Ethynylflavones, Highly Potent, and Selective Inhibitors of Cytochrome P450 1A1

The flavone backbone is a well-known pharmacophore present in a number of substrates and inhibitors of various P450 enzymes. In order to find highly potent and novel P450 family I enzyme inhibitors, an acetylene group was incorporated into six different positions of flavone. The introduction of an acetylene group at certain locations of the flavone backbone lead to time-dependent inhibitors of P450 1A1. 3′-Ethynylflavone, 4′-ethynylflavone, 6-ethynylflavone, and 7-ethynylflavone (<i>K</i>_I values of 0.035–0.056 μM) show strong time-dependent inhibition of P450 1A1, while 5-ethynylflavone (<i>K</i>_I value of 0.51 μM) is a moderate time-dependent inhibitor of this enzyme. Meanwhile, 4′-ethynylflavone and 6-ethynylflavone are highly selective inhibitors toward this enzyme. Especially, 6-ethynylflavone possesses a <i>K</i>_i value of 0.035 μM for P450 1A1 177- and 15-fold lower than those for P450s 1A2 and 1B1, respectively. The docking postures observed in the computational simulations show that the orientation of the acetylene group determines its capability to react with P450s 1A1 and 1A2. Meanwhile, conformational analysis indicates that the shape of an inhibitor determines its inhibitory selectivity toward these enzymes

SMN protein stability in whole blood: short term, long term, and freeze / thaw events.

<p>Whole blood of healthy subjects was used in the study. (A) SMN protein was measured in previously frozen, undiluted whole blood samples incubated at 4°C or at room temperature. (B) SMN protein was measured in undiluted whole blood samples of two subjects stored at -80°C or at -20°C. (C) SMN protein levels were measured in samples of two subjects that went through freeze-thaw cycles. *FDA acceptance criteria (below 85%).</p

Correlation of SMN protein levels between tissues.

<p>SMN protein levels were correlated in WT and C/C-allele SMA mice between (A) whole blood and brain and (B) whole blood and spinal cord.</p

SMN protein levels in SMA patient and control whole blood samples.

<p>(A) SMN levels with respect to age in all subjects. (B) SMN protein levels were measured in SMA patients with 2, 3 and 4 copies of <i>SMN2</i>. In patients over 2 months of age, SMN levels were significantly greater in SMA patient samples with 4 <i>SMN2</i> copies relative to those with 2 and 3 <i>SMN2</i> copies (p = 0.0001). (C) SMN was also measured in three control samples and SMN levels were found to be significantly greater in the control samples relative to levels in SMA patients over 2 months of age (p < 0.0001).</p

SMN protein levels in tissues of C/C-allele and WT mice measured by SMN-ECL and SMN-ELISA.

<p>Protein levels were measured in the spinal cord of C/C-allele and WT mice using (A) SMN-ECL and (B) SMN-ELISA. Both assays showed a statistically significant difference in SMN levels between WT and C/C-allele mice (p < 0.0001). (C) SMN protein levels in the whole blood of C/C-allele, WT and heterozygous mice measured by SMN-ECL.</p

SMN protein levels in WT and C/C-allele SMA mice of different ages.

<p>SMN protein levels were measured by SMN-ECL at various days post-birth in (A) brain, (B) whole blood, (C) spinal cord, and (D) gastrocnemius muscle.</p

SMN protein was found in various cell types in whole blood.

<p>Platelets, red blood cells, PBMCs and reticulocytes were separated by a fractionation procedure.</p

SMN protein levels in capillary and venous blood obtained over time from healthy volunteers did not vary significantly.

<p>Venous (A, B) and capillary (C) whole blood samples were obtained at 0, 4, 6, 24, 48, 72 hours and 1, 2, 3, 4 weeks from five healthy individuals. Fig 2B is an expanded version of Fig 2A. (D) SMN protein levels in capillary blood correlated significantly with SMN levels in venous blood (r² = 0.76, p < 0.0001).</p

SMN only detected in cerebral spinal fluid samples containing hemoglobin.

<p>The CSF samples obtained from healthy volunteers were concentrated prior to analysis, and the sensitivity of the SMN-ECL immunoassay was 0.3 pg/mL. Hemoglobin was measured using a hemoglobin immunoassay (Bethyl Laboratories E88-135). Approximately 3 pg of SMN correspond to 10,000 ng of hemoglobin in 1 mL of whole blood from a healthy adult. LLQ: lower limit of quantification.</p