Nanoscale Metal-Organic Layer Reprograms Cellular Metabolism to Enhance Photodynamic Therapy and Antitumor Immunity.

Abnormal cancer metabolism causes hypoxic and immunosuppressive tumor microenvironment (TME), which limits the antitumor efficacy of photodynamic therapy (PDT). Herein, we report a photosensitizing nanoscale metal-organic layer (MOL) with anchored 3-bromopyruvate (BrP), BrP@MOL, as a metabolic reprogramming agent to enhance PDT and antitumor immunity. BrP@MOL inhibited mitochondrial respiration and glycolysis to oxygenate tumors and reduce lactate production. This metabolic reprogramming enhanced reactive oxygen species generation during PDT and reshaped the immunosuppressive TME to enhance antitumor immunity. BrP@MOL-mediated PDT inhibited tumor growth by >90 % with 40 % of mice being tumor-free, rejected tumor re-challenge, and prevented lung metastasis. Further combination with immune checkpoint blockade potently regressed the tumors with >98 % tumor inhibition and 80 % of mice being tumor-free.

Genetically programmable cell membrane-camouflaged nanoparticles for targeted combination therapy of colorectal cancer.

Cell membrane-camouflaged nanoparticles possess inherent advantages derived from their membrane structure and surface antigens, including prolonged circulation in the bloodstream, specific cell recognition and targeting capabilities, and potential for immunotherapy. Herein, we introduce a cell membrane biomimetic nanodrug platform termed MPB-3BP@CM NPs. Comprising microporous Prussian blue nanoparticles (MPB NPs) serving as both a photothermal sensitizer and carrier for 3-bromopyruvate (3BP), these nanoparticles are cloaked in a genetically programmable cell membrane displaying variants of signal regulatory protein α (SIRPα) with enhanced affinity to CD47. As a result, MPB-3BP@CM NPs inherit the characteristics of the original cell membrane, exhibiting an extended circulation time in the bloodstream and effectively targeting CD47 on the cytomembrane of colorectal cancer (CRC) cells. Notably, blocking CD47 with MPB-3BP@CM NPs enhances the phagocytosis of CRC cells by macrophages. Additionally, 3BP, an inhibitor of hexokinase II (HK2), suppresses glycolysis, leading to a reduction in adenosine triphosphate (ATP) levels and lactate production. Besides, it promotes the polarization of tumor-associated macrophages (TAMs) towards an anti-tumor M1 phenotype. Furthermore, integration with MPB NPs-mediated photothermal therapy (PTT) enhances the therapeutic efficacy against tumors. These advantages make MPB-3BP@CM NPs an attractive platform for the future development of innovative therapeutic approaches for CRC. Concurrently, it introduces a universal approach for engineering disease-tailored cell membranes for tumor therapy.

A systemic analysis of monocarboxylate transporters in ovarian cancer and possible therapeutic interventions.

Monocarboxylate transporters (MCTs) play an immense role in metabolically active solid tumors by regulating concentration-dependent transport of different important monocarboxylates including pyruvate and lactate and are encoded by the SLC16A family of genes. Given the vast array of functions, these transporters play in oncogenesis, our objective was to look into the association of MCT1 (SLC16A1), MCT2 (SLC16A7), MCT3 (SLC16A8), and MCT4 (SLC16A3) with Epithelial ovarian cancer (EOC) pathophysiology by exploiting various publicly available databases and web resources. Few of the in silico findings were confirmed via in vitro experiments in EOC cell lines, SKOV3 and OAW-42. MCT1 and MCT4 were found to be upregulated at the mRNA level in OC tissues compared to normal. However, only higher level of MCT4 mRNA was found to be associated with poor patient survival. MCT4 was positively correlated with gene families responsible for invasion, migration, and immune modification, proving it to be one of the most important MCTs for therapeutic intervention. We compared the effects of MCT1/2 blocker SR13800 and a broad-spectrum MCT blocker α-Cyano Hydroxy Cinnamic Acid (α-CHCA) and discovered that α-CHCA has a greater effect on diminishing the invasive behavior of the cancer cells than MCT1/2 blocker SR13800. From our study, MCT4 has emerged as a prospective marker for predicting poor patient outcomes and a potential therapeutic target.

Targeting cancer lactate metabolism with synergistic combinations of synthetic catalysts and monocarboxylate transporter inhibitors.

Synthetic anticancer catalysts offer potential for low-dose therapy and the targeting of biochemical pathways in novel ways. Chiral organo-osmium complexes, for example, can catalyse the asymmetric transfer hydrogenation of pyruvate, a key substrate for energy generation, in cells. However, small-molecule synthetic catalysts are readily poisoned and there is a need to optimise their activity before this occurs, or to avoid this occurring. We show that the activity of the synthetic organometallic redox catalyst [Os(p-cymene)(TsDPEN)] (1), which can reduce pyruvate to un-natural D-lactate in MCF7 breast cancer cells using formate as a hydride source, is significantly increased in combination with the monocarboxylate transporter (MCT) inhibitor AZD3965. AZD3965, a drug currently in clinical trials, also significantly lowers the intracellular level of glutathione and increases mitochondrial metabolism. These synergistic mechanisms of reductive stress induced by 1, blockade of lactate efflux, and oxidative stress induced by AZD3965 provide a strategy for low-dose combination therapy with novel mechanisms of action.

Kinetic and structural studies of the reaction of Escherichia coli dihydrodipicolinate synthase with (S)-2-bromopropionate.

Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step in the lysine-biosynthetic pathway converting pyruvate and L-aspartate-ß-semialdehyde to dihydrodipicolinate. Kinetic studies indicate that the pyruvate analog (S)-2-bromopropionate inactivates the enzyme in a pseudo-first-order process. An initial velocity pattern indicates that (S)-2-bromopropionate is a competitive inhibitor versus pyruvate, with an inhibition constant of about 8 mM. Crystals of DHDPS complexed with (S)-2-bromopropionate formed in a solution consisting of 50 mM HEPES pH 7.5, 18% polyethylene glycol 3350, 8 mM spermidine, 0.2 M sodium tartrate and 5.0 mg ml-1 DHDPS. The crystals diffracted to 2.15 Šresolution and belonged to space group P1. The crystal structure confirms the displacement of bromine and the formation of a covalent attachment between propionate and Lys161 at the active site of the enzyme. Lys161 is the active-site nucleophile that attacks the carbonyl C atom of pyruvate and subsequently generates an imine adduct in the first half-reaction of the ping-pong enzymatic reaction. A comparison of the crystal structures of DHDPS complexed with pyruvate or (S)-2-bromopropionate indicates the covalent adduct formed from (S)-2-bromopropionate leads to a rotation of about 180° of the ß-δ C atoms of Lys61 that aligns the covalently bound propionate fairly closely with the imine adduct formed with pyruvate.

Human lactate dehydrogenase A undergoes allosteric transitions under pH conditions inducing the dissociation of the tetrameric enzyme.

The aerobic energetic metabolism of eukaryotic cells relies on the glycolytic generation of pyruvate, which is subsequently channelled to the oxidative phosphorylation taking place in mitochondria. However, under conditions limiting oxidative phosphorylation, pyruvate is coupled to alternative energetic pathways, e.g. its reduction to lactate catalyzed by lactate dehydrogenases (LDHs). This biochemical process is known to induce a significant decrease in cytosolic pH, and is accordingly denoted lactic acidosis. Nevertheless, the mutual dependence of LDHs action and lactic acidosis is far from being fully understood. Using human LDH-A, here we show that when exposed to acidic pH this enzyme is subjected to homotropic allosteric transitions triggered by pyruvate. Conversely, human LDH-A features Michaelis-Menten kinetics at pH values equal to 7.0 or higher. Further, citrate, isocitrate, and malate were observed to activate human LDH-A, both at pH 5.0 and 6.5, with citrate and isocitrate being responsible for major effects. Dynamic light scattering (DLS) experiments revealed that the occurrence of allosteric kinetics in human LDH-A is mirrored by a consistent dissociation of the enzyme tetramer, suggesting that pyruvate promotes tetramer association under acidic conditions. Finally, using the human liver cancer cell line HepG2 we isolated cells featuring cytosolic pH equal to 7.3 or 6.5, and we observed a concomitant decrease in cytosolic pH and lactate secretion. Overall, our observations indicate the occurrence of a negative feedback between lactic acidosis and human LDH-A activity, and a complex regulation of this feedback by pyruvate and by some intermediates of the Krebs cycle.

Mitochondria-targeted nanoplatforms for enhanced photodynamic therapy against hypoxia tumor.

BACKGROUND: Photodynamic therapy (PDT) is a promising therapeutic modality that can convert oxygen into cytotoxic reactive oxygen species (ROS) via photosensitizers to halt tumor growth. However, hypoxia and the unsatisfactory accumulation of photosensitizers in tumors severely diminish the therapeutic effect of PDT. In this study, a multistage nanoplatform is demonstrated to overcome these limitations by encapsulating photosensitizer IR780 and oxygen regulator 3-bromopyruvate (3BP) in poly (lactic-co-glycolic acid) (PLGA) nanocarriers. RESULTS: The as-synthesized nanoplatforms penetrated deeply into the interior region of tumors and preferentially remained in mitochondria due to the intrinsic characteristics of IR780. Meanwhile, 3BP could efficiently suppress oxygen consumption of tumor cells by inhibiting mitochondrial respiratory chain to further improve the generation of ROS. Furthermore, 3BP could abolish the excessive glycolytic capacity of tumor cells and lead to the collapse of ATP production, rendering tumor cells more susceptible to PDT. Successful tumor inhibition in animal models confirmed the therapeutic precision and efficiency. In addition, these nanoplatforms could act as fluorescence (FL) and photoacoustic (PA) imaging contrast agents, effectuating imaging-guided cancer treatment. CONCLUSIONS: This study provides an ideal strategy for cancer therapy by concurrent oxygen consumption reduction, oxygen-augmented PDT, energy supply reduction, mitochondria-targeted/deep-penetrated nanoplatforms and PA/FL dual-modal imaging guidance/monitoring. It is expected that such strategy will provide a promising alternative to maximize the performance of PDT in preclinical/clinical cancer treatment.

Biocompatible photoinduced CuAAC using sodium pyruvate.

Sodium pyruvate, a natural intermediate produced during cellular metabolism, is commonly used in buffer solutions and media for biochemical applications. Here we show the use of sodium pyruvate (SP) as a reducing agent in a biocompatible aqueous photoinduced azide-alkyne cycloaddition (CuAAC) reaction. This copper(I)-catalyzed 1,3-dipolar cycloaddition is triggered by SP under UV light irradiation, exhibits oxygen tolerance and temporal control, and provides a convenient alternative to current CuAAC systems, particularly for biomolecular conjugations.

ParaHydrogen Polarized Ethyl-[1-13 C]pyruvate in Water, a Key Substrate for Fostering the PHIP-SAH Approach to Metabolic Imaging.

An efficient synthesis of vinyl-[1-13 C]pyruvate has been reported, from which 13 C hyperpolarized (HP) ethyl-[1-13 C]pyruvate has been obtained by means of ParaHydrogen Induced Polarization (PHIP). Due to the intrinsic lability of pyruvate, which leads quickly to degradation of the reaction mixture even under mild reaction conditions, the vinyl-ester has been synthesized through the intermediacy of a more stable ketal derivative. 13 C and 1 H hyperpolarizations of ethyl-[1-13 C]pyruvate, hydrogenated using ParaHydrogen, have been compared to those observed on the more widely used allyl-derivative. It has been demonstrated that the spin order transfer from ParaHydrogen protons to 13 C, is more efficient on the ethyl than on the allyl-esterdue to the larger J-couplings involved. The main requirements needed for the biological application of this HP product have been met, i. e. an aqueous solution of the product at high concentration (40 mM) with a good 13 C polarization level (4.8 %) has been obtained. The in vitro metabolic transformation of the HP ethyl-[1-13 C]pyruvate, catalyzed by an esterase, has been observed. This substrate appears to be a good candidate for in vivo metabolic investigations using PHIP hyperpolarized probes.

Construction and Evaluation of Folic Acid-Modified 3-Bromopyruvate Cubosomes.

BACKGROUND Direct 3-bromopyruvate chemotherapy often causes side effects. We thus aimed to construct and evaluate folic acid-modified 3-bromopyruvate liquid crystalline nanoparticles (3BP-LCNP-FA) and assess their targeted antitumor effects in tumor-bearing nude mice. MATERIAL AND METHODS A liquid crystalline nanoparticle formulation was screened, and the structure was characterized using polarizing light- and transmission electron microscopy. The folate target was then synthesized and characterized using differential scanning calorimetry and proton nuclear magnetic resonance spectroscopy. In vitro, human CNE-2Z and MDA-MB-231 tumor cells were used to evaluate 3BP-LCNP-FA effects on tumor cell morphology and proliferation. Different drug formulations were administered to tumor-bearing nude mice to observe the treatment effects. Hepatic and renal toxicities were assessed using hematoxylin and eosin-stained liver, kidney, and lung sections along with serological analysis of liver and kidney injury markers (e.g., aspartate aminotransferase, alanine transaminase, blood urea nitrogen, and creatinine). Tumor tissue was observed for changes using proliferating cell nuclear antigen immunohistochemistry and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. RESULTS We successfully prepared 3BP-LCNP-FA of spherical shape with uniform size using the aforementioned techniques; drug loading did not alter crystal morphology. These cubosomes exhibited more potent antitumor activity than 3-bromopyruvate alone or non-folic acid-conjugated 3-bromopyruvate liquid crystalline nanoparticles in vitro and in vivo without obvious toxic side effects. CONCLUSIONS It is possible to successfully construct 3BP-LCNP-FA as a drug delivery vehicle that is more efficacious than 3-bromopyruvate and has no obvious toxic effects. Thus, folic acid-modified cubosomes can be used as effective carriers for targeted drug administration.

Electroanalysis of Infection with Methyl Pyruvate.

The discovery of infection enzyme leukocyte esterase (LE) hydrolyzing a mitochondrial substrate methyl pyruvate (MP) was explored in the development of electroanalytical methods for LE in human biofluids. The LE + MP reaction was coupled with alcohol oxidase to produce hydrogen peroxide, which was then reduced at a nitrogen-doped carbon nanotube electrode at -0.20 V, yielding current proportional to the LE content in a sample. The kinetic assays revealed a fast turnover (kcat = 15 s-1) and high specificity constant (kcatKm-1 = 2.3 × 106 M-1 s-1) for the LE-triggered hydrolysis of MP. The analytical assays were short (5 min) and the quantified LE was in the clinically relevant range of 22-300 µg L-1 (R2, 0.985). The immuno-electroanalysis could detect the picomole quantity of LE and yielded linear calibration plots up to 150 µg L-1 of LE with the same slope regardless of the sample matrix (urine, saliva, and phosphate buffer). The spike-and-recovery experiments displayed an LE recovery of 99-104%. The amperometric immunoassay of LE was less laborious than traditional enzyme-linked immunosorbent assay (ELISA) for LE and reduced the required sample incubation time from 4 h (sandwich ELISA) to 30 min (immuno-electroanalysis). The proposed combination of immunosorption with internally calibrated amperometry can also be used for a selective determination of other enzymes, which form enzymatically active immune complexes.

Protection by ethyl pyruvate against gamma radiation induced damage in bovine serum albumin.

Environmental factors like ionizing radiation induced generation of reactive oxygen species (ROS) cause macromolecular damage under physiological conditions. Proteins are the potential targets of ROS induced oxidative damage because of their abundance and their critical functions in the biological systems. The present study investigates the protective potential of ethyl pyruvate (EP) against ionizing radiation induced oxidative damage of bovine serum albumin (BSA) using spectroscopic, biochemical and SDS-PAGE techniques. Spectroscopic data shows that EP prevents the build up of protein damage markers like bityrosine formation and oxidation of tryptophan. Protein melting studies shows that the melting temperature (Tm) of the irradiated protein does not change significantly in the presence of EP. Biochemical assays indicate that ionizing radiation causes the generation of carbonyls and malondialdehyde and the loss of thiol content in proteins that is prevented by EP. The SDS-PAGE profile of gamma irradiated BSA shows the radioprotective effect of EP. These results indicate the radiation induced oxidative and molecular changes in the protein and that the EP protected the BSA from these modifications. Therefore, these results imply that EP has a good antiradical property and hence it can be proposed as a good radioprotective agent.

The mechanism of a one-substrate transketolase reaction.

Transketolase catalyzes the transfer of a glycolaldehyde residue from ketose (the donor substrate) to aldose (the acceptor substrate). In the absence of aldose, transketolase catalyzes a one-substrate reaction that involves only ketose. The mechanism of this reaction is unknown. Here, we show that hydroxypyruvate serves as a substrate for the one-substrate reaction and, as well as with the xylulose-5-phosphate, the reaction product is erythrulose rather than glycolaldehyde. The amount of erythrulose released into the medium is equimolar to a double amount of the transformed substrate. This could only be the case if the glycol aldehyde formed by conversion of the first ketose molecule (the product of the first half reaction) remains bound to the enzyme, waiting for condensation with the second molecule of glycol aldehyde. Using mass spectrometry of catalytic intermediates and their subsequent fragmentation, we show here that interaction of the holotransketolase with hydroxypyruvate results in the equiprobable binding of the active glycolaldehyde to the thiazole ring of thiamine diphosphate and to the amino group of its aminopyrimidine ring. We also show that these two loci can accommodate simultaneously two glycolaldehyde molecules. It explains well their condensation without release into the medium, which we have shown earlier.

Structural basis of the mechanism of ß-methyl epimerization by enzyme MarH.

Diverse derivatives of amino acids with different steric configurations are important biosynthetic building blocks. In biology, epimerization is an important way to generate steric diversity. MarH catalyzes the epimerization of the ß-position of (3R)-ß-methyl-indolepyruvate (MeInPy), forming (3S)-ß-MeInPy. Both compounds are derivatives of l-tryptophan (l-Trp) and are important precursors of bioactive natural products. Here, we report the crystal structures of MarH and the NMR structure of its complex with l-Trp, an analogue of its native substrate, (3R)-ß-MeInPy. Structural analysis and mutagenesis studies indicated that His25 acts as a base to remove Hß and generate a planar carbanion intermediate, which is then putatively reprotonated on the opposite face by a water molecule to form (3S)-ß-MeInPy in a stereospecific manner. The details of ß-site isomerization at the atomic level provide deeper insights into the epimerization mechanism of MarH and will facilitate further enzyme design to extend the substrate scope.

Effect of Protein Isotope Labeling on the Catalytic Mechanism of Lactate Dehydrogenase.

We investigate how isotopic labeling of the enzyme lactate dehydrogenase (LDH) affects its function. LDH is of special interest because there exists a line of residues spanning the protein that are involved in the transition state (TS) of the chemical reaction coordinate (so-called promoting vibration). Hence, studies have been carried out on this protein (as well as others) using labeled protein (so-called heavy protein) along with measurements of single turnover kcat yielding a KIE (=kcatlight/kcatheavy) aimed at understanding the effect of labeling generally and more specifically this line of residues. Here, it is shown that 13C, 15N, and 2H atom labeling of hhLDH (human heart) affects its internal structure which in turn affects its dynamics and catalytic mechanism. Spectral studies employing advanced FTIR difference spectroscopy show that the height of the electronic potential surface of the TS is lowered (probably by ground state destabilization) by labeling. Moreover, laser-induced T-jump relaxation kinetic spectroscopy shows that the microsecond to millisecond nuclear motions internal to the protein are affected by labeling. While the effects are small, they are sufficient to contribute to the observed KIE values as well or even more than promoting vibration effects.

Pyruvate Substitutions on Glycoconjugates.

Glycoconjugates are the most diverse biomolecules of life. Mostly located at the cell surface, they translate into cell-specific "barcodes" and offer a vast repertoire of functions, including support of cellular physiology, lifestyle, and pathogenicity. Functions can be fine-tuned by non-carbohydrate modifications on the constituting monosaccharides. Among these modifications is pyruvylation, which is present either in enol or ketal form. The most commonly best-understood example of pyruvylation is enol-pyruvylation of N-acetylglucosamine, which occurs at an early stage in the biosynthesis of the bacterial cell wall component peptidoglycan. Ketal-pyruvylation, in contrast, is present in diverse classes of glycoconjugates, from bacteria to algae to yeast-but not in humans. Mild purification strategies preventing the loss of the acid-labile ketal-pyruvyl group have led to a collection of elucidated pyruvylated glycan structures. However, knowledge of involved pyruvyltransferases creating a ring structure on various monosaccharides is scarce, mainly due to the lack of knowledge of fingerprint motifs of these enzymes and the unavailability of genome sequences of the organisms undergoing pyruvylation. This review compiles the current information on the widespread but under-investigated ketal-pyruvylation of monosaccharides, starting with different classes of pyruvylated glycoconjugates and associated functions, leading to pyruvyltransferases, their specificity and sequence space, and insight into pyruvate analytics.

Pharmacological prospection and structural characterization of two purified sulfated and pyruvylated homogalactans from green algae Codium isthmocladum.

Two sulfated polysaccharides (SPs), F2 and F3, isolated from Codium isthmocladum were found to contain galactose, sulfate, and pyruvate. The apparent molecular weights of F2 and F3 were determined to be 62 and 61 kDa, respectively. NMR spectroscopy combined with chemical analysis showed that F2 and F3 have the same structural features. However, F3 showed higher sulfate/sugar ratio (1/2.6) than F2 (1/4). F2 and F3 are essentially (1 → 3)-ß-D-galactans with some branching at C6. Pyruvylation occurs at O3 and O4, forming 3,4-O-(1-carboxyethylidene)-ß-D-Galp residues; some of these pyruvylated residues contain sulfate groups at C6. Some non-branching residues contain sulfate at C4. None of the SPs exhibited antioxidant activity. MTT results indicated that 1 mg/mL of both SPs about 40% of PANC-1 cell viability. At 10 µg/mL, F2 and F3 had 1.7-fold longer clotting times compared to that of Clexane® at the same concentration. The higher sulfate content of F3 is not a determining factor for pharmacological activities of galactans, considering that both F2 and F3 exerted the effects.

The NIR inspired nano-CuSMn(II) composites for lactate and glycolysis attenuation.

The level of lactate and hypoxia inducible factor (HIF) in cells has effect on tumor growth and drug resistance. The glycolysis of tumors could be inhibited by reducing the expression of lactate dehydrogenase A (LDHA) or the mutual conversion of lactic acid and pyruvic acid. To develop a bifunctional nanoparticle as both the cleaner of lactate and attenuator of glycolysis, a NIR (near infrared) responsive nanoparticle (Mn-CuS@BSA-FA) was synthesized and characterized. The Mn-CuS@BSA-FA catalyzed the oxidation of lactate to pyruvate under NIR irradiation. In vitro assay results demonstrate that Mn-CuS@BSA-FA can decrease the activity of LDHA and attenuate the conversion of lactate to pyruvate. Moreover, Mn-CuS@BSA-FA can inhibit the expression of HIF-1 and decrease the ATP level in HepG-1 cells. Our work demonstrates that Mn-CuS@BSA-FA can be a NIR enhanced glycolytic inhibitor for cancer interventional treatment.

Chemoenzymatic Platform for Synthesis of Chiral Organofluorines Based on Type†II Aldolases.

Aldolases are C-C bond forming enzymes that have become prominent tools for sustainable synthesis of complex synthons. However, enzymatic methods of fluorine incorporation into such compounds are lacking due to the rarity of fluorine in nature. Recently, the use of fluoropyruvate as a non-native aldolase substrate has arisen as a solution. Here, we report that the type II HpcH aldolases efficiently catalyze fluoropyruvate addition to diverse aldehydes, with exclusive (3S)-selectivity at fluorine that is rationalized by DFT calculations on a mechanistic model. We also measure the kinetic parameters of aldol addition and demonstrate engineering of the hydroxyl group stereoselectivity. Our aldolase collection is then employed in the chemoenzymatic synthesis of novel fluoroacids and ester derivatives in high stereopurity (d.r. 80-98 %). The compounds made available by this method serve as precursors to fluorinated analogs of sugars, amino acids, and other valuable chiral building blocks.