Mammals express nine membranous adenylyl cyclases (AC 1-9) for the catalysis of the important second messenger cAMP in the intracellular signaling pathway. Inhibitors of AC1 and AC5 may be useful drugs for neuroprotection, heart failure and longevity. 2�,3�-O-(N-Methylanthraniloyl) (MANT)-substituted nucleotides are competitive AC inhibitors and were synthesized with different nucleobases in high purity after reversed phase HPLC separation. MANT-nucleotides were tested for their inhibitory potency at recombinant AC1, 2, and 5 expressed by a Sf9 insect cell system. In our primary investigations MANT-ITP was identified as the most potent AC1 and 5 inhibitor known so far with Ki values of 1 - 3 nM.
Interestingly, bis-substituted MANT-nucleotides were discovered as side products in the regular synthesis of MANT-nucleotides. The synthesis of new mono- and bis-substituted anthraniloyl-group derived purine nucleotides was expanded. The anthraniloyl moiety differ by halogens of chlorine and bromine and acetylated amino group in 5 position of the phenyl ring system. Moreover, substitution at the amino function of the ANT-group lead to propyl derivatives.
The immobile and aerobic bacterium Bordetella pertussis secretes the key virulence factor, the adenylyl cyclase toxin CyaA. Overall, 32 compounds were prepared to compare 16 pairs of mono- and bis-substituted (M)ANT-nucleotides for their potencies at inhibiting CyaA and membranous ACs (mAC). For the first time we found inhibitors combining high inhibition potency for CyaA toxin with selectivity towards mammalian ACs. Bis-substituted halogen anthraniloyl-derived purine nucleotides inhibited CyaA in the nanomolar range in a competitive manner (Ki = 13 - 20 nM). Moreover, bis-substituted halogen anthraniloyl-derived nucleotides of adenine displayed not only high affinity to the bacterial AC, but also revealed high selectivity by 50- to 150-fold depending on the chosen mAC 1, 2 or 5. The modeling study demonstrate an impression for the obvious alignment of the (M)ANT-nucleotide binding mode to CyaA. The docking of bis-substituted halogen anthraniloyl derivatives exposed additional hydrophobic interactions between enzyme and inhibitor causing higher potency for this class of compounds.
Bis-substituted (M)ANT-nucleotides offered the advantage of excellent signal to noise ratio in fluorescence spectroscopy, compared to mono-substituted (M)ANT-nucleotides, with applications in HTS for assessment of non-fluorescent inhibitor potencies
