Alterations in sphingolipid composition and mitochondrial bioenergetics represent synergistic therapeutic vulnerabilities linked to multidrug resistance in leukemia.

Modifications in sphingolipid (SL) metabolism and mitochondrial bioenergetics are key factors implicated in cancer cell response to chemotherapy, including chemotherapy resistance. In the present work, we utilized acute myeloid leukemia (AML) cell lines, selected to be refractory to various chemotherapeutics, to explore the interplay between SL metabolism and mitochondrial biology supportive of multidrug resistance (MDR). In agreement with previous findings in cytarabine or daunorubicin resistant AML cells, relative to chemosensitive wildtype controls, HL-60 cells refractory to vincristine (HL60/VCR) presented with alterations in SL enzyme expression and lipidome composition. Such changes were typified by upregulated expression of various ceramide detoxifying enzymes, as well as corresponding shifts in ceramide, glucosylceramide, and sphingomyelin (SM) molecular species. With respect to mitochondria, despite consistent increases in both basal respiration and maximal respiratory capacity, direct interrogation of the oxidative phosphorylation (OXPHOS) system revealed intrinsic deficiencies in HL60/VCR, as well as across multiple MDR model systems. Based on the apparent requirement for augmented SL and mitochondrial flux to support the MDR phenotype, we explored a combinatorial therapeutic paradigm designed to target each pathway. Remarkably, despite minimal cytotoxicity in peripheral blood mononuclear cells (PBMC), co-targeting SL metabolism, and respiratory complex I (CI) induced synergistic cytotoxicity consistently across multiple MDR leukemia models. Together, these data underscore the intimate connection between cellular sphingolipids and mitochondrial metabolism and suggest that pharmacological intervention across both pathways may represent a novel treatment strategy against MDR.

Regulatory mechanism for the transmembrane receptor that mediates bidirectional vitamin A transport.

Vitamin A has diverse biological functions and is essential for human survival at every point from embryogenesis to adulthood. Vitamin A and its derivatives have been used to treat human diseases including vision diseases, skin diseases, and cancer. Both insufficient and excessive vitamin A uptake are detrimental, but how its transport is regulated is poorly understood. STRA6 is a multitransmembrane domain cell-surface receptor and mediates vitamin A uptake from plasma retinol binding protein (RBP). STRA6 can mediate both cellular vitamin A influx and efflux, but what regulates these opposing activities is unknown. To answer this question, we purified and identified STRA6-associated proteins in a native mammalian cell type that takes up vitamin A through STRA6 using mass spectrometry. We found that the major protein repeatedly identified as STRA6-associated protein is calmodulin, consistent with the cryogenic electron microscopy (cryo-EM) study of zebrafish STRA6 associated with calmodulin. Using radioactivity-based, high-performance liquid chromatography (HPLC)-based and real-time fluorescence techniques, we found that calmodulin profoundly affects STRA6's vitamin A transport activity. Increased calcium/calmodulin promotes cellular vitamin A efflux and suppresses vitamin A influx through STRA6. Further mechanistic studies revealed that calmodulin enhances the binding of apo-RBP to STRA6, and this enhancement is much more pronounced for apo-RBP than holo-RBP. This study revealed that calmodulin regulates STRA6's vitamin A influx or efflux activity by modulating its preferential interaction with apo-RBP or holo-RBP. This molecular mechanism of regulating vitamin A transport may point to new directions to treat human diseases associated with insufficient or excessive vitamin A uptake.

Chemotherapy selection pressure alters sphingolipid composition and mitochondrial bioenergetics in resistant HL-60 cells.

The combination of daunorubicin (dnr) and cytarabine (Ara-C) is a cornerstone of treatment for acute myelogenous leukemia (AML); resistance to these drugs is a major cause of treatment failure. Ceramide, a sphingolipid (SL), plays a critical role in cancer cell apoptosis in response to chemotherapy. Here, we investigated the effects of chemotherapy selection pressure with Ara-C and dnr on SL composition and enzyme activity in the AML cell line HL-60. Resistant cells, those selected for growth in Ara-C- and dnr-containing medium (HL-60/Ara-C and HL-60/dnr, respectively), demonstrated upregulated expression and activity of glucosylceramide synthase, acid ceramidase (AC), and sphingosine kinase 1 (SPHK1); were more resistant to ceramide than parental cells; and displayed sensitivity to inhibitors of SL metabolism. Lipidomic analysis revealed a general ceramide deficit and a profound upswing in levels of sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P) in HL-60/dnr cells versus parental and HL-60/Ara-C cells. Both chemotherapy-selected cells also exhibited comprehensive upregulations in mitochondrial biogenesis consistent with heightened reliance on oxidative phosphorylation, a property that was partially reversed by exposure to AC and SPHK1 inhibitors and that supports a role for the phosphorylation system in resistance. In summary, dnr and Ara-C selection pressure induces acute reductions in ceramide levels and large increases in S1P and C1P, concomitant with cell resilience bolstered by enhanced mitochondrial remodeling. Thus, strategic control of ceramide metabolism and further research to define mitochondrial perturbations that accompany the drug-resistant phenotype offer new opportunities for developing therapies that regulate cancer growth.

Mitochondria inside acute myeloid leukemia cells hydrolyze ATP to resist chemotherapy.

Despite early optimism, therapeutics targeting oxidative phosphorylation (OxPhos) have faced clinical setbacks, stemming from their inability to distinguish healthy from cancerous mitochondria. Herein, we describe an actionable bioenergetic mechanism unique to cancerous mitochondria inside acute myeloid leukemia (AML) cells. Unlike healthy cells which couple respiration to the synthesis of ATP, AML mitochondria were discovered to support inner membrane polarization by consuming ATP. Because matrix ATP consumption allows cells to survive bioenergetic stress, we hypothesized that AML cells may resist cell death induced by OxPhos damaging chemotherapy by reversing the ATP synthase reaction. In support of this, targeted inhibition of BCL-2 with venetoclax abolished OxPhos flux without impacting mitochondrial membrane potential. In surviving AML cells, sustained polarization of the mitochondrial inner membrane was dependent on matrix ATP consumption. Mitochondrial ATP consumption was further enhanced in AML cells made refractory to venetoclax, consequential to downregulations in both the proton-pumping respiratory complexes, as well as the endogenous F1-ATPase inhibitor ATP5IF1. In treatment-naive AML, ATP5IF1 knockdown was sufficient to drive venetoclax resistance, while ATP5IF1 overexpression impaired F1-ATPase activity and heightened sensitivity to venetoclax. Collectively, our data identify matrix ATP consumption as a cancer-cell intrinsic bioenergetic vulnerability actionable in the context of mitochondrial damaging chemotherapy.

Differential and isomer-specific modulation of vitamin A transport and the catalytic activities of the RBP receptor by retinoids.

Retinoids are vitamin A derivatives with diverse biological functions. Both natural and artificial retinoids have been used as therapeutic reagents to treat human diseases, but not all retinoid actions are understood mechanistically. Plasma retinol binding protein (RBP) is the principal and specific carrier of vitamin A in the blood. STRA6 is the membrane receptor for RBP that mediates cellular vitamin A uptake. The effects of retinoids or related compounds on the receptor's vitamin A uptake activity and its catalytic activities are not well understood. In this study, we dissected the membrane receptor-mediated vitamin A uptake mechanism using various retinoids. We show that a subset of retinoids strongly stimulates STRA6-mediated vitamin A release from holo-RBP. STRA6 also catalyzes the exchange of retinol in RBP with certain retinoids. The effect of retinoids on STRA6 is highly isomer-specific. This study provides unique insights into the RBP receptor's mechanism and reveals that the vitamin A transport machinery can be a target of retinoid-based drugs.

Socially Mediated Shift in Neural Circuits Activation Regulated by Synergistic Neuromodulatory Signaling.

Animals exhibit context-dependent behavioral decisions that are mediated by specific motor circuits. In social species these decisions are often influenced by social status. Although social status-dependent neural plasticity of motor circuits has been investigated in vertebrates, little is known of how cellular plasticity translates into differences in motor activity. Here, we used zebrafish (Danio rerio) as a model organism to examine how social dominance influences the activation of swimming and the Mauthner-mediated startle escape behaviors. We show that the status-dependent shift in behavior patterns whereby dominants increase swimming and reduce sensitivity of startle escape while subordinates reduce their swimming and increase startle sensitivity is regulated by the synergistic interactions of dopaminergic, glycinergic, and GABAergic inputs to shift the balance of activation of the underlying motor circuits. This shift is driven by socially induced differences in expression of dopaminergic receptor type 1b (Drd1b) on glycinergic neurons and dopamine (DA) reuptake transporter (DAT). Second, we show that GABAergic input onto glycinergic neurons is strengthened in subordinates compared with dominants. Complementary neurocomputational modeling of the empirical results show that drd1b functions as molecular regulator to facilitate the shift between excitatory and inhibitory pathways. The results illustrate how reconfiguration in network dynamics serves as an adaptive strategy to cope with changes in social environment and are likely conserved and applicable to other social species.

Simultaneous Inhibition of Ceramide Hydrolysis and Glycosylation Synergizes to Corrupt Mitochondrial Respiration and Signal Caspase Driven Cell Death in Drug-Resistant Acute Myeloid Leukemia.

Acute myelogenous leukemia (AML), the most prevalent acute and aggressive leukemia diagnosed in adults, often recurs as a difficult-to-treat, chemotherapy-resistant disease. Because chemotherapy resistance is a major obstacle to successful treatment, novel therapeutic intervention is needed. Upregulated ceramide clearance via accelerated hydrolysis and glycosylation has been shown to be an element in chemotherapy-resistant AML, a problem considering the crucial role ceramide plays in eliciting apoptosis. Herein we employed agents that block ceramide clearance to determine if such a "reset" would be of therapeutic benefit. SACLAC was utilized to limit ceramide hydrolysis, and D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-threo-PDMP) was used to block the glycosylation route. The SACLAC D-threo-PDMP inhibitor combination was synergistically cytotoxic in drug-resistant, P-glycoprotein-expressing (P-gp) AML but not in wt, P-gp-poor cells. Interestingly, P-gp antagonists that can limit ceramide glycosylation via depression of glucosylceramide transit also synergized with SACLAC, suggesting a paradoxical role for P-gp in the implementation of cell death. Mechanistically, cell death was accompanied by a complete drop in ceramide glycosylation, concomitant, striking increases in all molecular species of ceramide, diminished sphingosine 1-phosphate levels, resounding declines in mitochondrial respiratory kinetics, altered Akt, pGSK-3ß, and Mcl-1 expression, and caspase activation. Although ceramide was generated in wt cells upon inhibitor exposure, mitochondrial respiration was not corrupted, suggestive of mitochondrial vulnerability in the drug-resistant phenotype, a potential therapeutic avenue. The inhibitor regimen showed efficacy in an in vivo model and in primary AML cells from patients. These results support the implementation of SL enzyme targeting to limit ceramide clearance as a therapeutic strategy in chemotherapy-resistant AML, inclusive of a novel indication for the use of P-gp antagonists.

STRA6-catalyzed vitamin A influx, efflux, and exchange.

Vitamin A has diverse biological functions and is essential for human survival. STRA6 is the high-affinity membrane receptor for plasma retinol binding protein (RBP), the principle and specific carrier of vitamin A (retinol) in the blood. It was previously shown that STRA6 couples to lecithin retinol acyltransferase (LRAT) and cellular retinol binding protein I (CRBP-I), but poorly to CRBP-II, for retinol uptake from holo-RBP. STRA6 catalyzes both retinol release from holo-RBP, which is responsible for its retinol uptake activity, and the loading of free retinol into apo-RBP, which can cause retinol efflux. Although STRA6-catalyzed retinol efflux into apo-RBP can theoretically deplete cells of retinoid, it is unclear to what extent this efflux happens and in what context. We show here that STRA6 can couple strongly to both CRBP-I and CRBP-II for retinol efflux to apo-RBP. Strikingly, pure apo-RBP can cause almost complete depletion of retinol taken up by CRBP-I in a STRA6-dependent manner. However, if STRA6 encounters both holo-RBP and apo-RBP (as in blood), holo-RBP blocks STRA6-mediated retinol efflux by competing with apo-RBP's binding to STRA6 and by counteracting retinol efflux with influx. We also found that STRA6 catalyzes efficient retinol exchange between intracellular CRBP-I and extracellular RBP, even in the presence of holo-RBP. STRA6's retinol exchange activity may serve to refresh the intracellular retinoid pool. This exchange is also a previously unknown function of CRBP-I and distinguishes CRBP-I from LRAT.

On the nature of ceramide-mitochondria interactions - Dissection using comprehensive mitochondrial phenotyping.

Sphingolipids are a unique class of lipids owing to their non-glycerol-containing backbone, ceramide, that is constructed from a long-chain aliphatic amino alcohol, sphinganine, to which a fatty acid is attached via an amide bond. Ceramide plays a star role in the initiation of apoptosis by virtue of its interactions with mitochondria, a control point for a downstream array of signaling cascades culminating in apoptosis. Many pathways converge on mitochondria to elicit mitochondrial outer membrane permeabilization (MOMP), a step that corrupts bioenergetic service. Although much is known regarding ceramides interaction with mitochondria and the ensuing cell signal transduction cascades, how ceramide impacts the elements of mitochondrial bioenergetic function is poorly understood. The objective of this review is to introduce the reader to sphingolipid metabolism, present a snapshot of mitochondrial respiration, elaborate on ceramides convergence on mitochondria and the upstream players that collaborate to elicit MOMP, and introduce a mitochondrial phenotyping platform that can be of utility in dissecting the fine-points of ceramide impact on cellular bioenergetics.

Social Experience Regulates Endocannabinoids Modulation of Zebrafish Motor Behaviors.

Social status-dependent modulation of neural circuits has been investigated extensively in vertebrate and invertebrate systems. However, the effects of social status on neuromodulatory systems that drive motor activity are poorly understood. Zebrafish form a stable social relationship that consists of socially dominant and subordinate animals. The locomotor behavior patterns differ according to their social ranks. The sensitivity of the Mauthner startle escape response in subordinates increases compared to dominants while dominants increase their swimming frequency compared to subordinates. Here, we investigated the role of the endocannabinoid system (ECS) in mediating these differences in motor activities. We show that brain gene expression of key ECS protein pathways are socially regulated. Diacylglycerol lipase (DAGL) expression significantly increased in dominants and significantly decreased in subordinates relative to controls. Moreover, brain gene expression of the cannabinoid 1 receptor (CB1R) was significantly increased in subordinates relative to controls. Secondly, increasing ECS activity with JZL184 reversed swimming activity patterns in dominant and subordinate animals. JZL184 did not affect the sensitivity of the startle escape response in dominants while it was significantly reduced in subordinates. Thirdly, blockage of CB1R function with AM-251 had no effect on dominants startle escape response sensitivity, but startle sensitivity was significantly reduced in subordinates. Additionally, AM-251 did not affect swimming activities in either social phenotypes. Fourthly, we demonstrate that the effects of ECS modulation of the startle escape circuit is mediated via the dopaminergic system specifically via the dopamine D1 receptor. Finally, our empirical results complemented with neurocomputational modeling suggest that social status influences the ECS to regulate the balance in synaptic strength between excitatory and inhibitory inputs to control the excitability of motor behaviors. Collectively, this study provides new insights of how social factors impact nervous system function to reconfigure the synergistic interactions of neuromodulatory pathways to optimize motor output.

Intrinsic OXPHOS limitations underlie cellular bioenergetics in leukemia.

Currently there is great interest in targeting mitochondrial oxidative phosphorylation (OXPHOS) in cancer. However, notwithstanding the targeting of mutant dehydrogenases, nearly all hopeful 'mito-therapeutics' cannot discriminate cancerous from non-cancerous OXPHOS and thus suffer from a limited therapeutic index. Using acute myeloid leukemia (AML) as a model, herein, we leveraged an in-house diagnostic biochemical workflow to identify 'actionable' bioenergetic vulnerabilities intrinsic to cancerous mitochondria. Consistent with prior reports, AML growth and proliferation was associated with a hyper-metabolic phenotype which included increases in basal and maximal respiration. However, despite having nearly 2-fold more mitochondria per cell, clonally expanding hematopoietic stem cells, leukemic blasts, as well as chemoresistant AML were all consistently hallmarked by intrinsic OXPHOS limitations. Remarkably, by performing experiments across a physiological span of ATP free energy, we provide direct evidence that leukemic mitochondria are particularly poised to consume ATP. Relevant to AML biology, acute restoration of oxidative ATP synthesis proved highly cytotoxic to leukemic blasts, suggesting that active OXPHOS repression supports aggressive disease dissemination in AML. Together, these findings argue against ATP being the primary output of leukemic mitochondria and provide proof-of-principle that restoring, rather than disrupting, OXPHOS may represent an untapped therapeutic avenue for combatting hematological malignancy and chemoresistance.

Differential Dopamine D1 and D3 Receptor Modulation and Expression in the Spinal Cord of Two Mouse Models of Restless Legs Syndrome.

Restless Legs Syndrome (RLS) is often and successfully treated with dopamine receptor agonists that target the inhibitory D3 receptor subtype, however there is no clinical evidence of a D3 receptor dysfunction in RLS patients. In contrast, genome-wide association studies in RLS patients have established that a mutation of the MEIS1 gene is associated with an increased risk in developing RLS, but the effect of MEIS1 dysfunction on sensorimotor function remain unknown. Mouse models for a dysfunctional D3 receptor (D3KO) and Meis1 (Meis1KO) were developed independently, and each animal expresses some features associated with RLS in the clinic, but they have not been compared in their responsiveness to treatment options used in the clinic. We here confirm that D3KO and Meis1KO animals show increased locomotor activities, but that only D3KO show an increased sensory excitability to thermal stimuli. Next we compared the effects of dopaminergics and opioids in both animal models, and we assessed D1 and D3 dopamine receptor expression in the spinal cord, the gateway for sensorimotor processing. We found that Meis1KO share most of the tested behavioral properties with their wild type (WT) controls, including the modulation of the thermal pain withdrawal reflex by morphine, L-DOPA and D3 receptor (D3R) agonists and antagonists. However, Meis1KO and D3KO were behaviorally more similar to each other than to WT when tested with D1 receptor (D1R) agonists and antagonists. Subsequent Western blot analyses of D1R and D3R protein expression in the spinal cord revealed a significant increase in D1R but not D3R expression in Meis1KO and D3KO over WT controls. As the D3R is mostly present in the dorsal spinal cord where it has been shown to modulate sensory pathways, while activation of the D1Rs can activate motoneurons in the ventral spinal cord, we speculate that D3KO and Meis1KO represent two complementary animal models for RLS, in which the mechanisms of sensory (D3R-mediated) and motor (D1R-mediated) dysfunctions can be differentially explored.

An unlikely DNA cleaving agent: A photo-active trinuclear Cu(II) complex based on hexaazatriphenylene.

This paper describes the synthesis of a trinuclear Cu(II) complex (4) containing a central 1,4,5,8,9,12-hexaazatriphenylene-hexacarboxylate (hat) core (3). Low, micromolar concentrations of the negatively charged parent ligand 3 and the neutral trinuclear complex 4 were found to photocleave negatively charged pUC19 plasmid DNA with high efficiency at neutral pH (350nm, 50min, 22°C). The interactions of complex 4 with double-helical DNA were studied in detail. Scavenger and colorimetric assays pointed to the formation of Cu(I), superoxide anion radicals, hydrogen peroxide, and hydroxyl radicals during photocleavage reactions. UV-visible absorption, circular dichroism, DNA thermal denaturation, and fluorescence data suggested that the Cu(II) complex contacts double-stranded DNA in an external fashion. The persistent association of ligand 3 and complex 4 with Na(I) and/or other cations in aqueous solution might facilitate electrostatic DNA interactions.

Vitamin A Transport Mechanism of the Multitransmembrane Cell-Surface Receptor STRA6.

Vitamin A has biological functions as diverse as sensing light for vision, regulating stem cell differentiation, maintaining epithelial integrity, promoting immune competency, regulating learning and memory, and acting as a key developmental morphogen. Vitamin A derivatives have also been used in treating human diseases. If vitamin A is considered a drug that everyone needs to take to survive, evolution has come up with a natural drug delivery system that combines sustained release with precise and controlled delivery to the cells or tissues that depend on it. This "drug delivery system" is mediated by plasma retinol binding protein (RBP), the principle and specific vitamin A carrier protein in the blood, and STRA6, the cell-surface receptor for RBP that mediates cellular vitamin A uptake. The mechanism by which the RBP receptor absorbs vitamin A from the blood is distinct from other known cellular uptake mechanisms. This review summarizes recent progress in elucidating the fundamental molecular mechanism mediated by the RBP receptor and multiple newly discovered catalytic activities of this receptor, and compares this transport system with retinoid transport independent of RBP/STRA6. How to target this new type of transmembrane receptor using small molecules in treating diseases is also discussed.

Identification of PLXDC1 and PLXDC2 as the transmembrane receptors for the multifunctional factor PEDF.

Pigment Epithelium Derived Factor (PEDF) is a secreted factor that has broad biological activities. It was first identified as a neurotrophic factor and later as the most potent natural antiangiogenic factor, a stem cell niche factor, and an inhibitor of cancer cell growth. Numerous animal models demonstrated its therapeutic value in treating blinding diseases and diverse cancer types. A long-standing challenge is to reveal how PEDF acts on its target cells and the identities of the cell-surface receptors responsible for its activities. Here we report the identification of transmembrane proteins PLXDC1 and PLXDC2 as cell-surface receptors for PEDF. Using distinct cellular models, we demonstrate their cell type-specific receptor activities through loss of function and gain of function studies. Our experiments suggest that PEDF receptors form homooligomers under basal conditions, and PEDF dissociates the homooligomer to activate the receptors. Mutations in the intracellular domain can have profound effects on receptor activities.

Vitamin A transport and the transmembrane pore in the cell-surface receptor for plasma retinol binding protein.

Vitamin A and its derivatives (retinoids) play diverse and crucial functions from embryogenesis to adulthood and are used as therapeutic agents in human medicine for eye and skin diseases, infections and cancer. Plasma retinol binding protein (RBP) is the principal and specific vitamin A carrier in the blood and binds vitamin A at 1:1 ratio. STRA6 is the high-affinity membrane receptor for RBP and mediates cellular vitamin A uptake. STRA6 null mice have severely depleted vitamin A reserves for vision and consequently have vision loss, even under vitamin A sufficient conditions. STRA6 null humans have a wide range of severe pathological phenotypes in many organs including the eye, brain, heart and lung. Known membrane transport mechanisms involve transmembrane pores that regulate the transport of the substrate (e.g., the gating of ion channels). STRA6 represents a new type of membrane receptor. How this receptor interacts with its transport substrate vitamin A and the functions of its nine transmembrane domains are still completely unknown. These questions are critical to understanding the molecular basis of STRA6's activities and its regulation. We employ acute chemical modification to introduce chemical side chains to STRA6 in a site-specific manner. We found that modifications with specific chemicals at specific positions in or near the transmembrane domains of this receptor can almost completely suppress its vitamin A transport activity. These experiments provide the first evidence for the existence of a transmembrane pore, analogous to the pore of ion channels, for this new type of cell-surface receptor.

Retina, retinol, retinal and the natural history of vitamin A as a light sensor.

Light is both the ultimate energy source for most organisms and a rich information source. Vitamin A-based chromophore was initially used in harvesting light energy, but has become the most widely used light sensor throughout evolution from unicellular to multicellular organisms. Vitamin A-based photoreceptor proteins are called opsins and have been used for billions of years for sensing light for vision or the equivalent of vision. All vitamin A-based light sensors for vision in the animal kingdom are G-protein coupled receptors, while those in unicellular organisms are light-gated channels. This first major switch in evolution was followed by two other major changes: the switch from bistable to monostable pigments for vision and the expansion of vitamin A's biological functions. Vitamin A's new functions such as regulating cell growth and differentiation from embryogenesis to adult are associated with increased toxicity with its random diffusion. In contrast to bistable pigments which can be regenerated by light, monostable pigments depend on complex enzymatic cycles for regeneration after every photoisomerization event. Here we discuss vitamin A functions and transport in the context of the natural history of vitamin A-based light sensors and propose that the expanding functions of vitamin A and the choice of monostable pigments are the likely evolutionary driving forces for precise, efficient, and sustained vitamin A transport.

Hydrolysis of phosphatidylcholine by cerium(IV) releases significant amounts of choline and inorganic phosphate at lysosomal pH.

Niemann-Pick disease and drug-induced phospholipidosis are examples of lysosomal storage disorders in which serious respiratory infections are brought on by high levels of the phospholipid phosphatidylcholine in the acidic lamellar bodies and lysosomes of pulmonary cells. One approach to developing an effective therapeutic agent could involve the use of a metal to preferentially hydrolyze phospholipid phosphate ester bonds at mildly acidic, lysosomal pH values (~pH 4.8). Towards this end, here we have investigated phosphatidylcholine hydrolysis by twelve metal ion salts at 60°C. Using a malachite green/molybdate-based colorimetric assay to detect inorganic phosphate released upon metal-assisted phosphate ester bond hydrolysis, Ce(IV) was shown to possess outstanding reactivity in comparison to the eleven other metals. We then utilized cerium(IV) to hydrolyze phosphatidylcholine at normal, core body temperature (37°C). The malachite green/molybdate assay was used to quantitate free phosphate and an Amplex® Red-based colorimetric assay and matrix-assisted laser desorption ionization time-of-flight mass spectrometry were employed to detect choline. Ce(IV) hydrolyzed phosphatidylcholine more efficiently at lysosomal pH: i.e., at a Triton X-100:phosphatidylcholine molar mixing ratio of 1.57, yields of choline and phosphate were 51±4% and 40±4% at ~pH 4.8, compared to 28±4% and 27±5% at ~pH 7.2.