Selective Inhibitors of Human Liver Carboxylesterase Based on a β‑Lapachone Scaffold: Novel Reagents for Reaction Profiling

Carboxylesterases (CEs) are ubiquitous enzymes that are responsible for the metabolism of xenobiotics, including drugs such as irinotecan and oseltamivir. Inhibition of CEs significantly modulates the efficacy of such agents. We report here that β-lapachone is a potent, reversible CE inhibitor with <i>K</i>_i values in the nanomolar range. A series of amino and phenoxy analogues have been synthesized, and although the former are very poor inhibitors, the latter compounds are highly effective in modulating CE activity. Our data demonstrate that tautomerism of the amino derivatives to the imino forms likely accounts for their loss in biological activity. A series of N-methylated amino derivatives, which are unable to undergo such tautomerism, were equal in potency to the phenoxy analogues and demonstrated selectivity for the liver enzyme hCE1. These specific inhibitors, which are active in cell culture models, will be exceptionally useful reagents for reaction profiling of esterified drugs in complex biological samples

HB1.F3.C1 cells injected intravenously localized to microscopic bone marrow disease.

<div><p>Concordant detection of v-<i>myc</i> (HB1.F3.C1 cells) by PCR and TH expression (neuroblastoma cells) by RT-PCR in bone marrow specimens.</p> <p>Bone marrow samples isolated from animals injected with HB1.F3.C1 cells were analyzed for the presence of v-<i>myc</i> (HB1.F3.C1 cells) or the expression of TH (NB-1643 cells).</p> <p>HB1.F3.C1 cells were present in the bone marrow only when tumor cells were also present.</p> <p>HB1.F3.C1 cells were not detected in the bone marrow of non-tumor-bearing animals.</p> <p>The positive controls (+) were DNA extracted from HB1.F3.C1 cells for v-<i>myc</i>, and RNA extracted from NB-1691 cells for TH.</p> <p>The negative controls (−) contained no DNA or RNA template, respectively.</p></div

Es1^e SCID mice injected intravenously with neuroblastoma cells develop multiple disseminated tumors.

<div><p>SK-N-AS cells (5×10⁵) transduced to express luciferase were injected into tail veins.</p> <p>One month following injection of tumor cells, mice were injected intraperitoneally with luciferin and imaged using a Xenogen IVIS imaging system, according the directions of the manufacturer.</p> <p>Multiple tumors were present in 100% of mice.</p> <p>Two representative mice are shown.</p></div

Human carboxylesterase 1 active site structure.

<p>Active site of human carboxylesterase 1 covalently inhibited via S221 with cyclosarin (magenta) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017441#pone.0017441-Hemmert1" target="_blank">[8]</a>. The other catalytic residues, in addition to S221, are H468 and E354 (yellow), and are surrounded by hydrophobic residues (grey surface) including V146 and L363 (light blue), as well as the oxyanion hole (white).</p

HB1.F3.C1 cells target macroscopic metastatic neuroblastoma in the bone marrow.

<div><p>X-ray image of hind limb of a mouse with advanced stage neuroblastoma (Day 82; left panel, scale bar: 1 cm).</p> <p>Confirmation of the tumor mass as human SK-N-AS neuroblastoma cells was performed by immunohistochemistry using anti-human mitochondria antibody (not shown).</p> <p>The CM-DiI-labeled HB1.F3.C1 cells (red cells, injected into the tail vein 3 days prior to sacrifice) localized to tumor in the marrow (right panel; scale bar: 200 µm).</p></div

Treatment protocol of HB1.F3.C1/AdCMVrCE/CPT-11 NDEPT.

<div><p>One day prior to being used in treatments, cells were transduced with the AdCMVrCE construct (see text).</p> <p>The treatment schedule was based on the time-course of expression of the secreted form of rCE, following adenoviral transduction of HB1.F3.C1 cells (Danks, unpublished observation) and on a schedule of administration of CPT-11 that has been shown to be relatively effective for neuroblastoma patients <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000023#pone.0000023-Furman1" target="_blank">[35]</a>.</p></div

Organophosphate (OP) inhibition of human carboxylesterase 1 (hCE1).

<p><b>A</b>. Three G-type OP nerve agents and OP model compound (R represents respective <i>O</i>-alkoxy groups). Wild-type hCE1 preferentially binds the stereoisomers shown (7). <b>B</b>. Schematic mechanism of OP hydrolysis by hCE1. X represents the leaving group and * denotes a non-reactive state.</p

Schematic diagram of the protocol for NDEPT.

<div><p>Human neuroblastoma tumor cells are injected intravenously to produce disseminated tumors.</p> <p>At an appropriate time after injection of neuroblastoma cells, neural stem cells or neural progenitor cells transduced with adenovirus to express a prodrug-activating enzyme (in this study, a secreted form of rabbit carboxylesterase [rCE]) are injected intravenously.</p> <p>Following migration of stem cells or progenitor cells to tumor foci and a delay of 3–4 days to allow relatively high level expression of the prodrug-activating enzyme into the extracellular milieu at the tumor sites, mice are treated with the prodrug (in this study, CPT-11).</p> <p>The prodrug is activated selectively at tumor foci, to increase the therapeutic index of the prodrug.</p></div

Catalytic efficiencies (<i>k</i>_cat/<i>K</i>_m) of engineered enzymes towards hemisubstrates.

a<p>(16).</p>b<p>(32).</p>c<p>(32).</p

HB1.F3.C1 cells transduced with an adenoviral multiplicity of infection of 5, 10, or 20 retain their tumor-tropism toward conditioned medium from SK-N-AS neuroblastoma cells.

<p>Bars represent the average number of migrated cells±SEM of triplicate wells in modified Boyden chamber assays.</p