Development of a Retinal-Based Probe for the Profiling of Retinaldehyde Dehydrogenases in Cancer Cells

Abstract

Retinaldehyde dehydrogenases belong to a superfamily of enzymes that regulate cell differentiation and are responsible for detoxification of anticancer drugs. Chemical tools and methods are of great utility to visualize and quantify aldehyde dehydrogenase (ALDH) activity in health and disease. Here, we present the discovery of a first-in-class chemical probe based on retinal, the endogenous substrate of retinal ALDHs. We unveil the utility of this probe in quantitating ALDH isozyme activity in a panel of cancer cells via both fluorescence and chemical proteomic approaches. We demonstrate that our probe is superior to the widely used ALDEFLUOR assay to explain the ability of breast cancer (stem) cells to produce all-trans retinoic acid. Furthermore, our probe revealed the cellular selectivity profile of an advanced ALDH1A1 inhibitor, thereby prompting us to investigate the nature of its cytotoxicity. Our results showcase the application of substrate-based probes in interrogating pathologically relevant enzyme activities. They also highlight the general power of chemical proteomics in driving the discovery of new biological insights and its utility to guide drug discovery efforts.

STA‐55, an Easily Accessible, Broad‐Spectrum, Activity‐Based Aldehyde Dehydrogenase Probe

“…[12,16] NCT-505 is ar ecently reported ALDH1A1 inhibi-tor, [10] for which we showed, with LEI-945, that it inhibited ALDH1A3 to as imilar extenta st hat of ALDH1A1 in living cells. [14] CB7 is reporteda saselective ALDH3A1 inhibitor. [11] A549 cells were preincubated for 30 min with inhibitor (10 mm), after which time STA-55 (1 mm)w as added and incubated for 1h.…”

“…Comparison with the Expression Atlas [20,21] showed that all ALDH enzymes expressed in the A549 cell line were detected by the STA-55 broad-spectrum ALDH probe ( Figure 3C), whereas the more specific retinal-based LEI-945 did not detect ALDH1B1and ALDH3A1int his cell line. [14] The proteins significantly enriched by STA-55 were further analyzed by using the KEGG, [22] Panther, [23] and UniProtKB databases. [24] Proteins in glycolysis/gluconeogenesis, biosynthesis of antibiotics, carbon metabolism (ALDHs, lactate dehydrogenases, and phosphofructokinases),p athogenic Escherichia coli infection, gap junction (tubulins), and the proteasome were identified from the KEGG pathwayd atabase ( Figure 3D).…”

“…We recentlyd escribed the development of LEI-945, which is af irst-in-class retinal-based probe for the ABPP of retinaldehyde dehydrogenases, ALDH1A1, ALDH1A2, and ALDH1A3. [14] LEI-945e nabled the quantification of ALDH isozyme activities in ap anel of cancerc ell lines through both fluorescencea nd chemical proteomic approaches. The probe was superior to the widely used ALDEFLUOR assay in explaining the ability of breast cancerc ells to produce all-trans retinoic acid.…”

“…In addition, it revealed the cellular selectivity profile of NCT-505, an advanced ALDH1A1 inhibitor. [14] However,t he synthesis of LEI-945 is complex and challenging. We hypothesized that, by modifying the reported covalent pan-ALDH inhibitor Aldi-2, [15] an easily accessible, broad-spectrum probe for the ALDH family could be made (Figure 1).…”

Targeting Liver Cancer Stem Cells: An Alternative Therapeutic Approach for Liver Cancer

“…These proteins were first described as enzymes conferring resistance against cyclophosphamide in in stem cells and cancer [ 85 ]. In cancer cells, ALDH functions as a retinal dehydrogenase (RALDH) that is involved in a metabolic process of converting retinol (also known as vitamin A) into active ligand retinoic acid (RA) [ 86 ]. Retinoic acids are known to play many roles in the regulation of major embryonic growth and patterning decisions.…”