Development of Flavonoid-Based Inverse Agonists of the Key Signaling Receptor US28 of Human Cytomegalovirus

A series of 31 chalcone- and flavonoid-based derivatives were synthesized in good overall yields and screened for their inverse agonist activity on the US28 receptor of human cytomegalovirus (HCMV). With one exception (e.g., 2-(5-bromo-2-methoxyphenyl)-3-hydroxy-4<i>H</i>-chromen-4-one), halogen-substituted flavonoids were typically more potent inverse agonists than their related hydro derivatives. While toxicity could be used to partially explain the inverse agonist activity of some members of the series, 5-(benzyloxy)-2-(5-bromo-2-methoxyphenyl)-4<i>H</i>-chromen-4-one (<b>11b</b>) acted on the US28 receptor as a nontoxic, inverse agonist. The full inverse agonism (efficacy, −89%) and potency (EC₅₀ = 3.5 μM) observed with flavonoid <b>11b</b> is especially important as it provides both a new tool to study US28 signaling and a potential platform for the future development of HCMV-targeting drugs

Prediction of the phenotypic effects of the coding SNPs rs37369 (p.Val140Ile) and rs16899974 (p.Val498Leu) in AGXT2 using structural information from <i>in silico</i> homology modeling.

<p>(A) Three-dimensional model of the AGXT2 (V140I) dimer showing the subunits A (blue) and B (cyan) in space-filled presentation. Residues Q83 (chain A), I140 (chain B), and V498 (chain A) are colored in orange, yellow and magenta, respectively. The β-aminoisobutyrate substrate of subunit A is colored according to the atom types. This view shows that V498 is buried in the interior of the molecule, whereas I140 of chain B is located close to the substrate binding site of subunit A. (B) Enlargement of the site of the V140I mutation. Residues 60–120 of chain A (blue) and residues 120–150 of chain B (cyan) are shown in ribbon presentation. A red arrow denotes a clash between the side chains of I140 (chain B; yellow) and Q83 (chain A; orange) that is not observed for V140 in the wild-type. (C) Enlargement of the C-terminal residues 400–500 showing the effect of a V498L mutation. The larger side chain of L498 forms steric clashes with the methylene groups of the K417 side chain (denoted by a red arrow).</p

Biochemical data of the KPE19 volunteer cohort.

<p>Plasma and urinary concentrations were measured by HPLC-MS/MS.</p><p>* Plasma concentrations were determined for 395 volunteers.</p>†<p>Urinary concentrations were measured for 400 volunteers.</p>‡<p>Serum creatinine concentrations and creatinine clearance were determined for 390 volunteers.</p>§<p>Cockcroft-Gault formula was used for calculation of creatinine clearance.</p

<i>A</i>nalysis of both determined <i>AGXT2</i> SNPs with regard to plasma (in µmol/L) and urinary (in µmol/g creatinine) BAIB concentrations.

<p>Median values (25–75% percentile) are shown. Both SNPs were significant for plasma and urinary BAIB in two-way ANOVA models using log-transformed data. The p values in the last rows and lower columns are based on nested ANOVA tests of log-transformed data (significant if p<0.025).</p><p><i>AGXT2</i>, <i>alanine-glyoxylate aminotransferase 2</i>; BAIB, β-aminoisobutyrate; Hetero, heterozygous for minor allele; Homo, homozygous for minor allele; n.d., not determined; WT (wild-type), homozygous for major allele.</p

<i>AGXT2</i> SNPs and biochemical measures.

<p>Median values (25–75% percentile) are shown. The Kruskal-Wallis test was used for statistical analysis.</p><p>* Plasma concentrations of methylarginines, BAIB and L-arginine were determined for 395 volunteers.</p>†<p>Serum concentrations of creatinine were determined for 390 volunteers.</p>‡<p>Cockgroft-Gault formula was used for calculation of creatinine clearance.</p><p>ADMA, asymmetric dimethylarginine; <i>AGXT2</i>, <i>alanine-glyoxylate aminotransferase 2</i>; BAIB, β-aminoisobutyrate; Hetero, heterozygous for minor allele; Homo, homozygous for minor allele; SDMA, symmetric dimethylarginine; WT (wild-type), homozygous for major allele.</p

<i>Alanine-glyoxylate aminotransferase 2</i> (<i>AGXT2</i>) Polymorphisms Have Considerable Impact on Methylarginine and β-aminoisobutyrate Metabolism in Healthy Volunteers

<div><p>Elevated plasma concentrations of asymmetric (ADMA) and symmetric (SDMA) dimethylarginine have repeatedly been linked to adverse clinical outcomes. Both methylarginines are substrates of alanine-glyoxylate aminotransferase 2 (AGXT2). It was the aim of the present study to simultaneously investigate the functional relevance and relative contributions of common <i>AGXT2</i> single nucleotide polymorphisms (SNPs) to plasma and urinary concentrations of methylarginines as well as β-aminoisobutyrate (BAIB), a prototypic substrate of AGXT2. In a cohort of 400 healthy volunteers ADMA, SDMA and BAIB concentrations were determined in plasma and urine using HPLC-MS/MS and were related to the coding <i>AGXT2</i> SNPs rs37369 (p.Val140Ile) and rs16899974 (p.Val498Leu). Volunteers heterozygous or homozygous for the <i>AGXT2</i> SNP rs37369 had higher SDMA plasma concentrations by 5% and 20% (p = 0.002) as well as higher BAIB concentrations by 54% and 146%, respectively, in plasma and 237% and 1661%, respectively, in urine (both p<0.001). ADMA concentrations were not affected by both SNPs. A haplotype analysis revealed that the second investigated <i>AGXT2</i> SNP rs16899974, which was not significantly linked to the other <i>AGXT2</i> SNP, further aggravates the effect of rs37369 with respect to BAIB concentrations in plasma and urine. To investigate the impact of the amino acid exchange p.Val140Ile, we established human embryonic kidney cell lines stably overexpressing wild-type or mutant (p.Val140Ile) AGXT2 protein and assessed enzyme activity using BAIB and stable-isotope labeled [²H₆]-SDMA as substrate. <i>In vitro</i>, the amino acid exchange of the mutant protein resulted in a significantly lower enzyme activity compared to wild-type AGXT2 (p<0.05). <i>In silico</i> modeling of the SNPs indicated reduced enzyme stability and substrate binding. In conclusion, SNPs of <i>AGXT2</i> affect plasma as well as urinary BAIB and SDMA concentrations linking methylarginine metabolism to the common genetic trait of hyper-β-aminoisobutyric aciduria.</p></div

Demographic data of the KPE19 volunteer cohort.

<p>* Median value (25–75% percentile) is shown.</p>†<p>self-reported.</p

Characterization of HEK cell lines overexpressing human wild-type and mutant (rs37369, p.Val140Ile) AGXT2 protein.

<p>HEK cells overexpressing human wild-type (AGXT2 WT) and mutant (AGXT2 rs37369) protein showed similar AGXT2 protein expression levels determined by immunoblot (A, representative immunoblot; 20 µg total protein per line) and immunofluorescence (B) analysis. AGXT2 was localized to mitochondria in both cell lines. Cells transfected with the empty vector (VC) were used as negative control. Enzyme activity of mutant AGXT2 indicated significant reduction of [²H₆]-SDMA degradation (C; each n = 10), [²H₆]-DM’GV formation (D; each n = 10) and BAIB degradation (E; each n = 5) compared to wild-type protein normalized to total protein. Area ratio is defined as [²H₆]-DM’GV/DM’GV. Values are median ±1.5 IQR. Unpaired 2-tailed Student’s <i>t</i> test (* p<0.05, *** p<0.001) was used for statistical analysis. AGXT2, alanine-glyoxylate aminotransferase 2; DM’GV, (N,N’-dimethyl-guanidino)valeric acid (AGXT2-dependent metabolite of SDMA); HEK cells, human embryonic kidney 293 cells; VC, vector control; WT, wild-type.</p

Correlation of ADMA, SDMA, BAIB and creatinine concentrations measured in blood and urine.

<p>For calculations the Spearman’s test was used.</p><p>ADMA, asymmetric dimethylarginine; BAIB, β-aminoisobutyrate; Crea, creatinine; n.s., not significant; SDMA, symmetric dimethylarginine.</p