1,4-Dideoxy-1,4-imino-d- and l-lyxitol-based inhibitors bind to Golgi α-mannosidase II in different protonation forms

The development of effective inhibitors of Golgi alpha-mannosidase II (GMII, E.C.3.2.1.114) with minimal off-target effects on phylogenetically-related lysosomal alpha-mannosidase (LMan, E.C.3.2.1.24) is a complex task due to the complicated structural and chemical properties of their active sites. The pK(a) values (and also protonation forms in some cases) of several ionizable amino acids, such as Asp, Glu, His or Arg of enzymes, can be changed upon the binding of the inhibitor. Moreover, GMII and LMan work under different pH conditions. The pK(a) calculations on large enzyme-inhibitor complexes and FMO-PIEDA energy decomposition analysis were performed on the structures of selected inhibitors obtained from docking and hybrid QM/MM calculations. Based on the calculations, the roles of the amino group incorporated in the ring of the imino-d-lyxitol inhibitors and some ionizable amino acids of Golgi-type (Asp270-Asp340-Asp341 of Drosophila melanogaster alpha-mannosidase dGMII) and lysosomal-type enzymes (His209-Asp267-Asp268 of Canavalia ensiformis alpha-mannosidase, JBMan) were explained in connection with the observed inhibitory properties. The pyrrolidine ring of the imino-d-lyxitols prefers at the active site of dGMII the neutral form while in JBMan the protonated form, whereas that of imino-l-lyxitols prefers the protonation form in both enzymes. The calculations indicate that the binding mechanism of inhibitors to the active-site of alpha-mannosidases is dependent on the inhibitor structure and could be used to design new selective inhibitors of GMII. A series of novel synthetic N-substituted imino-d-lyxitols were evaluated with four enzymes from the glycoside hydrolase GH38 family (two of Golgi-type, Drosophila melanogaster GMIIb and Caenorhabditis elegans AMAN-2, and two of lysosomal-type, Drosophila melanogaster LManII and Canavalia ensiformis JBMan, enzymes). The most potent structures [N-9-amidinononyl and N-2-(1-naphthyl)ethyl derivatives] inhibited GMIIb (K-i = 40 nM) and AMAN-2 (K-i = 150 nM) with a weak selectivity index (SI) toward Golgi-type enzymes of IC50(LManII)/IC50(GMIIb) = 35 or IC50(JBMan)/IC50(AMAN-2) = 86. On the other hand, weaker micromolar inhibitors, such as N-2-naphthylmethyl or 4-iodobenzyl derivatives [IC50(GMIIb) = 2.4 mu M and IC50 (AMAN-2) = 7.6 mu M], showed a significant SI in the range from 111 to 812.

Automated summary

The development of effective inhibitors of Golgi alpha-mannosidase II (GMII) with minimal off-target effects is a complex task due to the complicated structural and chemical properties of their active sites. This study reveals that the binding mechanism of inhibitors to alpha-mannosidases is dependent on the inhibitor structure and can be used to design new selective inhibitors of GMII.