On the contrary the benzhydrol series showed

On the contrary, the benzhydrol series showed modest activity with EC of two digit micromole. The bulkiness of the nos abbreviation sale tail may be the reason for the low activity. Interestingly, introduction of H-bonding groups, such as OMe in and CF in , slightly improved the activity compared to the unsubstituted benzhydrol (). This was in agreement with what noticed in the PPAR-γ transactivation assay. The improvement of activity with these polar substituents might be attributed to a favored H-bonding interaction at the FFAR1 binding site. On general bases, four compounds (, , and ) showed moderate and balanced dual activities with EC <20 μM in both PPAR-γ and FFAR1 agonist assays. Remarkably, compounds and , furnished affinities in the lowmicromolar range on both targets. To obtain further insights into the differential activities of the members of both series, we examined their virtual binding within both receptors using GOLD Suite V.4.1.2. The co-crystal structures of hPPAR-γ (PDB code: ) and hFFAR1 (PDB code: ) were employed for this docking workflow. The first crystal form is an intact PPAR-γ/RXR-α nuclear receptor complex with rosiglitazone, 9--retinoic acid, and NCoA-2, while hFFAR1 is bound to the partial agonist fasiglifam (TAK-875). The ligands polar heads, TZDs, interacts significantly with the H-bonding triad (H323, H449 and Y473) within the hPPAR-γ polar arm containing the important AF-2helix. On similar bases, extensive polar network was predicted between the TZD war-head and both arginine residues (R183 and R258) in the hFFAR1 hydrophilic pocket. Further stabilization through complex charge networking with Y91, Y240, E172, S187, N241 and N244 was also noticed. Beside these polar networks, hydrophobic interactions with the ligands tail parts also contribute for effective occupancy of both receptors binding sites. However, many reports have suggested that interactions with such conserved H-bonding rich areas are of much greater importance for the activities of PPAR- and hFFAR1 agonists. These H-bonding network could stabilize the AF-2 helix in a conformation favoring the binding of co-activators to PPAR- and, consequently, enhancing their recruitment leading to gene transcription., , , Similarly, polar interactions with the critical arginines breaks the arginine-glutamate ionic locks at the extracellular surface of FFAR1 allows the receptor to adopt an active conformation through the outward movement of transmembrane (TM) helices. It is worth noting that the ligand hydrophobic tails could indirectly affect the activities through influencing the distances and angles of H-bonding interactions between the TZD heads and polar residues. The best docking modes were selected by considering both the GOLD docking scores as well as visual investigation. Throughout our virtual studies, the predicted poses regarding the ligand’s hydrophobic tail represents the significant mark for each receptor. In case of hPPAR-γ, the hydrophobic tails are extended deep into either of the two hydrophobic arms of the Y-shaped orthosteric site (a). Typically, compounds of series occupied the larger hydrophobic arm (II) favoring the least sterically hindered binding mode. They, with their branched benzhydrol-based tails, manage to make contacts with I326, L228, M329, F226 and P227. Surprisingly, compound , despite its large tail fragment, could manage to accommodate the tighter arm I (b). Through careful examination, the methoxy substituent was found to be directed into the exit terminal of arm (I) (ligand entrance site). Such orientation could allow a good chance of forming water-mediated H-bonding. Moreover, the involvement of methoxy-substituted benzhydrol in π-cation interaction with R280 and π–π stacking with F287, could stabilize this unexpected fitting. Despite this significant complementary of the tails with either hydrophobic cavities, only and showed predicted H-bondings with crucial residues plus an extra bonding with S289, only for . This gives more emphasis on the importance of H-bonding interactions with the AF-2 helix for PPAR-γ activation.