Unraveling the Impact of Six Pentacyclic Triterpenes Regulating Metabolic Pathways on Lung Carcinoma Cells.

Recently, there has been great interest in plant-derived compounds known as phytochemicals. The pentacyclic oleanane-, ursane-, and lupane-type triterpenes are phytochemicals that exert significant activity against diseases like cancer. Lung cancer is the leading cause of cancer-related death worldwide. Although chemotherapy is the treatment of choice for lung cancer, its effectiveness is hampered by the dose-limiting toxic effects and chemoresistance. Herein, we investigated six pentacyclic triterpenes, oleanolic acid, ursolic acid, asiatic acid, betulinic acid, betulin, and lupeol, on NSCLC A549 cells. These triterpenes have several structural variations that can influence the activation/inactivation of key cellular pathways. From our results, we determined that most of these triterpenes induced apoptosis, S-phase and G2/M-phase cycle arrest, the downregulation of ribonucleotide reductase (RR), reactive oxygen species, and caspase 3 activation. For chemoresistance markers, we found that most triterpenes downregulated the expression of MAPK/PI3K, STAT3, and PDL1. In contrast, UrA and AsA also induced DNA damage and autophagy. Then, we theoretically determined other possible molecular targets of these triterpenes using the online database ChEMBL. The results showed that even slight structural changes in these triterpenes can influence the cellular response. This study opens up promising perspectives for further research on the pharmaceutical role of phytochemical triterpenoids.

Association of congenital heart defects (CHD) with factors related to maternal health and pregnancy in newborns in Puerto Rico.

Background: Given the pervasive issues of obesity and diabetes both in Puerto Rico and the broader United States, there is a compelling need to investigate the intricate interplay among BMI, pregestational, and gestational maternal diabetes, and their potential impact on the occurrence of congenital heart defects (CHD) during neonatal development. Methods: Using the comprehensive System of Vigilance and Surveillance of Congenital Defects in Puerto Rico, we conducted a focused analysis on neonates diagnosed with CHD between 2016 and 2020. Our assessment encompassed a range of variables, including maternal age, gestational age, BMI, pregestational diabetes, gestational diabetes, hypertension, history of abortion, and presence of preeclampsia. Results: A cohort of 673 patients was included in our study. The average maternal age was 26 years, within a range of 22 to 32 years. The mean gestational age measured 39 weeks, with a median span of 38 to 39 weeks. Of the 673 patients, 274 (41%) mothers gave birth to neonates diagnosed with CHD. Within this group, 22 cases were linked to pre-gestational diabetes, while 202 were not; 20 instances were associated with gestational diabetes, compared to 200 without; and 148 cases exhibited an overweight or obese BMI, whereas 126 displayed a normal BMI. Conclusion: We identified a statistically significant correlation between pre-gestational diabetes mellitus and the occurrence of CHD. However, our analysis did not show a statistically significant association between maternal BMI and the likelihood of CHD. These results may aid in developing effective strategies to prevent and manage CHD in neonates.

A Synergistic pH-Responsive Serum Albumin-Based Drug Delivery System Loaded with Doxorubicin and Pentacyclic Triterpene Betulinic Acid for Potential Treatment of NSCLC.

Nanosized drug delivery systems (DDS) have been studied as a novel strategy against cancer due to their potential to simultaneously decrease drug inactivation and systemic toxicity and increase passive and/or active drug accumulation within the tumor(s). Triterpenes are plant-derived compounds with interesting therapeutic properties. Betulinic acid (BeA) is a pentacyclic triterpene that has great cytotoxic activity against different cancer types. Herein, we developed a nanosized protein-based DDS of bovine serum albumin (BSA) as the drug carrier combining two compounds, doxorubicin (Dox) and the triterpene BeA, using an oil-water-like micro-emulsion method. We used spectrophotometric assays to determine protein and drug concentrations in the DDS. The biophysical properties of these DDS were characterized using dynamic light scattering (DLS) and circular dichroism (CD) spectroscopy, confirming nanoparticle (NP) formation and drug loading into the protein structure, respectively. The encapsulation efficiency was 77% for Dox and 18% for BeA. More than 50% of both drugs were released within 24 h at pH 6.8, while less drug was released at pH 7.4 in this period. Co-incubation viability assays of Dox and BeA alone for 24 h demonstrated synergistic cytotoxic activity in the low µM range against non-small-cell lung carcinoma (NSCLC) A549 cells. Viability assays of the BSA-(Dox+BeA) DDS demonstrated a higher synergistic cytotoxic activity than the two drugs with no carrier. Moreover, confocal microscopy analysis confirmed the cellular internalization of the DDS and the accumulation of the Dox in the nucleus. We determined the mechanism of action of the BSA-(Dox+BeA) DDS, confirming S-phase cell cycle arrest, DNA damage, caspase cascade activation, and downregulation of epidermal growth factor receptor (EGFR) expression. This DDS has the potential to synergistically maximize the therapeutic effect of Dox and diminish chemoresistance induced by EGFR expression using a natural triterpene against NSCLC.

Oxidative Stress- and Autophagy-Inducing Effects of PSI-LHCI from Botryococcus braunii in Breast Cancer Cells.

Botryococcus braunii (B. braunii) is a green microalga primarily found in freshwater, reservoirs, and ponds. Photosynthetic pigments from algae have shown many bioactive molecules with therapeutic potential. Herein, we report the purification, characterization, and anticancer properties of photosystem I light-harvesting complex I (PSI-LHCI) from the green microalga B. braunii UTEX2441. The pigment-protein complex was purified by sucrose density gradient and characterized by its distinctive peaks using absorption, low-temperature (77 K) fluorescence, and circular dichroism (CD) spectroscopic analyses. Protein complexes were resolved by blue native-PAGE and two-dimensional SDS-PAGE. Triple-negative breast cancer MDA-MB-231 cells were incubated with PSI-LHCI for all of our experiments. Cell viability was assessed, revealing a significant reduction in a time- and concentration-dependent manner. We confirmed the internalization of PSI-LHCI within the cytoplasm and nucleus after 12 h of incubation. Cell death mechanism by oxidative stress was confirmed by the production of reactive oxygen species (ROS) and specifically superoxide. Furthermore, we monitored autophagic flux, apoptotic and necrotic features after treatment with PSI-LHCI. Treated MDA-MB-231 cells showed positive autophagy signals in the cytoplasm and nucleus, and necrotic morphology by the permeabilization of the cell membrane. Our findings demonstrated for the first time the cytotoxic properties of B. braunii PSI-LHCI by the induction of ROS and autophagy in breast cancer cells.

Key genes and drug delivery systems to improve the efficiency of chemotherapy.

Cancer cells can develop resistance to anticancer drugs, thereby becoming tolerant to treatment through different mechanisms. The biological mechanisms leading to the generation of anticancer treatment resistance include alterations in transmembrane proteins, DNA damage and repair mechanisms, alterations in target molecules, and genetic responses, among others. The most common anti-cancer drugs reported to develop resistance to cancer cells include cisplatin, doxorubicin, paclitaxel, and fluorouracil. These anticancer drugs have different mechanisms of action, and specific cancer types can be affected by different genes. The development of drug resistance is a cellular response which uses differential gene expression, to enable adaptation and survival of the cell to diverse threatening environmental agents. In this review, we briefly look at the key regulatory genes, their expression, as well as the responses and regulation of cancer cells when exposed to anticancer drugs, along with the incorporation of alternative nanocarriers as treatments to overcome anticancer drug resistance.

Smart Targeting To Improve Cancer Therapeutics.

Cancer is the second largest cause of death worldwide with the number of new cancer cases predicted to grow significantly in the next decades. Biotechnology and medicine can and should work hand-in-hand to improve cancer diagnosis and treatment efficacy. However, success has been frequently limited, in particular when treating late-stage solid tumors. There still is the need to develop smart and synergistic therapeutic approaches to achieve the synthesis of strong and effective drugs and delivery systems. Much interest has been paid to the development of smart drug delivery systems (drug-loaded particles) that utilize passive targeting, active targeting, and/or stimulus responsiveness strategies. This review will summarize some main ideas about the effect of each strategy and how the combination of some or all of them has shown to be effective. After a brief introduction of current cancer therapies and their limitations, we describe the biological barriers that nanoparticles need to overcome, followed by presenting different types of drug delivery systems to improve drug accumulation in tumors. Then, we describe cancer cell membrane targets that increase cellular drug uptake through active targeting mechanisms. Stimulus-responsive targeting is also discussed by looking at the intra- and extracellular conditions for specific drug release. We include a significant amount of information summarized in tables and figures on nanoparticle-based therapeutics, PEGylated drugs, different ligands for the design of active-targeted systems, and targeting of different organs. We also discuss some still prevailing fundamental limitations of these approaches, eg, by occlusion of targeting ligands.

Data on cytotoxic pattern of cholesterol analogs for lung adenocarcinoma cells.

Cholesterol (Cho) is a sterol that plays an essential role in the maintenance of biologic cell membranes, and various lipoproteins are its carriers through blood circulation [1]. Some FDA-approved anticancer drugs (i.e., Lipoplatin and Myocet) are conjugated to Cho moieties to improve their pharmacokinetic properties, cellular uptake and target specificity [2]. Recently natural and synthetic sterol compounds have shown a broad spectrum of pharmacological activities [3,4]. Herein, we investigated the anticancer activity of various natural Cho analogs, ie. asiatic acid (AsA), betulinic acid (BeA), oleanolic acid (OleA), ursolic Acid (UrA), lupeol (Lupe) and ß-sitosterol (ß-Sito) against non-small cell lung adenocarcinoma (A549). We performed theoretical calculations of the biophysicochemical properties, and viability assays in a range of 5-100 µM in A549 cells of these Cho analogs. We used ChemSketch and ChemSpider to determine physical properties, and GraphPad Prism 8 software for the data analysis to determine the inhibitory concentrations at 50% (IC50) of each compound.

Magnetic resonance imaging contrast enhancement in vitro and in vivo by octanuclear iron-oxo cluster-based agents.

A water-soluble octanuclear cluster, [Fe8], was studied with regard to its properties as a potential contrast enhancing agent in magnetic resonance imaging (MRI) in magnetic fields of 1.3, 7.2 and 11.9 T and was shown to have transverse relaxivities r2 = 4.01, 10.09 and 15.83 mM s-1, respectively. A related hydrophobic [Fe8] cluster conjugated with 5 kDa hyaluronic acid (HA) was characterized by 57Fe-Mössbauer and MALDI-TOF mass spectroscopy, and was evaluated in aqueous solutions in vitro with regard to its contrast enhancing properties [r2 = 3.65 mM s-1 (1.3 T), 26.20 mM s-1 (7.2 T) and 52.18 mM s-1 (11.9 T)], its in vitro cellular cytotoxicity towards A-549 cells and COS-7 cells and its in vivo enhancement of T2-weighted images (4.7 T) of a human breast cancer xenografted on a nude mouse. The physiologically compatible [Fe8]-HA conjugate was i.v. injected to the tumor-bearing mouse, resulting in observable, heterogeneous signal change within the tumor, evident 15 min after injection and persisting for approximately 30 min. Both molecular [Fe8] and its HA-conjugate show a strong magnetic field dependence on r2, rendering them promising platforms for the further development of T2 MRI contrast agents in high and ultrahigh magnetic fields.

First Total Synthesis of ω-Phenyl Δ6 Fatty Acids and their Leishmanicidal and Anticancer Properties.

INTRODUCTION: The first total synthesis of ω-phenyl Δ6 fatty acids (FA) and their cytotoxicity (A549) and leishmanicidal (L. infantum) activities are described. The novel 16-phenyl-6-hexadecynoic acid (1) and the known 16-phenylhexadecanoic acid (2) were synthesized in 7-8 steps with overall yields of 46 % and 41 %, respectively. The syntheses of the unprecedented 10-phenyl-6-decynoic acid (3), 10-cyclohexyl-6-decynoic acid (4) and 10-(4-methoxyphenyl)-6-decynoic acid (5) was also performed in 3 steps with 73-76 % overall yields. The use of lithium acetylide coupling enabled the 4-step synthesis of 10-phenyl-6Z-decenoic acid (6) with a 100 % cis-stereochemistry. The cytotoxicity of these novel FA was determined against A549 cells and L. infantum promastigotes and amastigotes. Among the ω-phenylated FA, the best cytotoxicity towards A549 was displayed by 1, with an IC50 of 18 ± 1 µM. On the other hand, among the C10 acids, the ω-cyclohexyl acid 4 presented the best cytotoxicity (IC50 = 40 ± 2 µM) towards A549. RESULTS: Based on caspase-3/7 studies neither of the FA induced apoptosis in A549, thus implying other mechanisms of cell death. CONCLUSION: The antileishmanial studies were performed with the top Leishmania donovani topoisomerase IB (LdTopIB) inhibitors, namely 1 and 2 (EC50 between 14 and 36 µM, respectively), acids that did not stabilize the cleavage complexes between LdTopIB and DNA. Acids 1 and 2 displayed cytotoxicity towards L. infantum amastigotes (IC50 = 3-6 µM) and L. infantum promastigotes (IC50 = 60- 70 µM), but low toxicity towards murine splenocytes. Our results identified 1 as the optimum ω- phenylated acid of the series.

Inducing cell death in vitro in cancer cells by targeted delivery of cytochrome c via a transferrin conjugate.

One of the major drawbacks of many of the currently used cancer drugs are off-target effects. Targeted delivery is one method to minimize such unwanted and detrimental events. To actively target lung cancer cells, we have developed a conjugate of the apoptosis inducing protein cytochrome c with transferrin because the transferrin receptor is overexpressed by many rapidly dividing cancer cells. Cytochrome c and transferrin were cross-linked with a redox sensitive disulfide bond for the intra-cellular release of the protein upon endocytosis by the transferrin receptor. Confocal results demonstrated the cellular uptake of the cytochrome c-transferrin conjugate by transferrin receptor overexpressing A549 lung cancer cells. Localization studies further validated that this conjugate escaped the endosome. Additionally, an in vitro assay showed that the conjugate could induce apoptosis by activating caspase-3. The neo-conjugate not only maintained an IC50 value similar to the well known drug cisplatin (50 µM) in A549 cancer cells but also was nontoxic to the normal lung (MRC5) cells. Our neo-conjugate holds promise for future development to target cancers with enhanced transferrin receptor expression.

Expanding the Therapeutic Potential of the Iron Chelator Deferasirox in the Development of Aqueous Stable Ti(IV) Anticancer Complexes.

The recent X-ray structure of titanium(IV)-bound human serum transferrin (STf) exhibiting citrate as a synergistic anion reveals a difference in Ti(IV) coordination versus iron(III), the metal endogenously delivered by the protein to cells. This finding enriches our bioinspired drug design strategy for Ti(IV)-based anticancer therapeutics, which applies a family of Fe(III) chelators termed chemical transferrin mimetic (cTfm) ligands to inhibit Fe bioavailability in cancer cells. Deferasirox, a drug used for iron overload disease, is a cTfm ligand that models STf coordination to Fe(III), favoring Fe(III) binding versus Ti(IV). This metal affinity preference drives deferasirox to facilitate the release of cytotoxic Ti(IV) intracellularly in exchange for Fe(III). An aqueous speciation study performed by potentiometric titration from pH 4 to 8 with micromolar levels of Ti(IV) deferasirox at a 1:2 ratio reveals exclusively Ti(deferasirox)2 in solution. The predominant complex at pH 7.4, [Ti(deferasirox)2]2-, exhibits the one of the highest aqueous stabilities observed for a potent cytotoxic Ti(IV) species, demonstrating little dissociation even after 1 month in cell culture media. UV-vis and 1H NMR studies show that the stability is unaffected by the presence of biomolecular Ti(IV) binders such as citrate, STf, and albumin, which have been shown to induce dissociation or regulate cellular uptake and can alter the activity of other antiproliferative Ti(IV) complexes. Kinetic studies on [Ti(deferasirox)2]2- transmetalation with Fe(III) show that a labile Fe(III) source is required to induce this process. The initial step of this process occurs on the time scale of minutes, and equilibrium for the complete transmetalation is reached on a time scale of hours to a day. This work reveals a mechanism to deliver Ti(IV) compounds into cells and trigger Ti(IV) release by a labile Fe(III) species. Cellular studies including other cTfm ligands confirm the Fe(III) depletion mechanism of these compounds and show their ability to induce early and late apoptosis.

A ubiquitous metal, difficult to track: towards an understanding of the regulation of titanium(iv) in humans.

Despite the ubiquitous nature of titanium(iv) and several examples of its beneficial behavior in different organisms, the metal remains underappreciated in biology. There is little understanding of how the metal might play an important function in the human body. Nonetheless, a new insight is obtained regarding the molecular mechanisms that regulate the blood speciation of the metal to maintain it in a nontoxic and potentially bioavailable form for use in the body. This review surveys the literature on Ti(iv) application in prosthetics and in the development of anticancer therapeutics to gain an insight into soluble Ti(iv) influx in the body and its long-term impact. The limitation in analytical tools makes it difficult to depict the full picture of how Ti(iv) is transported and distributed throughout the body. An improved understanding of Ti function and its interaction with biomolecules will be helpful in developing future technologies for its imaging in the body.

Unusual Synergism of Transferrin and Citrate in the Regulation of Ti(IV) Speciation, Transport, and Toxicity.

Human serum transferrin (sTf) is a protein that mediates the transport of iron from blood to cells. Assisted by the synergistic anion carbonate, sTf transports Fe(III) by binding the metal ion in a closed conformation. Previous studies suggest sTf's role as a potential transporter of other metals such as titanium. Ti is a widely used metal in colorants, foods, and implants. A substantial amount of Ti is leached into blood from these implants. However, the fate of the leached Ti and its transport into the cells is not known. Understanding Ti interaction with sTf assumes a greater significance with our ever increasing exposure to Ti in the form of implants. On the basis of in vitro studies, it was speculated that transferrin can bind Ti(IV) assisted by a synergistic anion. However, the role and identity of the synergistic anion(s) and the conformational state in which sTf binds Ti(IV) are not known. Here we have solved the first X-ray crystal structure of a Ti(IV)-bound sTf. We find that sTf binds Ti(IV) in an open conformation with both carbonate and citrate as synergistic anions at the metal binding sites, an unprecedented role for citrate. Studies with cell lines suggest that Ti(IV)-sTf is transported into cells and that sTf and citrate regulate the metal's blood speciation and attenuate its cytotoxic property. Our results provide the first glimpse into the citrate-transferrin synergism in the regulation of Ti(IV) bioactivity and offers insight into the future design of Ti(IV)-based anticancer drugs.

The cytotoxicity of BAMLET complexes is due to oleic acid and independent of the α-lactalbumin component.

Lipid-protein complexes comprised of oleic acid (OA) non-covalently coupled to human/bovine α-lactalbumin, named HAMLET/BAMLET, display cytotoxic properties against cancer cells. However, there is still a substantial debate about the role of the protein in these complexes. To shed light into this, we obtained three different BAMLET complexes using varying synthesis conditions. Our data suggest that to form active BAMLET particles, OA has to reach critical micelle concentration with an approximate diameter of 250 nm. Proteolysis experiments on BAMLET show that OA protects the protein and is probably located on the surface, consistent with a micelle-like structure. Native or unfolded α-lactalbumin without OA lacked any tumoricidal activity. In contrast, OA alone killed cancer cells with the same efficiency at equimolar concentrations as its formulation as BAMLET. Our data show unequivocally that the cytotoxicity of the BAMLET complex is exclusively due to OA and that OA alone, when formulated as a micelle, is as toxic as the BAMLET complex. The contradictory literature results on the cytotoxicity of BAMLET might be explained by our finding that it was imperative to sonicate the samples to obtain toxic OA.

Activation of caspase-dependent apoptosis by intracellular delivery of Cytochrome c-based nanoparticles.

BACKGROUND: Cytochrome c is an essential mediator of apoptosis when it is released from the mitochondria to the cytoplasm. This process normally takes place in response to DNA damage, but in many cancer cells (i.e., cancer stem cells) it is disabled due to various mechanisms. However, it has been demonstrated that the targeted delivery of Cytochrome c directly to the cytoplasm of cancer cells selective initiates apoptosis in many cancer cells. In this work we designed a novel nano-sized smart Cytochrome c drug delivery system to induce apoptosis in cancer cells upon delivery. RESULTS: Cytochrome c was precipitated with a solvent-displacement method to obtain protein nanoparticles. The size of the Cytochrome c nanoparticles obtained was 100-300 nm in diameter depending on the conditions used, indicating good potential to passively target tumors by the Enhanced Permeability and Retention effect. The surface of Cytochrome c nanoparticles was decorated with poly (lactic-co-glycolic) acid-SH via the linker succinimidyl 3-(2-pyridyldithio) propionate to prevent premature dissolution during delivery. The linker connecting the polymer to the protein nanoparticle contained a disulfide bond thus allowing polymer shedding and subsequent Cytochrome c release under intracellular reducing conditions. A cell-free caspase-3 assay revealed more than 80% of relative caspase activation by Cytochrome c after nanoprecipitation and polymer modification when compared to native Cytochrome c. Incubation of HeLa cells with the Cytochrome c based-nanoparticles showed significant reduction in cell viability after 6 hours while native Cytochrome c showed none. Confocal microscopy confirmed the induction of apoptosis in HeLa cells when they were stained with 4',6-diamidino-2-phenylindole and propidium iodide after incubation with the Cytochrome c-based nanoparticles. CONCLUSIONS: Our results demonstrate that the coating with a hydrophobic polymer stabilizes Cytochrome c nanoparticles allowing for their delivery to the cytoplasm of target cells. After smart release of Cytochrome c into the cytoplasm, it induced programmed cell death.

Chemical glycosylation of cytochrome c improves physical and chemical protein stability.

BACKGROUND: Cytochrome c (Cyt c) is an apoptosis-initiating protein when released into the cytoplasm of eukaryotic cells and therefore a possible cancer drug candidate. Although proteins have been increasingly important as pharmaceutical agents, their chemical and physical instability during production, storage, and delivery remains a problem. Chemical glycosylation has been devised as a method to increase protein stability and thus enhance their long-lasting bioavailability. RESULTS: Three different molecular weight glycans (lactose and two dextrans with 1 kD and 10 kD) were chemically coupled to surface exposed Cyt c lysine (Lys) residues using succinimidyl chemistry via amide bonds. Five neo-glycoconjugates were synthesized, Lac4-Cyt-c, Lac9-Cyt-c, Dex5(10kD)-Cyt-c, Dex8(10kD)-Cyt-c, and Dex3(1kD)-Cyt-c. Subsequently, we investigated glycoconjugate structure, activity, and stability. Circular dichroism (CD) spectra demonstrated that Cyt c glycosylation did not cause significant changes to the secondary structure, while high glycosylation levels caused some minor tertiary structure perturbations. Functionality of the Cyt c glycoconjugates was determined by performing cell-free caspase 3 and caspase 9 induction assays and by measuring the peroxidase-like pseudo enzyme activity. The glycoconjugates showed ≥94% residual enzyme activity and 86 ± 3 to 95 ± 1% relative caspase 3 activation compared to non-modified Cyt c. Caspase 9 activation by the glycoconjugates was with 92 ± 7% to 96 ± 4% within the error the same as the caspase 3 activation. There were no major changes in Cyt c activity upon glycosylation. Incubation of Dex3(1 kD)-Cyt c with mercaptoethanol caused significant loss in the tertiary structure and a drop in caspase 3 and 9 activation to only 24 ± 8% and 26 ± 6%, respectively. This demonstrates that tertiary structure intactness of Cyt c was essential for apoptosis induction. Furthermore, glycosylation protected Cyt c from detrimental effects by some stresses (i.e., elevated temperature and humidity) and from proteolytic degradation. In addition, non-modified Cyt c was more susceptible to denaturation by a water-organic solvent interface than its glycoconjugates, important for the formulation in polymers. CONCLUSION: The results demonstrate that chemical glycosylation is a potentially valuable method to increase Cyt c stability during formulation and storage and potentially during its application after administration.

Delivery of chemically glycosylated cytochrome c immobilized in mesoporous silica nanoparticles induces apoptosis in HeLa cancer cells.

Cytochrome c (Cyt c) is a small mitochondrial heme protein involved in the intrinsic apoptotic pathway. Once Cyt c is released into the cytosol, the caspase mediated apoptosis cascade is activated resulting in programmed cell death. Herein, we explore the covalent immobilization of Cyt c into mesoporous silica nanoparticles (MSN) to generate a smart delivery system for intracellular drug delivery to cancer cells aiming at affording subsequent cell death. Cyt c was modified with sulfosuccinimidyl-6-[3'-(2-pyridyldithio)-propionamido] hexanoate (SPDP) and incorporated into SH-functionalized MSN by thiol-disulfide interchange. Unfortunately, the delivery of Cyt c from the MSN was not efficient in inducing apoptosis in human cervical cancer HeLa cells. We tested whether chemical Cyt c glycosylation could be useful in overcoming the efficacy problems by potentially improving Cyt c thermodynamic stability and reducing proteolytic degradation. Cyt c lysine residues were modified with lactose at a lactose-to-protein molar ratio of 3.7 ± 0.9 using mono(lactosylamido)-mono(succinimidyl) suberate linker chemistry. Circular dichroism (CD) spectra demonstrated that part of the activity loss of Cyt c was due to conformational changes upon its modification with the SPDP linker. These conformational changes were prevented in the glycoconjugate. In agreement with the unfolding of Cyt c by the linker, a proteolytic assay demonstrated that the Cyt c-SPDP conjugate was more susceptible to proteolysis than Cyt c. Attachment of the four lactose molecules reversed this increased susceptibility and protected Cyt c from proteolytic degradation. Furthermore, a cell-free caspase-3 assay revealed 47% and 87% of relative caspase activation by Cyt c-SPDP and the Cyt c-lactose bioconjugate, respectively, when compared to Cyt c. This again demonstrates the efficiency of the glycosylation to improve maintaining Cyt c structure and thus function. To test for cytotoxicity, HeLa cells were incubated with Cyt c loaded MSN at different Cyt c concentrations (12.5, 25.0, and 37.5 µg/mL) for 24-72 h and cellular metabolic activity determined by a cell proliferation assay. While MSN-SPDP-Cyt c did not induced cell death, the Cyt c-lactose bioconjugate induced significant cell death after 72 h, reducing HeLa cell viability to 67% and 45% at the 25 µg/mL and 37.5 µg/mL concentrations, respectively. Confocal microscopy confirmed that the MSN immobilized Cyt c-lactose bioconjugate was internalized by HeLa cells and that the bioconjugate was capable of endosomal escape. The results clearly demonstrate that chemical glycosylation stabilized Cyt c upon formulation of a smart drug delivery system and upon delivery into cancer cells and highlight the general potential of chemical protein glycosylation to improve the stability of protein drugs.

Low operational stability of enzymes in dry organic solvents: changes in the active site might affect catalysis.

The potential of enzyme catalysis in organic solvents for synthetic applications has been overshadowed by the fact that their catalytic properties are affected by organic solvents. In addition, it has recently been shown that an enzyme's initial activity diminishes considerably after prolonged exposure to organic media. Studies geared towards understanding this last drawback have yielded unclear results. In the present work we decided to use electron paramagnetic resonance spectroscopy (EPR) to study the motion of an active site spin label (a nitroxide free radical) during 96 h of exposure of the serine protease subtilisin Carlsberg to four different organic solvents. Our EPR data shows a typical two component spectra that was quantified by the ratio of the anisotropic and isotropic signals. The isotropic component, associated with a mobile nitroxide free radical, increases during prolonged exposure to all solvents used in the study. The maximum increase (of 43%) was observed in 1,4-dioxane. Based on these and previous studies we suggest that prolonged exposure of the enzyme to these solvents provokes a cascade of events that could induce substrates to adopt different binding conformations. This is the first EPR study of the motion of an active-site spin label during prolonged exposure of an enzyme to organic solvents ever reported.

Enantioselective Transesterification Catalysis by Nanosized Serine Protease Subtilisin Carlsberg Particles in Tetrahydrofuran.

Enzyme catalysis in organic solvents is a powerful tool for stereo-selective synthesis but the enantioselectivity is still hard to predict. To overcome this obstacle, we employed a nanoparticulate formulation of subtilisin Carlsberg (SC) and designed a series of 14 structurally related racemic alcohols. They were employed in the model transesterification reaction with vinyl butyrate and the enantioselectivities were determined. In general, short alcohol side chains led to low enantioselectivties, while larger and bulky side chains caused better discrimination of the enantiomers by the enzyme. With several bulky substrates high enantioselectivities with E>100 were obtained. Computational modeling highlighted that key to high enantioselectivity is the discrimination of the R and S substrates by the sole hydrophobic binding pocket based on their size and bulkiness. While bulky S enantiomer side chains could be accommodated within the binding pocket, bulky R enantiomer side chains could not. However, when also the S enantiomer side chain becomes too large and does not fit into the binding pocket anymore, enantioselectivity accordingly drops.

Effect of prolonged exposure to organic solvents on the active site environment of subtilisin Carlsberg.

The potential of enzyme catalysis as a tool for organic synthesis is nowadays indisputable, as is the fact that organic solvents affect an enzyme's activity, selectivity and stability. Moreover, it was recently realized that an enzyme's initial activity is substantially decreased after prolonged exposure to organic media, an effect that further hampers their potential as catalysts for organic synthesis. Regrettably, the mechanistic reasons for these effects are still debatable. In the present study we have made an attempt to explain the reasons behind the partial loss of enzyme activity on prolonged exposure to organic solvents. Fluorescence spectroscopic studies of the serine protease subtilisin Carlsberg chemically modified with polyethylene glycol (PEG-SC) and inhibited with a Dancyl fluorophore, and dissolved in two organic solvents (acetonitrile and 1,4-dioxane) indicate that when the enzyme is initially introduced into these solvents, the active site environment is similar to that in water; however prolonged exposure to the organic medium causes this environment to resemble that of the solvent in which the enzyme is dissolved. Furthermore, kinetic studies show a reduction on both V(max) and K(M) as a result of prolonged exposure to the solvents. One interpretation of these results is that during this prolonged exposure to organic solvents the active-site fluorescent label inhibitor adopts a different binding conformation. Extrapolating this to an enzymatic reaction we argue that substrates bind in a less catalytically favorable conformation after the enzyme has been exposed to organic media for several hours.