Minocycline and tigecycline form higher-order Ca2+ complexes of stronger affinity than tetracycline

Abstract

Tetracycline (TC), minocycline (MIN) and tigecycline (TIG) share a divalent metal ion binding site essential for mediating their antibiotic function. Isothermal titration calorimetry (ITC) and UV–Vis absorbance measurements combined with the Job’s Method were used to study their interaction with Ca2+. 1H NMR was also used to probe the interaction of TIG with Ca2+. Results from ITC showed that MIN and TIG bind Ca2+ more strongly than TC by 10–16 and 8–12 times, respectively, at pH 6.8, or by 23–56 and 20–42 times, respectively, at pH 7.5, in NEM and Tris buffers. The higher affinities can be attributed to the electron donating dimethylamino group at C7 of MIN and TIG which strengthened the Ca2+–antibiotic bonds. Both ITC and the Job’s Method showed that all three antibiotics formed higher order Ca2+ complexes at pH 6.8 in the form of 1Ca2+–3TC, 1Ca2+–3MIN and 2Ca2+–3TIG. As pH was increased to 7.5, TC preferred a lower order complex (1Ca2+–1TC) while MIN and TIG persisted in their respective higher order complex. The preference of MIN and TIG for higher order complexes is attributed to their stronger affinity for Ca2+ and more lipophilic nature. The different complexation mode in the 2Ca2+–3TIG versus 1Ca2+–3MIN complex is attributed to the N-t-butylglycylamido group, which is unique to TIG, participating in Ca2+ coordination along with the β-ketoenol moiety common to all three antibiotics. These results are consistent with TIG having substantially higher affinity for the bacterial ribosome, broader spectrum of antimicrobial activity and evasion of classic tetracycline-resistance mechanisms and with MIN being more effective than TC as an anti-inflammatory drug. These results do not correlate with their relative oral bioavailability. The current findings may aid in the rational design of novel tetracycline derivatives.