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Structure-activity relationships of pyrimidine nucleotides containing a 5'-α,β-methylene diphosphonate at the P2Y₆ receptor.

ABSTRACT: The G_q-coupled P2Y₆ receptor (P2Y₆R) is a component of the purinergic signaling system and functions in inflammatory, cardiovascular and metabolic processes. UDP, the native P2Y₆R agonist and P2Y₁₄R partial agonist, is subject to hydrolysis by ectonucleotidases. Therefore, we have synthesized UDP/CDP analogues containing a stabilizing α,β-methylene bridge as P2Y₆R agonists and identified compatible affinity-enhancing pyrimidine modifications. A distal binding region on the receptor was explored with 4-benzyloxyimino cytidine 5'-diphosphate analogues and their potency determined in a calcium mobilization assay. A 4-trifluoromethyl-benzyloxyimino substituent in 25 provided the highest human P2Y₆R potency (MRS4554, 0.57 µM), and a 5-fluoro substitution of the cytosine ring in 28 similarly enhanced potency, with >175- and 39-fold selectivity over human P2Y₁₄R, respectively. However, 3-alkyl (31-33, 37, 38), β-d-arabinofuranose (39) and 6-aza (40) substitution prevented P2Y₆R activation. Thus, we have identified new α,β-methylene bridged N⁴-extended CDP analogues as P2Y₆R agonists that are highly selective over the P2Y₁₄R.

SUBMITTER: Oliva P

PROVIDER: S-EPMC8276771 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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Structure-activity relationships of pyrimidine nucleotides containing a 5'-α,β-methylene diphosphonate at the P2Y6 receptor.

Oliva Paola P Scortichini Mirko M Dobelmann Clemens C Jain Shanu S Gopinatth Varun V Toti Kiran S KS Phung Ngan B NB Junker Anna A Jacobson Kenneth A KA

Bioorganic & medicinal chemistry letters 20210526

The Gq-coupled P2Y6 receptor (P2Y6R) is a component of the purinergic signaling system and functions in inflammatory, cardiovascular and metabolic processes. UDP, the native P2Y6R agonist and P2Y14R partial agonist, is subject to hydrolysis by ectonucleotidases. Therefore, we have synthesized UDP/CDP analogues containing a stabilizing α,β-methylene bridge as P2Y6R agonists and identified compatible affinity-enhancing pyrimidine mo ...[more]

PMID: 34048882

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Project description:Background and purposePlatelet function during inflammation is dependent on activation by endogenous nucleotides. Non-canonical signalling via the P2Y1 receptor is important for these non-thrombotic functions of platelets. However, apart from ADP, the role of other endogenous nucleotides acting as agonists at P2Y1 receptors is unknown. This study compared the effects of ADP, Ap3A, NAD+ , ADP-ribose, and Up4A on platelet functions contributing to inflammation or haemostasis.Experimental approachPlatelets obtained from healthy human volunteers were incubated with ADP, Ap3A, NAD+ , ADP-ribose, or Up4A, with aggregation and fibrinogen binding measured (examples of function during haemostasis) or before exposure to fMLP to measure platelet chemotaxis (an inflammatory function). In silico molecular docking of these nucleotides to the binding pocket of P2Y1 receptors was then assessed.Key resultsPlatelet aggregation and binding to fibrinogen induced by ADP was not mimicked by NAD+ , ADP-ribose, and Up4A. However, these endogenous nucleotides induced P2Y1 -dependent platelet chemotaxis, an effect that required RhoA and Rac-1 activity, but not canonical PLC activity. Analysis of molecular docking of the P2Y1 receptor revealed distinct differences of amino acid interactions and depth of fit within the binding pocket for Ap3A, NAD+ , ADP-ribose, or Up4A compared with ADP.Conclusion and implicationsPlatelet function (aggregation vs motility) can be differentially modulated by biased-agonist activation of P2Y1 receptors. This may be due to the character of the ligand-binding pocket interaction. This has implications for future therapeutic strategies aimed to suppress platelet activation during inflammation without affecting haemostasis as is the requirement of current ant-platelet drugs.Linked articlesThis article is part of a themed issue on Platelet purinergic receptor and non-thrombotic disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.4/issuetoc.

Project description:Protein secondary structure elements (PSSEs) such as α-helices, β-strands, and turns are the primary building blocks of the tertiary protein structure. Our primary interest here is to reveal the characteristics of the nanoenvironment formed by both PSSEs and their surrounding amino acid residues (AARs), which might contribute to the general understanding of how proteins fold. The characteristics of such nanoenvironments must be specific to each secondary structure element, and we have set our goal here to gather the fullest possible description of the α-helical nanoenvironment. In general, this postulate (the existence of specific nanoenvironments for specific protein substructures/neighbourhoods/regions with distinct functionality) was already successfully explored and confirmed for some protein regions, such as protein-protein interfaces and enzyme catalytic sites. Consequently, PSSEs were the obvious next choice for additional work for further evidence showing that specific nanoenvironments (having characteristics fully describable by means of structural and physical chemical descriptors) do exist for the corresponding and determined intraprotein regions. The nanoenvironment of α-helices (nEoαH) is defined as any region of the protein where this secondary structure element type is detected. The nEoαH, therefore, includes not only the α-helix amino acid residues but also the residues immediately around the α-helix. The hypothesis that motivated this work is that it might in fact be possible to detect a postulated "signal" or "signature" that distinguishes the specific location of α-helices. This "signal" must be discernible by tracking differences in the values of physical, chemical, physicochemical, structural and geometric descriptors immediately before (or after) the PSSE from those in the region along the α-helices. The search for this specific nanoenvironment "signal" was made possible by aligning previously selected α-helices of equal length. Afterward, we calculated the average value, standard deviation and mean square error at each aligned residue position for each selected descriptor. We applied Student's t-test, the Kolmogorov-Smirnov test and MANOVA statistical tests to the dataset constructed as described above, and the results confirmed that the hypothesized "signal"/"signature" is both existing/identifiable and capable of distinguishing the presence of an α-helix inside the specific nanoenvironment, contextualized as a specific region within the whole protein. However, such conclusion might rarely be reached if only one descriptor is considered at a time. A more accurate signal with broader coverage is achieved only if one applies multivariate analysis, which means that several descriptors (usually approximately 10 descriptors) should be considered at the same time. To a limited extent (up to a maximum of 15% of cases), such conclusion is also possible with only a single descriptor, and the conclusion is also possible in general for up to 50-80% of cases when no less than 5 nonlinear descriptors are selected and considered. Using all the descriptors considered in this work, provided all assumptions about data characteristics for this analysis are met, multivariate analysis regularly reached a coverage and accuracy above 90%. Understanding how secondary structure elements are formed and maintained within a protein structure could enable a more detailed understanding of how proteins reach their final 3D structure and consequently, their function. Likewise, this knowledge may also improve the tools used to determine how good a structure is by means of comparing the "signal" around a selected PSSE with the one obtained from the best (resolution and quality wise) protein structures available.

Dataset Information

Structure-activity relationships of pyrimidine nucleotides containing a 5'-α,β-methylene diphosphonate at the P2Y₆ receptor.

Publications

Structure-activity relationships of pyrimidine nucleotides containing a 5'-α,β-methylene diphosphonate at the P2Y<sub>6</sub> receptor.

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OmicsDI is part of the ELIXIR infrastructure

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Dataset Information

Structure-activity relationships of pyrimidine nucleotides containing a 5'-α,β-methylene diphosphonate at the P2Y6 receptor.

Publications

Structure-activity relationships of pyrimidine nucleotides containing a 5'-α,β-methylene diphosphonate at the P2Y&lt;sub&gt;6&lt;/sub&gt; receptor.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Structure-activity relationships of pyrimidine nucleotides containing a 5'-α,β-methylene diphosphonate at the P2Y₆ receptor.

Structure-activity relationships of pyrimidine nucleotides containing a 5'-α,β-methylene diphosphonate at the P2Y<sub>6</sub> receptor.