BODIPY-Caged Photoactivated Inhibitors of Cathepsin B Flip the Light Switch on Cancer Cell Apoptosis

Acquired resistance to apoptotic agents is a long-standing challenge in cancer treatment. Cathepsin B (CTSB) is an enzyme which, among many essential functions, promotes apoptosis during cellular stress through regulation of intracelllular proteolytic networks on the minute timescale. Recent data indicate that CTSB inhibition may be a promising method to steer cells away from apoptotic death towards necrosis, a mechanism of cell death that can overcome resistance to apoptotic agents, stimulate an immune response and promote anti-tumor immunity. Unfortunately, rapid and selective intracellular inactivation of CTSB has not been possible. However, here we report on the synthesis and characterization of photochemical and biological properties of BODIPY-caged inhibitors of CTSB that are cell permeable, highly selective and activated rapidly upon exposure to visible light. Intriguingly, these compounds display tunable photophysical and biological properties based on substituents bound directly to boron. Me2BODIPY-caged compound 8 displays the dual-action capability of light-accelerated CTSB inhibition and singlet oxygen production from a singular molecular entitiy. The dual-action capacity of 8 leads to a rapid necrotic response in MDA-MB-231 triple negative breast cancer cells with high phototherapeutic indexes (\u3e30) and selectivity vs. non-cancerous cells that neither CTSB inhibition nor photosensitization gives alone. Our work confirms that singlet oxygen production and CTSB inactivation is highly synergistic and a promising method for killing cancer cells. Furthermore, our ability to trigger intracellular inactivation of CTSB with light will provide researchers with a powerful photochemical tool for probing biochemical processes on short timescales

Synthesis, Characterization, and Glutathionylation of Cobalamin Model Complexes [Co(N4PyCO₂Me)Cl]Cl₂ and [Co(Bn-CDPy3)Cl]Cl₂

Synthetic Co­(III) complexes containing N5 donor sets undergo glutathionylation to generate biomimetic species of glutathionylcobalamin (GSCbl), an important form of cobalamin (Cbl) found in nature. For this study, a new Co­(III) complex was synthesized derived from the polypyridyl pentadentate N5 ligand N4PyCO₂Me (<b>1</b>). The compound [Co­(N4PyCO₂Me)­Cl]­Cl₂ (<b>3</b>) was characterized by X-ray crystallography, UV–vis, IR, ¹H NMR, and ¹³C NMR spectroscopies and mass spectrometry (HRMS). Reaction of <b>3</b> with glutathione (GSH) in H₂O generates the biomimetic species [Co­(N4PyCO₂Me)­(SG)]²⁺ (<b>5</b>), which was generated <i>in situ</i> and characterized by UV–vis and ¹H NMR spectroscopies and HRMS. ¹H NMR and UV–vis spectroscopic data are consistent with ligation of the cysteine thiolate of GSH to the Co­(III) center of <b>5</b>, as occurs in GSCbl. Kinetic analysis indicated that the substitution of chloride by GS^– occurs by a second-order process [<i>k</i>₁ = (10.1 ± 0.7) × 10^–2 M^–1 s^–1]. The observed equilibrium constant for formation of <b>5</b> (<i>K</i>_obs = 870 ± 50 M^–1) is about 3 orders of magnitude smaller than for GSCbl. Reaction of the Co­(III) complex [Co­(Bn-CDPy3)­Cl]­Cl₂ (<b>4</b>) with GSH generates glutathionylated species [Co­(Bn-CDPy3)­(GS)]²⁺ (<b>6</b>), analogous to <b>5</b>. Glutathionylation of <b>4</b> occurs at a similar rate [<i>k</i>₂ = (8.4 ± 0.5) × 10^–2 M^–1 s^–1], and the observed equilibrium constant (<i>K</i>_obs = 740 ± 47 M^–1) is slightly smaller than for <b>5</b>. Glutathionylation showed a significant pH dependence, where rates increased with pH. Taken together, these results suggest that glutathionylation is a general reaction for Co­(III) complexes related to Cbl

Heteroleptic Copper(I)-Based Complexes Incorporating BINAP and π-Extended Diimines: Synthesis, Catalysis and Biological Applications

A series of copper-based photocatalysts of the type Cu(NN)(BINAP)BF4 were synthesized bearing π-extended diimine ligands. Their behavior in several photocatalytic processes were evaluated and revealed acceptable levels of activity in an SET process, but negligible activity in PCET or ET processes. Suitable activity in ET processes could be restored through modification of the ligand. The BINAP-derived complexes were then evaluated for activity against triple-negative breast cancer cell lines. Controls indicated that copper complexes, and not their ligands, were responsible for activity. Encouraging activity was displayed by a homoleptic complex Cu(dppz)2BF4

Cumyl Ester as the C-Terminal Protecting Group in the Enantioselective Alkylation of Glycine Benzophenone Imine

Cumyl ester is an optimal C-terminal protecting group for glycine benzophenone imine in asymmetric alkylation reactions catalyzed by <i>Cinchona</i> chiral phase-transfer catalysts. High levels of enantioselectivity have been obtained (up to 94% ee) with this substrate, which provides an attractive alternative to the analogous <i>tert</i>-butyl ester. N-terminal imines and the C-terminal esters can be cleaved from alkylation products by hydrogenolysis, while maintaining acid-labile side chain protecting groups

Ir(III)-Based Agents for Monitoring the Cytochrome P450 3A4 Active Site Occupancy.

Cytochromes P450 (CYPs) are a superfamily of enzymes responsible for biosynthesis and drug metabolism. Monitoring the activity of CYP3A4, the major human drug-metabolizing enzyme, is vital for assessing the metabolism of pharmaceuticals and identifying harmful drug-drug interactions. Existing probes for CYP3A4 are irreversible turn-on substrates that monitor activity at specific time points in end-point assays. To provide a more dynamic approach, we designed, synthesized, and characterized emissive Ir(III) and Ru(II) complexes that allow monitoring of the CYP3A4 active-site occupancy in real time. In the bound state, probe emission is quenched by the active-site heme. Upon displacement from the active site by CYP3A4-specific inhibitors or substrates, these probes show high emission turn-on. Direct probe binding to the CYP3A4 active site was confirmed by X-ray crystallography. The lead Ir(III)-based probe has nanomolar Kd and high selectivity for CYP3A4, efficient cellular uptake, and low toxicity in CYP3A4-overexpressing HepG2 cells

Affinity-Enhanced Luminescent Re(I)- and Ru(II)-Based Inhibitors of the Cysteine Protease Cathepsin L

Two new Re­(I)- and Ru­(II)-based inhibitors were synthesized with the formulas [Re­(phen)­(CO)₃(<b>1</b>)]­(OTf) (<b>7</b>; phen = 1,10-phenanthroline, OTf = trifluoromethanesulfonate) and [Ru­(bpy)₂(<b>2</b>)]­(Cl)₂ (<b>8</b>; bpy = 2,2′-bipyridine), where <b>1</b> and <b>2</b> are the analogues of CLIK-148, an epoxysuccinyl-based cysteine cathepsin L inhibitor (CTSL). Compounds <b>7</b> and <b>8</b> were characterized using various spectroscopic techniques and elemental analysis; <b>7</b> and <b>8</b> both show exceptionally long excited state lifetimes. Re­(I)-based complex <b>7</b> inhibits CTSL in the low nanomolar range, affording a greater than 16-fold enhancement of potency relative to the free inhibitor <b>1</b> with a second-order rate constant of 211000 ± 42000 M^–1 s^–1. Irreversible ligation of <b>7</b> to papain, a model of CTSL, was analyzed with mass spectroscopy, and the major peak shown at 24283 au corresponds to that of papain-<b>1</b>-Re­(CO)₃(phen). Compound <b>7</b> was well tolerated by DU-145 prostate cancer cells, with toxicity evident only at high concentrations. Treatment of DU-145 cells with <b>7</b> followed by imaging via confocal microscopy showed substantial intracellular fluorescence that can be blocked by the known CTSL inhibitor CLIK-148, consistent with the ability of <b>7</b> to label CTSL in living cells. Overall this study reveals that a Re­(I) complex can be attached to an enzyme inhibitor and enhance potency and selectivity for a medicinally important target, while at the same time allowing new avenues for tracking and quantification due to long excited state lifetimes and non-native element composition