Lestaurtinib Inhibits Histone Phosphorylation and Androgen-Dependent Gene Expression in Prostate Cancer Cells

Background: Epigenetics is defined as heritable changes in gene expression that are not based on changes in the DNA sequence. Posttranslational modification of histone proteins is a major mechanism of epigenetic regulation. The kinase PRK1 (protein kinase C related kinase 1, also known as PKN1) phosphorylates histone H3 at threonine 11 and is involved in the regulation of androgen receptor signalling. Thus, it has been identified as a novel drug target but little is known about PRK1 inhibitors and consequences of its inhibition. Methodology/Principal Finding: Using a focused library screening approach, we identified the clinical candidate lestaurtinib (also known as CEP-701) as a new inhibitor of PRK1. Based on a generated 3D model of the PRK1 kinase using the homolog PKC-theta (protein kinase c theta) protein as a template, the key interaction of lestaurtinib with PRK1 was analyzed by means of molecular docking studies. Furthermore, the effects on histone H3 threonine phosphorylation and androgen-dependent gene expression was evaluated in prostate cancer cells. Conclusions/Significance: Lestaurtinib inhibits PRK1 very potently in vitro and in vivo. Applied to cell culture it inhibits histone H3 threonine phosphorylation and androgen-dependent gene expression, a feature that has not been known yet. Thus our findings have implication both for understanding of the clinical activity of lestaurtinib as well as for future PRK

Chlorflavonin Targets Acetohydroxyacid Synthase Catalytic Subunit IlvB1 for Synergistic Killing of <i>Mycobacterium tuberculosis</i>

The flavonoid natural compound chlorflavonin was isolated from the endophytic fungus <i>Mucor irregularis</i>, which was obtained from the Cameroonian medicinal plant <i>Moringa stenopetala</i>. Chlorflavonin exhibited strong growth inhibitory activity <i>in vitro</i> against <i>Mycobacterium tuberculosis</i> (MIC₉₀ 1.56 μM) while exhibiting no cytotoxicity toward the human cell lines MRC-5 and THP-1 up to concentrations of 100 μM. Mapping of resistance-mediating mutations employing whole-genome sequencing, chemical supplementation assays, and molecular docking studies as well as enzymatic characterization revealed that chlorflavonin specifically inhibits the acetohydroxyacid synthase catalytic subunit IlvB1, causing combined auxotrophies to branched-chain amino acids and to pantothenic acid. While exhibiting a bacteriostatic effect in monotreatment, chlorflavonin displayed synergistic effects with the first-line antibiotic isoniazid and particularly with delamanid, leading to a complete sterilization in liquid culture in combination treatment. Using a fluorescent reporter strain, intracellular activity of chlorflavonin against <i>Mycobacterium tuberculosis</i> inside infected macrophages was demonstrated and was superior to streptomycin treatment

Known and newly identified PRK1 inhibitors.

<p>Known and newly identified PRK1 inhibitors.</p

Details of the binding of (A) staurosporine (green), (B) K252a (orange), (C) lestaurtinib (cyan), and (D) Ro318220 (dark-yellow) to the PRK1 kinase domain.

<p>Common interactions of the inhibitors are hydrogen bonds involving Glu696 and Ser698 of the hinge region, and van-der-Waals interactions with the gatekeeper residue Met695, as well as with Val629, Phe626, Leu747, and Phe904. In addition, some of the inhibitors interact with Asp744, Asn745 and a conserved water molecule (red sphere) nearby the Mg²⁺ binding site of the kinase. The backbone is shown as a purple ribbon. Only relevant amino acids are displayed. Hydrogen bonds are shown as dashed orange colored lines.</p

Effects of lestaurtinib on the mRNA expression of the androgen receptor target genes.

<p>Expression of androgen receptor target genes <i>TMPRSS2</i>, <i>IGF1-R</i>, <i>NKX3.1</i>, <i>CXCR4</i>, <i>MAK</i>, <i>MAF</i>, <i>N4A1</i>, <i>GREB1</i> and <i>FKBP5</i> measured by qRT-PCR in androgen (R1881) stimulated LNCaP prostate cancer cells are lowered by the treatment with lestaurtinib (final concentration 5 µM). The mRNA expression of the <i>GAPDH</i> gene was used as a control. Bars represent mean + SD (n = 5). P-value: ns  =  non significant; *  =  <0.05; **<0.01; ***<0.001.</p

PRK1 in vitro binding.

<p>PRK1 in vitro binding.</p