pH-Sensitive Liposomes with Embedded 3-(isobutylamino)cholan-24-oic Acid: What Is the Possible Mechanism of Fast Cargo Release?

pH-sensitive liposomes have great potential for biomedical applications, in particular as nanocontainers for the delivery of biologically active compounds to specific areas of the human body. In this article, we discuss the possible mechanism of fast cargo release from a new type of pH-sensitive liposomes with embedded ampholytic molecular switch (AMS, 3-(isobutylamino)cholan-24-oic acid) with carboxylic anionic groups and isobutylamino cationic ones attached to the opposite ends of the steroid core. AMS-containing liposomes demonstrated the rapid release of the encapsulated substance when altering the pH of an outer solution, but the exact mechanism of the switch action has not yet been accurately determined. Here, we report on the details of fast cargo release based on the data obtained using ATR-FTIR spectroscopy as well as atomistic molecular modeling. The findings of this study are relevant to the potential application of AMS-containing pH-sensitive liposomes for drug delivery.

Phosphonate Inhibitors of Pyruvate Dehydrogenase Perturb Homeostasis of Amino Acids and Protein Succinylation in the Brain.

Mitochondrial pyruvate dehydrogenase complex (PDHC) is essential for brain glucose and neurotransmitter metabolism, which is dysregulated in many pathologies. Using specific inhibitors of PDHC in vivo, we determine biochemical and physiological responses to PDHC dysfunction. Dose dependence of the responses to membrane-permeable dimethyl acetylphosphonate (AcPMe2) is non-monotonous. Primary decreases in glutathione and its redox potential, methionine, and ethanolamine are alleviated with increasing PDHC inhibition, the alleviation accompanied by physiological changes. A comparison of 39 brain biochemical parameters after administration of four phosphinate and phosphonate analogs of pyruvate at a fixed dose of 0.1 mmol/kg reveals no primary, but secondary changes, such as activation of 2-oxoglutarate dehydrogenase complex (OGDHC) and decreased levels of glutamate, isoleucine and leucine. The accompanying decreases in freezing time are most pronounced after administration of methyl acetylphosphinate and dimethyl acetylphosphonate. The PDHC inhibitors do not significantly change the levels of PDHA1 expression and phosphorylation, sirtuin 3 and total protein acetylation, but increase total protein succinylation and glutarylation, affecting sirtuin 5 expression. Thus, decreased production of the tricarboxylic acid cycle substrate acetyl-CoA by inhibited PDHC is compensated by increased degradation of amino acids through the activated OGDHC, increasing total protein succinylation/glutarylation. Simultaneously, parasympathetic activity and anxiety indicators decrease.

Copper-catalyzed deacetonative Sonogashira coupling.

A convenient Pd- and phosphine-free protocol for assembling internal alkynes from tertiary propargyl alcohols and (het)aryl halides has been developed. The proposed tandem approach includes the base-promoted retro-Favorskii fragmentation followed by Cu-catalyzed C(sp)-C(sp2) cross-coupling. The use of inexpensive reagents (e.g. a catalyst, additives, a base, and a solvent) and good functional group tolerance make the procedure practical and cost-effective. The synthetic utility of the method was demonstrated by a smooth alkynylation of vinyl iodides derived from natural steroidal hormones.

Domino assembly of dithiocarbamates via Cu-catalyzed denitrogenative thiolation of iodotriazole-based diazo precursors.

An efficient domino approach to assemble benzoxazoles and anthranilamides bearing dithiocarbamate moieties has been developed. The proposed route represents a Cu-catalyzed three-component reaction between readily available 5-iodo-1,2,3-triazoles, amines, and CS2. The cascade transformation is based on a denitrogenative coupling of in situ formed dithiocarbamic acids with diazo intermediates, generated via annulation-triggered triazole ring-opening. This method is applicable to nucleophilic secondary amines and features good functional group compatibility.

Administration of Phosphonate Inhibitors of Dehydrogenases of 2-Oxoglutarate and 2-Oxoadipate to Rats Elicits Target-Specific Metabolic and Physiological Responses.

In vitro and in cell cultures, succinyl phosphonate (SP) and adipoyl phosphonate (AP) selectively target dehydrogenases of 2-oxoglutarate (OGDH, encoded by OGDH/OGDHL) and 2-oxoadipate (OADH, encoded by DHTKD1), respectively. To assess the selectivity in animals, the effects of SP, AP, and their membrane-penetrating triethyl esters (TESP and TEAP) on the rat brain metabolism and animal physiology are compared. Opposite effects of the OGDH and OADH inhibitors on activities of OGDH, malate dehydrogenase, glutamine synthetase, and levels of glutamate, lysine, citrulline, and carnosine are shown to result in distinct physiological responses. ECG is changed by AP/TEAP, whereas anxiety is increased by SP/TESP. The potential role of the ester moiety in the uncharged precursors of the 2-oxo acid dehydrogenase inhibitors is estimated. TMAP is shown to be less efficient than TEAP, in agreement with lower lipophilicity of TMAP vs. TEAP. Non-monotonous metabolic and physiological impacts of increasing OADH inhibition are revealed. Compared to the non-treated animals, strong inhibition of OADH decreases levels of tryptophan and beta-aminoisobutyrate and activities of malate dehydrogenase and pyruvate dehydrogenase, increasing the R-R interval of ECG. Thus, both metabolic and physiological actions of the OADH-directed inhibitors AP/TEAP are different from those of the OGDH-directed inhibitors SP/TESP, with the ethyl ester being more efficient than methyl ester.

Annulation-Triggered Denitrogenative Transformations of 2-(5-Iodo-1,2,3-triazolyl)benzoic Acids.

The ability of [1,2,3]triazolobenzoxazinones to act as a source of "hidden" diazo group was discovered. These diazo precursors can be easily prepared by the intramolecular cyclization of 2-(5-iodo-1,2,3-triazolyl)benzoic acids. The Cu-catalyzed capture of the hidden diazo group allows for further functionalization through the denitrogenative pathway. The transformations proceed via the formation of either diazoimine or diazoamide intermediates. Novel routes to various anthranilamides as well as thiolated benzoxazinones were developed using the one-pot cyclization/diazo capture procedure.

Domino Construction of Benzoxazole-Derived Sulfonamides via Metal-Free Denitrogenation of 5-Iodo-1,2,3-triazoles in the Presence of SO2 and Amines.

A straightforward domino approach to assemble benzoxazole-derived sulfonamides has been developed. The method is based on annulation-induced in situ generation of diazo compounds from readily available 2-(5-iodo-1,2,3-triazolyl)phenols, followed by metal-free denitrogenative transformation upon the action of 1,4-diazabicyclo[2.2.2]octane bis(sulfur dioxide) (DABSO) and amines. The protocol is operationally simple and features a broad substrate scope, furnishing a library of target compounds in generally good yields.

Assembly of Thiosubstituted Benzoxazoles via Copper-Catalyzed Coupling of Thiols with 5-Iodotriazoles Serving as Diazo Surrogates.

An efficient cascade approach to thiosubstituted benzoxazoles has been developed. The transformation starts with in situ generation of a diazo compound via annulation-triggered electrocyclic opening of the 1,2,3-triazole ring. The subsequent Cu-catalyzed trapping of diazo intermediates by various thiols affords the desired heterocycles in generally good yields of up to 91%. The protocol features very good functional group tolerance and is applicable to substrates with different electronic properties.

A Route to Triazole-Fused Sultams via Metal-Free Base-Mediated Cyclization of Sulfonamide-Tethered 5-Iodotriazoles.

An efficient direct approach to triazole-fused sultams has been developed. The key step of the proposed strategy is base-mediated cyclization of sulfonamide-tethered 5-iodo-1,2,3-triazoles which are readily available via an improved protocol for Cu-catalyzed 1,3-dipolar cycloaddition. The annulation of the sultam fragment to the triazole ring proceeds smoothly under transition-metal-free conditions in the presence of Cs2CO3 in dioxane at 100 °C and affords fused heterocycles in high yields up to 99%. The favorability of an SNAr-like mechanism for the cyclization was supported by DFT calculations. The applicability of the developed procedure to modification of natural compounds was demonstrated by preparation of a deoxycholic acid derivative.

A novel approach to a controlled opening of liposomes.

A novel strategy is described for preparing pH-sensitive liposomes which releases the encapsulated drug in response to the change in pH of surrounding solution. The liposomes, composed of conventional zwitter-ionic egg yolk lecithin (EL), additionally contains a pH-sensitive "activator" (AMS), a derivative of lithocholic acid with anionic and cationic groups attached to the opposite ends of the steroid core. AMS changes its orientation in the liposomal membrane thus adapting to acidity/basicity of the outer solution. The rotation of AMS induces disordering of the membrane and a fast release of the bioactive cargo. In particular, 50-60 % of the encapsulated antitumor drug, doxorubicin and cisplatin, leaks from the liposomes within the first minute after acidification of the surrounding solution. Low-toxic EL-AMS liposomes, loaded with doxorubicin, show themselves active towards multidrug resistant cells. Fast-acting and low-toxic EL-AMS liposomes can be used in the design of smart liposomal containers in the drug delivery field.

Synthetic analogues of 2-oxo acids discriminate metabolic contribution of the 2-oxoglutarate and 2-oxoadipate dehydrogenases in mammalian cells and tissues.

The biological significance of the DHTKD1-encoded 2-oxoadipate dehydrogenase (OADH) remains obscure due to its catalytic redundancy with the ubiquitous OGDH-encoded 2-oxoglutarate dehydrogenase (OGDH). In this work, metabolic contributions of OADH and OGDH are discriminated by exposure of cells/tissues with different DHTKD1 expression to the synthesized phosphonate analogues of homologous 2-oxodicarboxylates. The saccharopine pathway intermediates and phosphorylated sugars are abundant when cellular expressions of DHTKD1 and OGDH are comparable, while nicotinate and non-phosphorylated sugars are when DHTKD1 expression is order(s) of magnitude lower than that of OGDH. Using succinyl, glutaryl and adipoyl phosphonates on the enzyme preparations from tissues with varied DHTKD1 expression reveals the contributions of OADH and OGDH to oxidation of 2-oxoadipate and 2-oxoglutarate in vitro. In the phosphonates-treated cells with the high and low DHTKD1 expression, adipate or glutarate, correspondingly, are the most affected metabolites. The marker of fatty acid ß-oxidation, adipate, is mostly decreased by the shorter, OGDH-preferring, phosphonate, in agreement with the known OGDH dependence of ß-oxidation. The longest, OADH-preferring, phosphonate mostly affects the glutarate level. Coupled decreases in sugars and nicotinate upon the OADH inhibition link the perturbation in glucose homeostasis, known in OADH mutants, to the nicotinate-dependent NAD metabolism.

Selective Inhibition of 2-Oxoglutarate and 2-Oxoadipate Dehydrogenases by the Phosphonate Analogs of Their 2-Oxo Acid Substrates.

Phosphonate analogs of pyruvate and 2-oxoglutarate are established specific inhibitors of cognate 2-oxo acid dehydrogenases. The present work develops application of this class of compounds to specific in vivo inhibition of 2-oxoglutarate dehydrogenase (OGDH) and its isoenzyme, 2-oxoadipate dehydrogenase (OADH). The isoenzymes-enriched preparations from the rat tissues with different expression of OADH and OGDH are used to characterize their interaction with 2-oxoglutarate (OG), 2-oxoadipate (OA) and the phosphonate analogs. Despite a 100-fold difference in the isoenzymes ratio in the heart and liver, similar Michaelis saturations by OG are inherent in the enzyme preparations from these tissues ( K m O G = 0.45 ± 0.06 and 0.27 ± 0.026 mM, respectively), indicating no significant contribution of OADH to the OGDH reaction, or similar affinities of the isoenzymes to OG. However, the preparations differ in the catalysis of OADH reaction. The heart preparation, where OADH/OGDH ratio is ≈ 0.01, possesses low-affinity sites to OA ( K m O A = 0.55 ± 0.07 mM). The liver preparation, where OADH/OGDH ratio is ≈ 1.6, demonstrates a biphasic saturation with OA: the low-affinity sites ( K m , 2 O A = 0.45 ± 0.12 mM) are similar to those of the heart preparation; the high-affinity sites ( K m , 1 O A = 0.008 ± 0.001 mM), revealed in the liver preparation only, are attributed to OADH. Phosphonate analogs of C5-C7 dicarboxylic 2-oxo acids inhibit OGDH and OADH competitively to 2-oxo substrates in all sites. The high-affinity sites for OA are affected the least by the C5 analog (succinyl phosphonate) and the most by the C7 one (adipoyl phosphonate). The opposite reactivity is inherent in both the low-affinity OA-binding sites and OG-binding sites. The C6 analog (glutaryl phosphonate) does not exhibit a significant preference to either OADH or OGDH. Structural analysis of the phosphonates binding to OADH and OGDH reveals the substitution of a tyrosine residue in OGDH for a serine residue in OADH among structural determinants of the preferential binding of the bulkier ligands to OADH. The consistent kinetic and structural results expose adipoyl phosphonate as a valuable pharmacological tool for specific in vivo inhibition of the DHTKD1-encoded OADH, a new member of mammalian family of 2-oxo acid dehydrogenases, up-regulated in some cancers and associated with diabetes and obesity.

Annulation-Induced Cascade Transformation of 5-Iodo-1,2,3-triazoles to 2-(1-Aminoalkyl)benzoxazoles.

Base-mediated cyclization of (5-iodo-1,2,3-triazolyl)phenols was proposed as a new synthetic strategy for the in situ generation of diazoimines via electrocyclic ring opening of the fused heterocycle. Cu-catalyzed amination of the intermediate diazoalkanes was employed to develop an efficient cascade approach to functionalized benzoxazoles.

Pd- and Cu-catalyzed approaches in the syntheses of new cholane aminoanthraquinone pincer-like ligands.

Cu- and Pd-catalyzed arylation of aminocholanes has been described for the first time. While this Cu-catalyzed protocol provides high yields in reactions of aminocholanes with iodoarenes, Pd catalysis was found to be preferable for the reactions of aminocholanes with dichloroanthraquinones. UV-vis titration of bis(cholanylamino)anthraquinones with a series of cations demonstrated their high binding affinity to Cu2+, Al3+, and Cr3+.

Bioconversion of 6-(N-methyl-N-phenyl)aminomethyl androstane steroids by Nocardioides simplex.

The newly synthesized (α/ß)-diastereomers of 6-(N-methyl-N-phenyl)aminomethylandrost-4-ene-3,17-dione (5) and 6-(N-methyl-N-phenyl)aminomethylandrost-4-en-17ß-ol-3-one (6) were firstly investigated as substrates for the whole cells of Nocardioides simplex VKM Ac-2033D in comparison with their unsubstituted analogs, - androst-4-ene-3,17-dione (1) and androst-4-en-17ß-ol-3-one (2). 1(2)-Dehydroderivatives were identified as the major bioconversion products from all the substrates tested. When using the mixtures of (α/ß)-stereoisomers of 5 and 6 as the substrates, only ß-stereoisomers of the corresponding 1,4-diene-steroids were formed. Along with 1(2)-dehydrogenation, N. simplex VKM Ac-2033D promoted oxidation of the hydroxyl group at C-17 position of 6: both 6(α) and 6(ß) were transformed to the corresponding 17-keto derivatives. No steroid core destruction was observed during the conversion of the 6-substituted androstanes 5 and 6, while it was significant when 1 or 2 was used as the substrate. The results suggested high potentials of N. simplex VKM Ac-2033D for the generation of novel 1(2)-dehydroanalogs.

Specific inhibition by synthetic analogs of pyruvate reveals that the pyruvate dehydrogenase reaction is essential for metabolism and viability of glioblastoma cells.

The pyruvate dehydrogenase complex (PDHC) and its phosphorylation are considered essential for oncotransformation, but it is unclear whether cancer cells require PDHC to be functional or silenced. We used specific inhibition of PDHC by synthetic structural analogs of pyruvate to resolve this question. With isolated and intramitochondrial PDHC, acetyl phosphinate (AcPH, KiAcPH = 0.1 µM) was a much more potent competitive inhibitor than the methyl ester of acetyl phosphonate (AcPMe, KiAcPMe = 40 µM). When preincubated with the complex, AcPH also irreversibly inactivated PDHC. Pyruvate prevented, but did not reverse the inactivation. The pyruvate analogs did not significantly inhibit other 2-oxo acid dehydrogenases. Different cell lines were exposed to the inhibitors and a membrane-permeable precursor of AcPMe, dimethyl acetyl phosphonate, which did not inhibit isolated PDHC. Using an ATP-based assay, dependence of cellular viability on the concentration of the pyruvate analogs was followed. The highest toxicity of the membrane-permeable precursor suggested that the cellular action of charged AcPH and AcPMe requires monocarboxylate transporters. The relevant cell-specific transcripts extracted from Gene Expression Omnibus database indicated that cell lines with higher expression of monocarboxylate transporters and PDHC components were more sensitive to the PDHC inhibitors. Prior to a detectable antiproliferative action, AcPH significantly changed metabolic profiles of the investigated glioblastoma cell lines. We conclude that catalytic transformation of pyruvate by pyruvate dehydrogenase is essential for the metabolism and viability of glioblastoma cell lines, although metabolic heterogeneity causes different cellular sensitivities and/or abilities to cope with PDHC inhibition.

Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays.

Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay.

Alkynylation of steroids via Pd-free Sonogashira coupling.

Cu-catalyzed Pd-free Sonogashira coupling has been proposed as a straightforward and convenient route to valuable steroidal enynes. A biligand catalyst system based on Ph3P and TMEDA has been designed. The protocol was utilized for the efficient coupling of iodosteroids with diverse terminal alkynes and 1-trimethylsilylalkynes. A possible role of an auxiliary ligand as a phase-transfer catalyst for a sparingly soluble inorganic base (K2CO3) was revealed.

Synthesis of novel 1,2,3-triazolyl derivatives of pregnane, androstane and D-homoandrostane. Tandem "click" reaction/Cu-catalyzed D-homo rearrangement.

Copper-catalyzed 1,3-dipolar cycloaddition has been employed in the reaction of steroidal azides with various terminal alkynes. A number of novel 1,2,3-triazolyl derivatives of pregnane, androstane and D-homoandrostane were obtained in high yield (70-98%). The developed synthetic protocols allowed us to attach the triazolyl moiety to both the side chain and the steroidal backbone directly, despite the steric hindrance exerted by the polycyclic system. The presence of Cu(II) was shown to evoke d-homo rearrangement under mild conditions. A rational choice of the copper precatalyst permitted us to carry out the "click" reaction either along with tandem d-homo rearrangement or in the absence of this process. The tendency of 16-heterosubstituted steroids to undergo D-homo rearrangement under Cu(II) catalysis was studied.

Thiamin diphosphate-dependent enzymes: from enzymology to metabolic regulation, drug design and disease models.

Bringing a knowledge of enzymology into research in vivo and in situ is of great importance in understanding systems biology and metabolic regulation. The central metabolic significance of thiamin (vitamin B1 ) and its diphosphorylated derivative (thiamin diphosphate; ThDP), and the fundamental differences in the ThDP-dependent enzymes of metabolic networks in mammals versus plants, fungi and bacteria, or in health versus disease, suggest that these enzymes are promising targets for biotechnological and medical applications. Here, the in vivo action of known regulators of ThDP-dependent enzymes, such as synthetic structural analogs of the enzyme substrates and thiamin, is analyzed in light of the enzymological data accumulated during half a century of research. Mimicking the enzyme-specific catalytic intermediates, the phosphonate analogs of 2-oxo acids selectively inhibit particular ThDP-dependent enzymes. Because of their selectivity, use of these compounds in cellular and animal models of ThDP-dependent enzyme malfunctions improves the validity of the model and its predictive power when compared with the nonselective and enzymatically less characterized oxythiamin and pyrithiamin. In vitro studies of the interaction of thiamin analogs and their biological derivatives with potential in vivo targets are necessary to identify and attenuate the analog selectivity. For both the substrate and thiamin synthetic analogs, in vitro reactivities with potential targets are highly relevant in vivo. However, effective concentrations in vivo are often higher than in vitro studies would suggest. The significance of specific inihibition of the ThDP-dependent enzymes for the development of herbicides, antibiotics, anticancer and neuroprotective strategies is discussed.