Bisubstrate Inhibitors for the Enzyme Catechol O‐Methyltransferase (COMT): Dramatic Effects of Ribose Modifications on Binding Affinity and Binding Mode

Abstract

Inhibition of the enzyme catechol O‐methyltransferase (COMT) is of significant interest in the therapy of Parkinson's disease. Described herein are structural analogs of the potent bisubstrate inhibitor (−)‐1 (IC50=9 nM; Table 1) for COMT, with target modifications of the central ribose moiety. Their synthesis involves, as key intermediates, adenosine derivatives, which are transformed to the potential bisubstrate inhibitors by a similar sequence of six steps (Schemes 1–4). The compounds were submitted to an enzymatic assay for determination of their in vitro inhibitory activity against COMT, and the inhibition mechanism with respect to the binding side of the cofactor S‐adenosylmethionine (SAM) was analyzed by kinetics measurements (Fig. 3). Both binding affinity and binding mode were exceedingly sensitive towards modifications of the ribose moiety (Table 1). Removal of the 2′‐OH group upon changing from (−)‐1 to (−)‐2 (IC50=28 μM) led to a reduction in binding affinity by more than three orders of magnitude. At the same time, competitive inhibition kinetics with respect to the SAM binding site was maintained, thereby supporting a bisubstrate binding mode. Unlike (−)‐2, the dideoxyribose inhibitor (−)‐3 (IC50=3 μM) showed a mixed and the cyclopentane derivative (+)‐4 (IC50=1 μM) an uncompetitive inhibition mechanism with respect to the SAM binding site. In the complex of the latter, the adenine‐substituted cyclopentane ring orients most probably towards the surface of the enzyme into the surrounding solution. The enantiomeric compounds (−)‐5 (IC50=43 μM) and (+)‐5 (IC50=141 μM), wherein the ribose had been replaced by a pyrrolidine ring, showed only low binding affinity.

Separation of astaxanthin from cells of Phaffia rhodozyma using colloidal gas aphrons in a flotation column

“…In contrast to the substitution of the catechol substructure, for which a variety of lipophilic groups are tolerated, we found in earlier studies6c, 7, 8 with bisubstrate inhibitors (−)‐ 1 (IC 50 =9 n M ), (−)‐ 2 (40 n m) , (−)‐ 3 (28 μ M ), (−)‐ 4 (3 μ M ), and (+)‐ 5 (1 μ M ) that the ribose moiety seems to be very sensitive to even minor structural modifications. This observation led to the comprehensive investigation of the effects of ribose modification on ligand affinity and binding mode reported herein.…”

Molecular Recognition at the Active Site of Catechol‐O‐methyltransferase (COMT): Adenine Replacements in Bisubstrate Inhibitors

“…Synthesis: In contrast to earlier approaches, [14][15][16] in which adenine was introduced in the first step, the nucleobase substitutes in the new bisubstrate inhibitors 2 were introduced at a later stage of the synthesis (Scheme 1). [12,19] The general protocol involved amide coupling of allylic amine 3 with known activated catechol ester 4.…”

“…[12] The X-ray crystal structure of the ternary complex of ligand 1 (median inhibitory concentration IC 50 = 9 nm, inhibitory constant K i = 2 nm, [13] see Figure 1) with COMT and a Mg 2 + ion (PDB code: 1JR4) [14] was used as the basis for a detailed exploration of the molecular recognition properties of the entire active site of COMT. [15,16] Previously, we were able to demonstrate that 5-nitrocatechol anchors are not required for high-affinity bisubstrate inhibition and can be replaced with lipophilic substituents, such as a 4-fluorophenyl ring, without loss in activity against COMT. [17,18] The predicted orientation of the 4-fluorophenyl ring in the active site was recently confirmed by a series of X-ray cocrystal structure analyses.…”

Bisubstrate Inhibitors of Catechol O‐Methyltransferase (COMT): the Crucial Role of the Ribose Structural Unit for Inhibitor Binding Affinity

“…A similar noncompetitive binding had been previously observed for cyclopentane (+)-5, while furan (À)-4 had shown a mixed competitive inhibition mechanism. [8] After the results obtained with the ribose, deoxyribose, and ammonium derivatives, it came as quite a surprise that the reestablishment of both 2'-OH and 3'-OH groups in the series of carbocyclic inhibitors 13-20 led to completely inactive compounds in most of the cases studied (Tables 3 and 4). The seemingly conservative mutation of the ribose ether oxygen atom to a CH 2 unit leads to a dramatic decrease in inhibitory activity of five orders of magnitude (!)…”

“…[8] Most interestingly, the newly prepared 3'-deoxy derivatives (À)-6 and (À)-7 showed IC 50 values in the nanomolar range (40 and 180 nm, respectively), revealing only a % 4.5-fold decrease in biological activity relative to the parent ribose derivatives (À)-1 and (À)-2. The 200-fold difference in potency ( % 13.7 kJ mol À1 , Table 4) between the 2'-deoxy (À)-3 and the 3'-deoxy (À)-6 ribose derivatives is equally remarkable and unexpected.…”