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  • rgs protein A range of inhibitors were therefore synthesized

    2020-09-29

    A range of inhibitors were therefore synthesized containing either carboxylic rgs protein or ester functionality at the ‘tail’ group as well as variations in the ‘head.’ These were assayed in vitro using the DCIP assay for binding affinity against and human DHODH at 50μM, respectively (). Analysis of the affinity data in reveals some interesting trends. Specifically, the majority of carboxylic acid-containing compounds (, entries 7, 8, 11, 12, 15, and 16) bind with similar affinities to both enzymes except in the case of entries 7 and 8 where a degree of selectivity is observed for the human enzyme. With the exception of entries 1 and 6, (which have low affinity for both enzymes), compounds containing an ester substituent in the ‘tail’ portion (, entries 2, 5, 9, 10, 13, and 14), generally bind somewhat more tightly to the enzyme. This is consistent with our simple design criteria which predicts that such systems, when bound DHODH in the same orientation as found for A77 1726, and containing a carbonyl group at either the - or -positions of the aryl moiety, will show enhanced binding to DHODH compared to that with HsDHODH due to H-bonding between this carbonyl group and the backbone N–H of Met536 within DHODH. Interestingly, with the exception of entry 5 versus entry 13 (), addition of a chloro-substituent in the ‘tail’ portion generally led to increased enzyme affinity compared to the non-chlorinated homologue (1 vs 9; 2 vs 10; 6 vs 14). However, these trends are not consistent with a single binding mode for compounds – () to the human and enzymes, in which all compounds bind exclusively in an ‘A77-like’ orientation in which the ‘head’ group is located in the polar region of the binding cavity () and not in the reverse orientation (). In order to probe the predicted binding modes of these inhibitors, the compounds were docked using eHiTS, and AutoDock, , into crystal structures of DHODH (1TV5) and HsDHODH (1D3H). Interestingly, both docking programs predict that the reversed ‘head’ group conformation is preferred for binding of these inhibitors in both the human and (to a lesser extent) the parasite forms of DHODH when the substituent on the aromatic ring is acidic (). This is not surprising since this binding mode features a salt bridge between Arg136 and the free carboxylate anion, a feature which is present in the structures of many tight bonding inhibitors of HsDHODH which contain carboxyl groups., This suggests that the binding mode of compounds featuring R as a carboxyl group is conserved between different DHODH species, which in turn, could lead to the non specific binding which is observed for these derivatives. Inspection of the binding cavities for both the human and enzymes reveals that, in addition to a large hydrophobic cavity in which the bulk of the hydrophobic portions of inhibitors are predicted to reside, there is also a smaller hydrophobic cavity located near to residue Arg265 in the enzyme (Arg136 in HsDHODH). The docking studies indicated that, for binding of the diethylmalonyl-based inhibitors in an ‘A77-like’ orientation, the volume of the small hydrophobic cavity is such that only one of the two ester-derived ethyl groups is accommodated, the remaining ethyl group being stacked alongside the aromatic portion of the molecule, forming a compact arrangement within the large hydrophobic cavity (). This role of flexibility within the diethyl portion of these molecules was probed via creation of a set of enamines derived from Meldrum’s acid, in which the alkyl portions of the ‘head’ group now form part of a rigid cyclic framework and cannot adopt the conformation shown in . The rgs protein results of a study of the ability of these systems to inhibit both Pf- and HsDHODH are summarized in . As expected, these results show that a rigid ‘head’ group is disadvantageous for binding, particularly in the cases where the ‘tail’ group features an ester (entries 19, 20, 23 and 24, ) as these rigid systems generally show poorer inhibition of both enzymes compared to that observed for the simple diester-based molecules (entries 1, 2, 9, and 10, ). This observation is in keeping with the prediction that, by locating one of the ethyl groups alongside the aromatic core within the large hydrophobic cavity, the diester-based systems are better able to form a tighter packing arrangement within the larger hydrophobic cavity of DHODH compared to that possible for the Meldrum’s acid derivatives. Significant binding is observed in this series only when R is a carboxyl group. Docking studies suggest that, once again, a ‘reverse’ orientation involving a salt bridge to Arg265 in DHODH and Arg136 in HsDHODH is a dominant interaction.