Crafting Molecular Tools for 15-Lipoxygenase-1 in a Single Step

Abstract

Small molecule modulators are powerful tools for selectively probing and manipulating proteins in native biological systems. However, the development of versatile modulators that exhibit desired properties is hindered by the lack of a rapid and robust synthetic strategy. Here, we develop a facile and reliable one-step methodology for the generation of multifunctional toolboxes encompassing a wide variety of chemical modulators with different desired features. These modulators bind irreversibly to the protein target via a selective warhead. Key elements are introduced onto the warhead in a single step using multi-component reactions. To illustrate the power of this new technology, we synthesized a library of diverse modulators designed to explore a highly challenging and poorly understood protein, human 15-lipoxygenase-1. Modulators made include; activity-based/photoaffinity probes, chemosensors, photocrosslinkers, as well as light-controlled and high-affinity inhibitors. The efficacy of our compounds was successfully established through the provision of on demand inhibition and labeling of our target protein in vitro, in cellulo and in vivo; thus, proving that this technology has promising potential for applications in many complex biological systems.

Keywords: covalent inhibitors; human 15-lipoxygenase-1; photopharmacology; probes; small molecule modulators.

Figure 1

Strategy for the development of BAM compounds. The covalent anchor (BA) was incorporated through a reactive group (aldehyde) in 2CRs and MCRs to yield BAM probes, sensors, photocrosslinkers, as well as light‐controlled and high‐affinity inhibitors. Modulation of 15‐LOX‐1 activity occurred following a time‐dependent binding of the respective BAM compound, accomplished in a single step.

Figure 2

Rational development of potent binders. a) 2D plot analysis of previously identified 15‐LOX‐1 inhibitors, based on lipophilicity (clogP) and shape index, as well as the molecular weight (MW) and the polar surface area. The size and the color of every dot represents the quantity of similar scaffolds and the inhibitory value (nM), respectively. b) Representation of the inhibitory potency of amine (nM), acid (nM) and isocyanide (at 50 μΜ) fragments against 15‐LOX‐1. The active fragments are presented and the common structural component is highlighted. The color gradient represents the inhibitory potency, ranging from high (green) to low (red).

Figure 3

Synthesis of BAM compounds. a) Reagents and conditions: a) PCC, dry DCM, rt, 2 h; b) 1‐bromo‐2‐butyne, CuI, NaI, K₂CO₃, DMF, rt, 48 h; c) hydrazine, MeOH, rt or benzohydrazide, THF, rt, 16 h; d) 2‐aminopyridine, isocyanide, Sc(OTf)₃, MeOH, r.t. or 45 °C, 16–24 h; e) carboxylic acid, isocyanide, DCM, rt, 12 h; f) amine, isocyanide, carboxylic acid, MeOH, r.t., 16–24 h; g) amine, isocyanide, TMSN₃, MeOH, 45 °C, 16–24 h. b) The libraries of the synthesized BAM compounds obtained from different reactions with their corresponding yields. The color represents the inhibitory value (IC₅₀ in nM scale, 20 min preincubation) as shown. The symbol represents the potential application of the compound, lightning bolt: photo‐switchable; magnet: inhibitor; star: probe; anchor: cross‐linker.

Figure 4

Exploring the nature of inhibition. a) Lineweaver–Burk plot of BAM1. b) Inhibition of 15‐LOX‐1 with the IC₅₀ graphs of 10 and 20 minutes of incubation with BAM2 (left) and C6 (right). c) The active site of human 15‐LOX‐1. Residues Y395, T396 and L397, identified by MS experiments, are shown in orange sticks. d) Table of IC₅₀ values (10 and 20 minutes) of the synthesized BAM compounds. All values are reported with their standard deviation. e) Chemical structures of control non‐covalent compounds, with key structural elements highlighted. f) IC₅₀ curves showing the different inhibitory potencies of BAM5 (left) and BAM10 (right), using AA or LA as substrates in the enzymatic reaction. g) Inhibition of 15‐LOX‐1 and soybean LOX with the IC₅₀ curves after 30 min incubation with the compound BAM3. All experiments were performed in triplicates (n=3), and the standard error is reported.

Figure 5

The photo‐induced features of BAMs. a) The absorption/emission spectra of BAM14. b) The absorption/emission spectra of BAM10. c) The fluorescence of BAM14 (left) or BAM10 (right) after labeling of 15‐LOX‐1. Emission spectra for the supernatant (yellow) and resuspended pellet (cyan) after acetone precipitation. A blank sample of acetone is represented with black (left). Dot‐blot (fluorescence) of BAM10‐treated 15‐LOX‐1, with or without preincubation with ThioLox. Control experiments in the absence of 15‐LOX‐1 is shown in the bottom. Experiments were performed in duplicates (n=2). d) Emission spectra of BAM14 in presence of 5 and 20 equivalents of Fe³⁺. e) Absorption spectra of BAM12 showing the conversion from the E to Z conformation upon increasing exposure time of UV light, and return to the E conformation after heat treatment. f) Reversible photochromism of BAM12 (5 cyclic irradiations). g) Inhibitory potency of BAM12 before and after UV irradiation. h) Inhibitory potency of BAM13 before and after UV irradiation. All IC₅₀ experiments were performed in triplicates (n=3), and the standard error is reported. i) SDS‐page reveals increasing crosslinked protein species, after incubation of BAM13 (or DMSO: C) with cell lysate (with overexpressed 15‐LOX‐1) and application of UV irradiation for 0, 10, 20 and 30 min (L: protein ladder).

Figure 6

BAM10 in live cell and in vivo imaging applications. a) Workflows for competition and irreversibility assays to assess in cellulo target engagement of 15‐LOX‐1 by BAM10, using confocal microscopy. RAW 264.7 cells were seeded on glass‐bottom dishes and treated in situ. b) Representative images of live cells pre‐treated with either DMSO or BAM3 (50 μM) or Thiolox (50 μM) and subsequently labelled with 5 μM BAM10. c) Boxplot showing the quantified intensity per cell area from experiments in b. d) Irreversibility assay where live cells were initially incubated with BAM10 for various durations (3.75 to 120 minutes) and then treated with DMSO or ThioLox (50 μM) for 1 hour before imaging. e) BLAST results showing C. elegans genes with high sequence identity to 15‐LOX‐1. Colored sequence regions correspond to the highlighted colored areas in the 15‐LOX‐1 structure. f) 16‐hour incubation of C. elegans with BAM10 leads to significantly higher fluorescence intensity on the pharynx and the intestine. g) Stereoscope images of C. elegans on NGM plates treated with DMSO (Ctr), paraquat (PQ) or BAM10. C. elegans grown on BAM10 reached similar sizes and showed normal egg development in their gonads, comparable to control samples. Additionally, unlike the negative control (PQ‐treated plates), BAM10 does not appear to repel animals from the food source. h) Box‐plot of the quantified intensity per area from experiments using 5 μM BAM10 (left) pre‐incubated with DMSO or BAM3 (50 μM) or Thiolox (50 μM), (right) on RNAi constructs. Cell culture experiments were performed in triplicates (n=3). Seven or more cell areas were acquired per replicate. For the C. elegans experiments, up to 25 animals per condition were used. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.