specifically. Finally, they described their method of selecting drugs as a “high-throughput screen” without providing any explanation for how they chose the compounds they screened or how the compounds’ structures were derived.
    D/C: The findings were very similar to previous allosteric studies described in previous literature, such as the shift of dose response curves. However, one striking result was in Fig. 3A and even more so, Fig. 3B; both compounds displayed agonist-like activity for forskolin-stimulated cAMP accumulation (although BMS-986122 showed higher %cAMP inhibition). By definition; allosteric modulators do not show any activity alone, only in combination with the binding of an orthosteric agonist. This makes me question the accuracy of characterizing these compounds as allosteric modulators and the authors fail to address this result in the discussion or summary.

  7. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.
    – The hypothesis of the study is that novel positive allosteric modulators (PAMs) for the mu-opioid receptor will modulate the activity of mu-agonists and yield new therapeutics with lower side-effect profiles.
    – In figure 1, the authors use a high-throughput screening process to identify novel MOR PAMs. Two compounds were identified (BMS-986121 and BMS-986122). They show that neither of these compounds had significant beta-arrestin recruitment activity on their own, but they did augment the response produced by a low concentration of endomorphin-1. BMS121 and BMS122 each respectively increased the activity level from low effect to 76% and 83% of the maximal effect of endomorphin-1. They also show in this figure that the compounds are selective for mu- receptors over delta- receptors. Lastly, they do not rule out that these compounds could be silent allosteric modulators at the delta-opioid receptors, meaning that they bind to an allosteric site but do not augment the response of orthosteric ligands. Next the authors test the two novel compounds for receptor binding and also in assays measuring beta-arrestin recruitment, inhibition of adenylyl cyclase, and for G-protien activation using GTPgammaS binding. In the cAMP-accululation assay they show that there is some agonist activity produced by these compounds but that it was at concentrations higher than those required to produce PAM-like effects. In addition, the structure activity relationship of BMS-122 was tested in the beta-arestin recruitment assay. Their results suggest that modifications in the chemical structure can effect the selectivity of these analogs to mu vs delta opioid receptors. They also show that some of the analogs (BMS-123 and BMS-124) may have SAM activity at the receptor, however since they do show some weak efficacy depending on whether tested with a partial or full agonist suggesting these compounds may not be truly “silent.” The authors also test for probe-dependence of the BMS-121 compound and find that it is not probe dependent for the orthosteric agonist.
    – In conclusion the authors state they have identified two novel mu-opioid receptor PAMs and that this study provides rational for the development of opioid receptor PAMs for therapeutic use.
    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.
    – The authors use a wide array of in vitro pharmacological techniques and it was nice to see the addition of multiple types of binding assays to determine selectivity and for the SAR studies. It would have also been nice to include some behavioral studies since the authors stress that these compounds could produce enhanced pain therapy with reduces side-effects such as dependence and tolerance. They could have added some rodent studies in which an orthosteric ligand is administered and the augmentation of the dose-response curve is recorded in the presence of the PAM or vehicle. Also, to support their claim these compounds would have less dependence potentials they could’ve looked at them in certain operant models such as self-administration or ICSS. Regardless, their data is strong, however the addition of in vivo studies could’ve made this a much higher impact publication. It would have also been nice to see controls such as binding studies looking to see if the BMS compounds augmented the responses of non-mu opioid drugs. The data analysis is clear and it is nice that they included the confidence intervals in the text so the reader does not have to go searching for them. Overall their results are substantiated by their data.
    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.
    – The introduction is somewhat brief but does give a solid background on why they are targeting mu-opioid receptors and there is some general background on allosteric modulators. They do not discuss any previous publications that may have explored the idea allosteric modulation of opioid receptors specifically (although it seems they may be the first to identify a PAM, there must’ve been some effort before that resulted in non-specific effects), although they do cite studies for AMs of other receptor types. Their conclusions re-state the significance of their discovery in that it would increase analgesic effects but reduce side effects. However, this statement would have been better supported with the addition of behavioral experiments to explore this in whole organisms.

  8. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.
    Burford et al. hypothesized that μ-opioid receptors have a binding site other than the orthosteric site where positive allosteric modulators (PAM) and silent allosteric modulators (SAM) could bind to either enhance the effect of the orthosteric ligand or produce no additive effect. Here they describe their findings of the existence of μ-opioid receptor selective PAM and SAMs that might have a potential in the discovery of further pain therapies. Their major results are the discovery of the PAM by the high-throughput screen (HTS) BMS-986121 and BMS-976122 and further screening by different assays. The overall conclusions are that their work shows that opioid allosteric modulators may have therapeutic uses in treating pain.
    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.
    Method/result1: was the identification and characterization of the μ-opioid receptor PAMs – to identify ligands that could bind to the μ-opioid receptor Burford et al. started with the high-throughput screen (HTS) using a μ-selective agonist endomorphin-I. They used U2OS cells that expressed β-arrestin2 and a PK donor that was fused to the OPRM1 receptor expressed in those cells. This allowed to determine the recruitment activity of the receptor (fig. 1C-D). From this they identified the two potential μ-opioid receptor PAMs BMS-986121 and BMS-976122 (fig. 1A-B). The results shown indicate that the compounds don’t recruit β-arrestin2 alone but do so in the presence of the endomorphin-I only in OPRM1 cells not OPRD1. I like the fact that they included delta data to show the selectivity of these compounds, but I guess I would have also liked to see other recptor types (kappa, ORL1).
    Method/result2: Next they wanted to better categorize the compounds at the μ-opioid receptor so to do this they tested three different assays. First, β-arrestin recruitment by generating concentration-response curves (CRCs) (fig. 2A/B). Concentration-dependent leftward shifts can be seen in both the CRC produced by the ligands. They provide EC50 values of endomorphin-I as the concentration of each agonist increases as well as Kb and the cooperativity factor (α). The second assay was inhibition of adenylyl cyclase (AC) activity. I thought this was a good assay for them to show because of opioid receptor activity that modulates AC. I also like the fact that they state why they chose to work with different cells (from U2OS to CHO-μ cells). Another thing I really like about this section is that Burford et al. went into detail about the distinction of both compounds and the lack of reproducibility of BMS-986121. Figure 3 shows %cAMP inhibition curves of these ligands alone and in the presence of endomorphin-I. Alone both compounds showed some what of agonist activity, but this increased in the presence of endomorphin-I. The third assay ran was [35S]GTPγS binding to determine G protein activation in a different cell line (C6-μ glioma). To compare binding activity of the ligands they were maximized to the % effect of DAMGO. BMS-986121 produced a fourfold shift in the CRC and BMS-976122 produced a sevenfold shift in the CRC (fig. 4). They also went on to perform saturation binding assay, they looked at the activity of the ligands in mouse brain membranes, they identified SAMs.
    Overall, I think that the in vitro data Burford et al. showed was good enough to prove their point, although like I said I would have been interested to see activity at other receptor types. What they did not show but they make a point in their conclusion to is in vivo pharmacology. How can we know about these being potential therapeutics with improved side effects without seeing any evidence of this in animal models?
    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.
    The introduction was vast, and Burford et al. did a good job at introducing what opioids are, where they bind to, and the effects they have. They also introduce the concept of allosteric modulators in good detail. They had a lot of references cited to work of others in the field which is always good. Their scientific premise is good, they are precise as to why it is important that PAMs be identified but I do think their premise falls short due to the lack of behavioral data. The PAMs would be great to test to see if the effects in the in vitro assays could be seen too. Their conclusions were described as each section was done and they did have an overall summary. I would have liked to know more about any future directions they were thinking of taking these conclusions in.

  9. Donald Jessup
    1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.

    Hypothesis: a HTS assay will reveal the presence of μ-selective PAMs and SAMs which will offer new potential therapeutic targets

    Results/Methods: 1.) HTS using PathHunter enzyme complementation assay technology identified two potential PAMs for the u-opioid receptor, BMS-986121 and BMS-986122 . subsequent experiments revealed that neither compound recruited β -arrestin alone, however both compounds significantly augmented the β-arrestin recruitment response produced by a low concentration of endomorphin-I. . Neither compound had any significant effect in the presence or absence of the δ agonist leu-enkephalin indicating a potential interaction with the u-receptor specifically. 2.) BMS-986121 and BMS-986122 were tested in three functional assays, β-arrestin recruitment, inhibition of adenylyl cyclase activity, and G protein activation using [35S]GTPγS binding. β-arrestin recruitment experiments demonstrated that BMS-986121 andBMS-986122 produced concentration-dependent, saturable leftward shifts in the potency of endomorphin-I responses. The cAMP-accumulation assay in CHO cells when stimulated, endomorphin-I produced a 17-fold reduction in forskolin-stimulated accumulation. BMS-986121 and BMS-986122 significantly increased the inhibition of forskolin-stimulated adenylyl cyclase activity produced endomorphin-I. Additionally both μ-PAMs also exhibited some intrinsic agonist activity causing inhibition of cAMP accumulation in the absence of any orthosteric agonist. These experiments also suggested the presence of a potential receptor reserve – as a discrepancy between the agonist activity of the PAMs could be seen in this assay as well as a lack of agonist activity seen in the β-arrestin–recruitment assay. Lastly μ-PAMs were characterized in G protein activation [35S]GTPγS-binding studies in membranes from C6 glioma cells expressing recombinant μ-opioid receptors and were compared to DAMGO activation. BMS-986121 produced a sevon-fold leftward shift and BMS-986122 produced a four-fold leftward shift in potency. Enhancement of the maximal response to morphine (partial agonist) in the [35S]GTPγS-binding assay would suggested that the μ-PAMs
    were also able to modulate agonist efficacy. 3.) To determine whether μ-PAM activity can be observed in native tissues, DAMGO-stimulated [35S]GTPγS binding in membranes from mouse brain was also assessed, and a 4.5-fold leftward in the presence of BMS-986122 was observed. Suggesting it was possible to examine these effects in a physiologically relevant tissue with endogenous receptors. 4.) Two analogs (BMS-986123 and BMS-986124) were assessed for their ability to inhibit orthosteric agonist activity or for their ability to inhibit BMS-986122 PAM activity n the β-arrestin–recruitment assay in U2OS-OPRM1 cells and in the [35S]GTPγS assay. The results of which showed that in the β-arrestin assay, neither compound significantly inhibited an EC80 concentration of endomorphin-I, and in the [35S]GTPγS assay, the coaddition of BMS-986122 with BMS-986124 resulted in an inhibition of the PAM effect, with DAMGO potency enhanced less than twofold suggesting that these analogs are SAMs although both analongs increased morphine Emax slightly in a subsequent experiment.

    Conclusion: BMS-986121 and BMS-986122 are PAM’s of the μ -opioid receptor as indicated by their ability to enhance orthosteric agonist activity. Additionally analogs of these compounds may act as SAMs at the same site.

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.

    This paper had an exhaustive and purely pharmacological approach to characterizing and quantifying these compound identified in the HTS. Overall it’s very compelling to see multiple in-vivo and the one in-vitro experiment in mouse brain, as the authors went about establishing binding affinity and quantifying the degree of positive allosteric modulation with DAMGO and morphine potency changes. Overall the experiments were well controlled, with most being compared to what the full or partial agonist activity would look like in the absence and then subsequently the presence of the presumptive PAMs. All that said, I would have liked to see just one experiment that had a functional measure of these PAMs to evaluate, because presently this paper presented strong data for the binding, interaction, and secondary messenger activation of the PAMs but some in-vivo or in-vitro functional consequences would have been nice to see. Although to be fair the paper stands well enough without them.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    The Introduction strongly establishes the scientific rational and relevant background knowledge for the study and was overall very informative and well-constructed. However it would have been good to see the authors point specifically to the field specific implications by more directly referencing the other published work that sort of lead up to this. As it stands that’s kind of left to the reader to take that away from the background, rather than being specifically worded and addressed. The discussion likewise avoids similar language, but overall this is a minor criticism as the discussion thoroughly explores the implications of PAMs and their clinical significance based on the compelling data presented in the study. Overall the conclusion is well within the bounds of the presented results and the authors addressed several potential limitations both in the results and discussion section.

  10. Please answer the following. Do not exceed 1 page for your answer; you may use outline/bullet points.
    1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.
    The hypothesis of this study is that an allosteric approach can be employed in the development of opioid receptor ligands, positive allosteric modulators (PAMs) in particular. A high throughput screen using Path-Hunter enzyme complementation assay in human osteosarcoma (U2OS) cells was used to identify PAMs at MOR. PAMs identified by the screening were further characterized through assays measuring beta-arrestin 2 recruitment, inhibition of forskolin-stimulated cAMP accumulation, and GTPS binding. DAMGO stimulated GTPS binding was measured in native mouse brain tissue to show PAM activity in physiologically relevant tissues at relevant receptor concentrations. Structure activity relationship (SAR) studies employing the same assays were repeated using structural analogs of the previously identified PAMs to try and develop more selective PAMs or even silent allosteric modulators (SAMs). Two compounds identified in the initial screen, BMS-986121 and BMS-986122 displayed PAM activity at OPRM1 and neither PAM nor agonist activity at OPRD1. Both compounds augmented enomorphin-1 (EC10) mediated inhibition of adenylyl cyclase, with some intrinsic agonist activity at higher doses. Both compounds showed showed PAM activity with respect to GTPS binding in both heterologous and native tissue systems, without intrinsic agonist activity. In the SAR studies, analogs of BMS-986122, BMS-986123 and BMS-986124 were shown to be SAMs that dose-dependently decrease BMS-986122 PAM activity in DAMGO stimulated GTPS binding assays.

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.
    The initial enzyme complementation assay looked for PAM activity with respect to endomorphin-1 stimulated beta-arrestin recruitment (PAM detection mode). A low (EC10) dose of endomorphin-1 seems appropriate for this assay since the idea is to find a PAM; a higher dose of endomorphin would give a lower margin for detecting PAM compounds. To control for agonists that might be detected by the screening, compounds were also tested in the absence of endomorphin-1 (agonist detection mode). The two PAMs that were identified in the screen were tested again in a beta-arrestin recruitment assay except now multiple doses of endomorphin-1 were used. This showed that BMS-986121 and BMS-986122 dose-dependently produce leftward shifts in beta-arrestin recruitment response from endomorphin-1. The forskolin assay was performed in CHO cells rather than U2OS cells because cAMP-inhibition responses in U2OS cells were too small to measure. This consideration explains why beta-arrestin recruitment instead of Gi/o activation (forskolin or GTPS binding assay) was measured in the initial drug screening. Again, PAM and agonist detection modes were used in the forskolin assay to control for intrinsic agonist activity of the PAMs under study. C6 glioma cells expressing recombinant OPRM1 were used as a heterologous platform for measuring GTPS binding. GTPS binding was also measured in native mouse brain tissue and this was an important control to show that the PAMs being studied are physiologically relevant; they bind in the right tissues expressing normal concentrations of receptor. The SAR studies tested similar analogs against BMS-986122 and not BMS-986121 because the former had the greatest PAM activity. They showed that BMS-986123 and BMS-986124 are SAMs, and controlled for NAM activity. Overall I think the results clearly show that allosterism can be applied to opioid receptors and that this presents a promising avenue for the development of opioid ligands in the future.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    The introduction concisely lays out the rationale for the study. Allosterism has been applied to many GPCRs, and MOR, being a GPCR must have some positive or negative allosteric modulators. I think they could have explicitly stated what ligands have been developed prior to the study which have been shown to affect opioid receptors allosterically. It is not clear whether this study is the first to identify MOR PAMs. Regardless, I think the approach to this study was effective and employed the appropriate controls. The results and discussion are quite clear and I think support the overall conclusion.

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