Discovery of ML358, a Selective Small Molecule Inhibitor of the SKN‑1 Pathway Involved in Drug Detoxification and Resistance in Nematodes

Nematodes parasitize ∼1/3 of humans worldwide, and effective treatment via administration of anthelmintics is threatened by growing resistance to current therapies. The nematode transcription factor SKN-1 is essential for development of embryos and upregulates the expression of genes that result in modification, conjugation, and export of xenobiotics, which can promote resistance. Distinct differences in regulation and DNA binding relative to mammalian Nrf2 make SKN-1 a promising and selective target for the development of anthelmintics with a novel mode of action that targets stress resistance and drug detoxification. We report <b>17</b> (<b>ML358</b>), a first in class small molecule inhibitor of the SKN-1 pathway. Compound <b>17</b> resulted from a vanillamine-derived hit identified by high throughput screening that was advanced through analog synthesis and structure–activity studies. Compound <b>17</b> is a potent (IC₅₀ = 0.24 μM, <i>E</i>_max = 100%) and selective inhibitor of the SKN-1 pathway and sensitizes the model nematode <i>C. elegans</i> to oxidants and anthelmintics. Compound <b>17</b> is inactive against Nrf2, the homologous mammalian detoxification pathway, and is not toxic to <i>C. elegans</i> (LC₅₀ > 64 μM) and Fa2N-4 immortalized human hepatocytes (LC₅₀ > 5.0 μM). In addition, <b>17</b> exhibits good solubility, permeability, and chemical and metabolic stability in human and mouse liver microsomes. Therefore, <b>17</b> is a valuable probe to study regulation and function of SKN-1 <i>in vivo</i>. By selective targeting of the SKN-1 pathway, <b>17</b> could potentially lead to drug candidates that may be used as adjuvants to increase the efficacy and useful life of current anthelmintics

Overexpression of tissue-nonspecific alkaline phosphatase (TNAP) in endothelial cells accelerates coronary artery disease in a mouse model of familial hypercholesterolemia

<div><p>Objective</p><p>Overexpression of tissue-nonspecific alkaline phosphatase (TNAP) in endothelium leads to arterial calcification in mice. The purpose of this study was to examine the effect of elevated endothelial TNAP on coronary atherosclerosis. In addition, we aimed to examine endogenous TNAP activity in human myocardium.</p><p>Approach and results</p><p>A vascular pattern of TNAP activity was observed in human non-failing, ischemic, and idiopathic dilated hearts (5 per group); no differences were noted between groups in this study. Endothelial overexpression of TNAP was achieved in mice harboring a homozygous recessive mutation in the low density lipoprotein receptor (<i>whc</i> allele) utilizing a <i>Tie2-cre</i> recombinase (WHC-eTNAP mice). WHC-eTNAP developed significant coronary artery calcification at baseline compared WHC controls (4312 vs 0μm² alizarin red area, p<0.001). Eight weeks after induction of atherosclerosis, lipid deposition in the coronary arteries of WHC-eTNAP was increased compared to WHC controls (121633 vs 9330μm² oil red O area, p<0.05). Coronary lesions in WHC-eTNAP mice exhibited intimal thickening, calcifications, foam cells, and necrotic cores. This was accompanied by the reduction in body weight and left ventricular ejection fraction (19.5 vs. 23.6g, p<0.01; 35% vs. 47%, p<0.05). In a placebo-controlled experiment under atherogenic conditions, pharmacological inhibition of TNAP in WHC-eTNAP mice by a specific inhibitor SBI-425 (30mg*kg^-1*d^-1, for 5 weeks) reduced coronary calcium (78838 vs.144622μm²) and lipids (30754 vs. 77317μm²); improved body weight (22.4 vs.18.8g) and ejection fraction (59 vs. 47%). The effects of SBI-425 were significant in the direct comparisons with placebo but disappeared after TNAP-negative placebo-treated group was included in the models as healthy controls.</p><p>Conclusions</p><p>Endogenous TNAP activity is present in human cardiac tissues. TNAP overexpression in vascular endothelium in mice leads to an unusual course of coronary atherosclerosis, in which calcification precedes lipid deposition. The prevalence and significance of this mechanism in human atherosclerosis requires further investigations.</p></div

Effects of SBI-425 on plasma alkaline phosphatase activity, plasma pyrophosphate (PP_i), and atherosclerosis in WHC-eTNAP mice.

<p><b>(A)</b> Plasma alkaline phosphatase activity was measured in plasma from non-fasted mice collected one to three weeks after initiation of the treatment protocol. <b>(B)</b> Plasma PP_i was measured in plasma from non-fasted mice collected from a subset of animals one week after initiation of the treatment protocol. <b>(C)</b> Alizarin red staining for calcium is shown, representative images. <b>(D)</b> Oil red O staining, hematoxylin counterstained; representative images. <b>(E)</b> Quantification of calcium (based on alizarin red staining) and lipids (Oil red O staining) in coronary arteries. <b>(F)</b> Quantification of calcium (alizarin red staining) and lipids (Oil red O staining) in aortic roots. (C-F) Data were collected at 13 weeks of age; *, p < 0.05; **, p < 0.01; ***, p < 0.001.</p

Endothelial TNAP activity in human myocardium.

<p><b>(A)</b> Fifteen samples were analyzed—five from non-failing donor hearts (#1–5); five from ischemic HF (#6–10), and five from idiopathic dilated HF patients (#11–15); serial sections from each sample were stained with oil red O (hematoxylin counterstain), AP activity, and alizarin red; microscopic fields containing arteries were identified and captured; arrows, small arteries. <b>(B)</b> Quantification of AP activity in each category of samples expressed as AP-positive area per high power field (HPF). <b>(C)</b> Representative consecutive sections from sample #5 (non-failing) stained for AP activity in the absence (left) or presence (right) of 12.5 mM of L-homoarginine, a specific TNAP inhibitor. <b>(D)</b> A photomerged overlay image of AP activity (white) and alizarin red staining (brown) from sample #15 (idiopathic dilated HF); <b>(E)</b> Top panels, AP activity (dark blue) in combination with α-smooth muscle actin (SMA) immunohistochemistry (brown); bottom panels, same vessels stained with SMA antibody (red) in combination with endothelial specific isolectin B4 (IB4, green); arrow, AP activity.</p

Physiological characteristics of WHC and WHC-eTNAP mice (Mean ± SD).

<p>Physiological characteristics of WHC and WHC-eTNAP mice (Mean ± SD).</p

Effect of SBI-425 on plasma calcium, phosphorus, and lipids (Mean ± SD).

<p>Effect of SBI-425 on plasma calcium, phosphorus, and lipids (Mean ± SD).</p

Blood chemistry of WHC and WHC-eTNAP mice (Mean ± SD).

<p>Blood chemistry of WHC and WHC-eTNAP mice (Mean ± SD).</p

Physiologic parameters of WHC and WHC-eTNAP mice in the SBI-425 study (Mean ± SD).

<p>Physiologic parameters of WHC and WHC-eTNAP mice in the SBI-425 study (Mean ± SD).</p

Coronary artery lesions in WHC-eTNAP mice.

<p><b>(A)</b> Consecutive sections of a left coronary artery (LCA) and a septal branch of the right coronary artery (RCA) from a 16-weeks-old WHC mouse with hypercholesterolemia (H&E staining). Both vessels are unaffected by atherosclerosis. <b>(B)</b> Consecutive sections of LCA and RCA from a 16-weeks-old WHC-eTNAP mouse with hypercholesterolemia (H&E staining). Both vessels are affected by atherosclerosis, with the RCA being severely stenotic. <b>(C)</b> Immunohistochemical detection of osteocalcin (green) in 16-weeks-old WHC and WHC-eTNAP mice with hypercholesterolemia. Sections were counterstain with DAPI (blue). <b>(D)</b> Sections containing the RCA from 13-weeks-old WHC and WHC-eTNAP mice with hypercholesterolemia were stained with picrosirius red for collagen. <b>(E)</b> Oil red O staining (red) of the LCA from 16-weeks-old WHC and WHC-eTNAP mice with hypercholesterolemia, counterstained with hematoxylin (purple).</p

Expression of osteogenic and chondrogenic markers in aortas of WHC and WHC-eTNAP mice at 16 weeks of age (arbitrary units; Mean ± SD).

<p>Expression of osteogenic and chondrogenic markers in aortas of WHC and WHC-eTNAP mice at 16 weeks of age (arbitrary units; Mean ± SD).</p