AMPD2 inhibitor 1

The discovery of 3,3-dimethyl-1,2,3,4-tetrahydroquinoxaline-1-carboxamides as AMPD2 inhibitors with a novel mechanism of action

AMP deaminase 2 (AMPD2) has been thought to play an important role in energy homeostasis and immuno-oncology, while selective AMPD2 inhibitors are highly demanded to clarify the physiological function of AMPD2. In this report, we describe selective AMPD2 inhibitors inducing allosteric modulation. Based on hypothesis that compounds that exhibit increased inhibition by preincubation would cause conformational change of the enzyme, starting from HTS hit compound 4, we discovered compound 8 through the SAR study. From X-ray structural information of 8, this chemical series has a novel mechanism of action that changes the substrate pocket to prevent AMP from binding. Further elaboration of compound 8 led to the tool compound 21 which exhibited potent inhibitory activity of AMPD2 in ex vivo evaluation of mouse liver.

Metformin in vitro and in vivo increases adenosine signaling in rabbit corpora cavernosa

Introduction: In subjects with erectile dysfunction responding poorly to sildenafil, metformin was reported to improve erections.
Aims: The aim of this study is to investigate metformin's mechanism of action on erectile function, particularly focusing on adenosine (ADO) and nitric oxide (NO) signaling in an animal model of high-fat diet (HFD)-induced metabolic syndrome.
Methods: In vitro contractility studies of penile strips. Penile expression of genes related to ADO or NO signaling was also evaluated.
Main outcome measure: In vitro contractility studies were used to investigate the effect of in vivo and ex vivo metformin administration on ADO- or acetylcholine (Ach)-induced relaxation of penile strips from HFD as compared with animals fed a regular diet (RD).
Results: Expression of ADO receptor type 3 (A3 R), ADO deaminase (ADA), AMP deaminase type 1 (AMPD1), and 2 (AMPD2) was decreased in HFD as compared with RD. Accordingly, in HFD the ADO relaxant effect was potentiated as compared with RD (P < 0.02). In vivo metformin treatment in both RD and HFD significantly increased the ADO relaxing effect (P < 0.0001 and P < 0.01, respectively, vs. relative untreated groups) although to a different extent. In fact, the half-maximal inhibitory concentration (IC50 )/IC50 ratio in RD increased fourfold vs. HFD (RD IC50 ratio = 13.75 ± 2.96; HFD IC50 ratio = 2.85 ± 0.52). In corpora cavernosa (CC) from HFD, in vivo metformin (i) normalized A3 R, ADA, and AMPD1; (ii) further decreased AMPD2; (iii) increased dimethylarginine dimethylamino-hydrolase; and (iv) partially restored impaired Ach-induced relaxation. Ex vivo metformin time and dose dependently increased the relaxant effect of ADO in RD. The potentiating effect of metformin on ADO-induced relaxation was significantly reduced by preincubation with NO synthase inhibitor N(ω) -Nitro-L-arginine methyl ester hydrochloride (L-NAME). Interestingly, in vivo testosterone supplementation in HFD rabbits (i) increased penile expression of endothelial NO synthase and AMPD2 and (ii) restored metformin's potentiating effect on ADO-induced relaxation up to RD level.
Conclusion: Metformin in vivo and ex vivo increases ADO signaling in CC, most probably interfering with NO formation and ADO breakdown.

Synthesis and Biochemical Testing of 3-(Carboxyphenylethyl)imidazo[2,1-f][1,2,4]triazines as Inhibitors of AMP Deaminase

C-Ribosyl imidazo[2,1-f][1,2,4]triazines and 3-[2-(3-carboxyphenyl)ethyl]-3,6,7,8-tetrahydroimidazo[4,5-d][1,3]diazepin-8-ols represent two classes of known AMP deaminase inhibitors. A combination of the aglycone from the former class with the ribose phosphate mimic from the latter led to the 3-[2-(3-carboxyphenyl)ethyl]imidazo[2,1-f][1,2,4]triazines, which represent a new class of AMP deaminase inhibitors. The best compound, 3-[2-(3-carboxy-5,6,7,8-tetrahydronaphthyl)ethyl]imidazo[2,1-f][1,2,4]triazine (8), was a good inhibitor of all three human AMPD recombinant isozymes (AMPD1, AMPD2, and AMPD3; IC50 = 0.9-5.7 μM) but a poor inhibitor of the plant recombinant enzyme (Arabidopsis FAC1; IC50 = 200 μM).

Reversion in Chinese hamster lines amplified at the AMPD2 locus: spontaneous and benzamide-stimulated gradual loss of amplified alleles of marker genes

The HC47 and HC474 cell lines of Chinese hamster fibroblasts resist coformycin through the intrachromosomal amplification of the AMP deaminase 2 (AMPD2) gene. Due to the coamplification of a mu glutathione S-transferase (GST) gene, these mutant lines are more sensitive than GMA32 wild-type parental cells to buthionine sulfoximine (BSO), an inhibitor of glutathione biosynthesis. This property was exploited to select revertants of amplification from HC474 cells. Reversion in that line is frequently a gradual process that does not involve extrachromosomal intermediates. The terminal products of this process are commonly cells with a complete deletion of the amplified allele of marker genes and are therefore haploid for these loci on the homologous chromosome. Exposing HC474 cells to benzamide (BA), an inhibitor of polyADP-ribosylation, increased the recovery of revertants to an extent allowing the detection of reverting cells without BSO selection. This effect of BA was used to isolate revertant cells from the HC47 line that is extremely stable and to demonstrate that the mechanism of gradual reversion also occurs in this line. The gradual deletion of amplified copies within the chromosomes suggests that breakage-fusion-bridge (BFB) cycles drive this process.