Uncoupling Activity Research Articles

Design, synthesis, and biological evaluation of imidazo[4,5-b]pyridine mitochondrial uncouplers for the treatment of metabolic dysfunction-associated steatohepatitis (MASH)

Small molecule mitochondrial uncouplers have gained traction for their potential therapeutic use against metabolic dysfunction-associated steatohepatitis (MASH). Herein, we report a novel imidazo[4,5-b]pyridine scaffold derived from iterative modifications of the potent uncoupler BAM15. Our structure-activity relationship (SAR) study demonstrated that this promising scaffold has a range of tolerated substitutions that allows for the modulation of uncoupling activity and in vivo pharmacokinetic properties. Specifically, compound SHS206 displayed an EC50 of 830 nM in L6 myoblasts and, importantly, showed no cytotoxicity in vitro or adverse effects in mice up to 1000 mg/kg. SHS206 was administered orally at 100 and 300 mg/kg in a GAN mouse model of MASH and was observed to lower liver triglyceride levels while food intake, body weight, temperature, organ weights, and cholesterol levels remained unaltered. Together, these findings illuminate imidazo[4,5-b]pyridine as a promising scaffold for the future development of mitochondrial uncouplers.

Common Mitochondrial Targets of Curcumin and Cinnamic Acid, the Membrane-Active Natural Phenolic Compounds

Background: Research has shown the multiple actions of curcumin on different cell systems, including enzymes and mitochondria. The detected effects of curcumin on mitochondria are diverse, ranging from protective to toxic. Objectives: In this present work, the influence of curcumin, as well as cinnamic acid, which is a microbial metabolite and a possible product of the microbial breakdown of curcumin, on isolated mitochondria, was investigated. Methods: Membrane potential, swelling, respiration, and calcium retention capacity were studied using selective electrodes, fluorescence and spectral methods. Results: It was found that curcumin at low concentrations (10–20 μM) activated the opening of the calcium-dependent permeability transition pore (mPTP) and decreased the calcium retention capacity and threshold concentrations necessary for the mPTP opening. Moreover, curcumin caused a concentration-dependent stepwise decrease in the membrane potential, accompanied by the activation of respiration and a decrease in oxidative phosphorylation, which indicates that curcumin is a typical mitochondrial uncoupler. The uncoupling effect strongly depended on the concentration of curcumin, which also increased, stepwise, from weak uncoupling at 25 µM to complete uncoupling at 75–100 µM. Cinnamic acid had similar effects, with the exception of the depolarizing effect, at concentrations that were an order of magnitude higher. Conclusions: Presumably, the uncoupling action of curcumin is a priming event that modulates any energy- and redox-dependent mitochondrial functions, from positive stimulation to toxic disorder. This effect can also underlie the curcumin-induced changes in different cellular processes and be achieved by targeted delivery of curcumin to certain cells, bypassing the microbiota.

Mitochondrial uncoupling caused by a wide variety of protonophores is differently sensitive to carboxyatractyloside in rat heart and liver mitochondria

Mitochondrial uncoupling by small-molecule protonophores is generally accepted to proceed via transmembrane proton shuttling. The idea of facilitating this process by the adenine nucleotide translocase ANT originated primarily from the partial reversal of the DNP-induced mitochondrial uncoupling by the ANT inhibitor carboxyatractyloside (CATR). Recently, the sensitivity to CATR was also observed for the action of such potent OxPhos uncouplers as BAM15, SF6847, FCCP and niclosamide. Here, we report measurements of the CATR effect on the activity of a large number of conventional and novel uncouplers in isolated mammalian mitochondria. Despite the broad variety of chemical structures, CATR attenuated the uncoupling efficacy of all the anionic protonophores in rat heart mitochondria with high abundance of ANT, whereas the effect was much less pronounced or even absent, e.g. for SF6847, in rat liver mitochondria with low ANT content. The fact that the uncoupling action is tissue specific for a broad spectrum of anionic protonophores is highlighted here for the first time. Only with the cationic uncoupler ellipticine and the channel-forming peptide gramicidin A, no sensitivity to CATR was found even in rat heart mitochondria. By contrast, with the recently described ester-stabilized ylidic protonophores [Kirsanov et al. Bioelectrochemistry 2023], the stimulating effect of CATR was discovered both in liver and heart mitochondria.

Expanding the π-system of Fatty Acid-Anion Transporter Conjugates Modulates Their Mechanism of Proton Transport and Mitochondrial Uncoupling Activity.

Mitochondrial uncoupling by small molecule protonophores is a promising strategy for developing novel anticancer agents. Recently, aryl urea substituted fatty acids (aryl ureas) were identified as a new class of protonophoric anticancer agents. To mediate proton transport these molecules self-assemble into membrane-permeable anionic dimers in which intermolecular hydrogen bonds between the carboxylate and aryl-urea anion receptor delocalise the negative charge across the aromatic π-system. In this work, we extend the aromatic π-system by introducing a second phenyl substituent to the aryl urea scaffold and compare the proton transport mechanisms and mitochondrial uncoupling actions of these compounds to their monoaryl analogues. It was found that incorporation of meta-linked phenyl substituents into the aryl urea scaffold enhanced proton transport in vesicles and demonstrated superior capacity to depolarise mitochondria, inhibit ATP production and reduce the viability of MDA-MB-231 breast cancer cells. In contrast, diphenyl ureas linked through a 1,4-distribution across the phenyl ring displayed diminished proton transport activity, despite both diphenyl urea isomers possessing similar binding affinities for carboxylates. Mechanistic studies suggest that inclusion of a second aryl ring changes the proton transport mechanism, presumably due to steric factors that impose higher energy penalties for dimer formation.

Phytopathogenic Fungicidal Activity and Mechanism Approach of Three Kinds of Triphenylphosphonium Salts.

The triphenylphosphonium (TPP) cation has been widely used as a carrier for mitochondria-targeting molecules. We synthesized two commonly employed targeting systems, namely, ω-triphenylphosphonium fatty acids (group 2) and ω-triphenylphosphonium fatty alcohols (group 3), to assess the impact of the TPP module on the biological efficacy of mitochondria-targeting molecules. We evaluated their fungicidal activities against nine plant pathogenic fungi in comparison to alkyl-1-triphenylphosphonium compounds (group 1). All three compound groups exhibited fungicidal activity and displayed a distinct "cut-off effect", which depended on the length of the carbon chain. Specifically, group 1 compounds showed a cut-off point at C10 (compound 1-7), while group 2 and 3 compounds exhibited cut-off points at C15 (compound 2-12) and C14 (compound 3-11), respectively. Notably, group 1 compounds displayed significantly higher fungicidal activity compared to groups 2 and 3. However, group 2 and 3 compounds showed similar activity to each other, although susceptibility may depend on the pathogen tested. Initial investigations into the mechanism of action of the most active compounds suggested that their fungicidal performance may be primarily attributed to their ability to damage the membrane, as well as uncoupling activity and inhibition of fungal respiration. Our findings suggest that the TPP module used in delivery systems as aliphatic acyl or alkoxyl derivatives with carbon chains length < 10 will contribute negligible fungicidal activity to the TPP-conjugate compared to the effect of high level of accumulation in mitochondria due to its mitochondria-targeting ability. These results provide a foundation for utilizing TPP as a promising carrier in the design and development of more effective mitochondria-targeting drugs or pesticides.

High-energy pacing inhibits slow-wave dysrhythmias in the small intestine.

The motility of the gastrointestinal tract is coordinated in part by rhythmic slow waves, and disrupted slow-wave patterns are linked to functional motility disorders. At present, there are no treatment strategies that primarily target slow-wave activity. This study assessed the use of pacing to suppress glucagon-induced slow-wave dysrhythmias in the small intestine. Slow waves in the jejunum were mapped in vivo using a high-resolution surface-contact electrode array in pigs (n = 7). Glucagon was intravenously administered to induce hyperglycemia. Slow-wave propagation patterns were categorized into antegrade, retrograde, collision, pacemaker, and uncoupled activity. Slow-wave characteristics such as period, amplitude, and speed were also quantified. Postglucagon infusion, pacing was applied at 4 mA and 8 mA and the resulting slow waves were quantified spatiotemporally. Antegrade propagation was dominant throughout all stages with a prevalence of 55 ± 38% at baseline. However, glucagon infusion resulted in a substantial and significant increase in uncoupled slow waves from 10 ± 8% to 30 ± 12% (P = 0.004) without significantly altering the prevalence of other slow-wave patterns. Slow-wave frequency, amplitude, and speed remained unchanged. Pacing, particularly at 8 mA, significantly suppressed dysrhythmic slow-wave patterns and achieved more effective spatial entrainment (85%) compared with 4 mA (46%, P = 0.039). This study defined the effect of glucagon on jejunal slow waves and identified uncoupling as a key dysrhythmia signature. Pacing effectively entrained rhythmic activity and suppressed dysrhythmias, highlighting the potential of pacing for gastrointestinal disorders associated with slow-wave abnormalities.NEW & NOTEWORTHY Glucagon was infused in pigs to induce hyperglycemia and the resulting slow-wave response in the intact jejunum was defined in high resolution for the first time. Subsequently, with pacing, the glucagon-induced dysrhythmias were suppressed and spatially entrained for the first time with a success rate of 85%. The ability to suppress slow-wave dysrhythmias through pacing is promising in treating motility disorders that are associated with intestinal dysrhythmias.

Structural investigations on the mitochondrial uncouplers niclosamide and FCCP.

There has been renewed interest in using mitochondrial uncoupler compounds such as niclosamide and carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) for the treatment of obesity, hepatosteatosis and diseases where oxidative stress plays a role. However, both FCCP and niclosamide have undesirable effects that are not due to mitochondrial uncoupling, such as inhibition of mitochondrial oxygen consumption by FCCP and induction of DNA damage by niclosamide. Through structure-activity analysis, we identified FCCP analogues that do not inhibit mitochondrial oxygen consumption but still provided good, although less potent, uncoupling activity. We also characterized the functional role of the niclosamide 4'-nitro group, the phenolic hydroxy group and the anilide amino group in mediating uncoupling activity. Our structural investigations provide important information that will aid further drug development.

Abstract 3011: Parkin Mediated Mitophagy Deficiency In VSMCs Exacerbates Abdominal Aortic Aneurysm

Background: Parkin-mediated mitophagy eliminates dysfunctional mitochondria to reduce inflammatory responses caused by mtDAMPs. Given that reduced mitophagy is a hallmark of aging and AAA is an aging-associated disease, we sought to investigate the role of Parkin-mediated mitophagy in the development of AAAs. Methods: Groups of young (8-10 wks, n= 6/group) and aged (18-20 months, n=6/group) male Mito-QC mice either without intervention or underwent AAV.mPCSK9 D377Y infection and western diet feeding followed by Angiotensin II (AngII) or saline pump infusion for 28 days. Separately, young male Myh11-creER T2 Parkin fl/fl mice underwent tamoxifen (Parkin SMCKO) or vehicle injections (n=35-39/group), AAV.mPCSK9 D377Y infection, western diet feeding and AngII infusion. In vitro , MOVAs were treated with AngII and the USP30 inhibitor, a mitophagy enhancer, or vehicle followed by assessment of Parkin and mtROS. Results: VSMCs in aged Mito-QC mice, identified by a CD45 - CD90 - α-actin + profile, exhibit diminished mitophagy activity (p=0.017) and decreased Parkin expression (p=0.029). In young AAAs, VSMCs characterized as CD45 - CD90 - α-actin high and α-actin low also demonstrated decreased mitophagy (p=0.0017, p=0.0020), decreased Parkin(p=0.029), and a decreased trend in SDHB in Complex II (p=0.103) and NDUFB8 in Complex I (p=0.040) of the electron transport chain. Parkin SMCKO mice demonstrated increased AAA rupture (p=0.038), greater aneurysm size (p=0.044), reduced mtDNA copy numbers (p=0.026) and elevated mtROS compared to their controls. Parkin SMCKO mice demonstrated trends of diminished expression of respiratory complexes (p=0.070) and an exacerbated decrease in the maximal OCR involving both coupled and uncoupled activities of Complex I plus II (p=0.045, 0.028). In vitro experiments demonstrated treatment with the USP30 inhibitor reversed the reduction in mitophagy activity and mtROS increasement induced by AngII in MOVAS. Conclusions: In conclusion, mitophagy activity in aortic VSMCs and Parkin decreased with aging and separately, in AAAs and Parkin SMCKO mice, associated with exacerbation of AAA progression. These studies suggest stabilization of Parkin-dependent mitophagy could represent a novel AAA therapeutic target.

Conjugating uncoupler compounds with hydrophobic hydrocarbon chains to achieve adipose tissue selective drug accumulation

One potential approach for treating obesity is to increase energy expenditure in brown and white adipose tissue. Here we aimed to achieve this outcome by targeting mitochondrial uncoupler compounds selectively to adipose tissue, thus avoiding side effects from uncoupling in other tissues. Selective drug accumulation in adipose tissue has been observed with many lipophilic compounds and dyes. Hence, we explored the feasibility of conjugating uncoupler compounds with a lipophilic C8-hydrocarbon chain via an ether bond. We found that substituting the trifluoromethoxy group in the uncoupler FCCP with a C8-hydrocarbon chain resulted in potent uncoupling activity. Nonetheless, the compound did not elicit therapeutic effects in mice, likely as a consequence of metabolic instability resulting from rapid ether bond cleavage. A lipophilic analog of the uncoupler compound 2,6-dinitrophenol, in which a C8-hydrocarbon chain was conjugated via an ether bond in the para-position (2,6-dinitro-4-(octyloxy)phenol), exhibited increased uncoupling activity compared to the parent compound. However, in vivo pharmacokinetics studies suggested that 2,6-dinitro-4-(octyloxy)phenol was also metabolically unstable. In conclusion, conjugation of a hydrophobic hydrocarbon chain to uncoupler compounds resulted in sustained or improved uncoupling activity. However, an ether bond linkage led to metabolic instability, indicating the need to conjugate lipophilic groups via other chemical bonds.

Vascular protein disulfide isomerase A1 mediates endothelial dysfunction induced by angiotensin II in mice.

Protein disulfide isomerases (PDIs) are involved in platelet aggregation and intravascular thrombosis, but their role in regulating endothelial function is unclear. Here, we characterized the involvement of vascular PDIA1 in angiotensin II (Ang II)-induced endothelial dysfunction in mice. Endothelial dysfunction was induced in C57BL/6JCmd male mice via Ang II subcutaneous infusion, and PDIA1 was inhibited with bepristat. Endothelial function was assessed invivo with magnetic resonance imaging and exvivo with a myography, while arterial stiffness was measured as pulse wave velocity. Nitric oxide (NO) bioavailability was measured in the aorta (spin-trapping electron paramagnetic resonance) and plasma (NO2 - and NO3 - levels). Oxidative stress, eNOS uncoupling (DHE-based aorta staining), and thrombin activity (thrombin-antithrombin complex; calibrated automated thrombography) were evaluated. The inhibition of PDIA1 by bepristat in Ang II-treated mice prevented the impairment of NO-dependent vasodilation in the aorta as evidenced by the response to acetylcholine invivo, increased systemic NO bioavailability and the aortic NO production, and decreased vascular stiffness. Bepristat's effect on NO-dependent function was recapitulated exvivo in Ang II-induced endothelial dysfunction in isolated aorta. Furthermore, bepristat diminished the Ang II-induced eNOS uncoupling and overproduction of ROS without affecting thrombin activity. In Ang II-treated mice, the inhibition of PDIA1 normalized the NO-ROS balance, prevented endothelial eNOS uncoupling, and, thereby, improved vascular function. These results indicate the importance of vascular PDIA1 in regulating endothelial function, but further studies are needed to elucidate the details of the mechanisms involved.

Association of a high rate of glycolysis and the activity of the uncoupling of oxidation and phosphorylation in the blood cells of patients with late-stage knee osteoarthritis and the development of postoperative pain

Objective: to investigate the relationship between the expression of genes mediating cellular energy production and the development of chronic postoperative pain (CPP) after total knee arthroplasty (TKA) in patients with osteoarthritis (OA).Material and methods. Prior to TKA, the blood of 50 patients with stage III–IV knee OA and complaints of constant pain and joint dysfunction was analyzed. The control group consisted of 26 healthy individuals. Pain intensity was assessed using a visual analogue scale (VAS), a short BPI questionnaire and the WOMAC index, and the presence of neuropathic pain was assessed using the DN4 and PainDETECT questionnaires. The development of CPP was determined 3 and 6 months after TKA. Total RNA isolated from blood was used to determine the expression of PKM2, LDH, SDH, AMPKα, PDH, IDH, MDH and ATP synthase genes by real-time quantitative reverse transcriptase-polymerase chain reaction.Results and discussion. CPP ≥30 mm according to VAS was detected in 17 patients. Before TKA, the expression of all analyzed genes was significantly increased compared to that of the control group. However, there were no differences in clinical, pain-related and functional indicators in the analyzed group of patients with OA. Before surgery, patients who subsequently developed CPP had significantly higher expression of genes related to glycolysis (PKM2, LDH), Krebs cycle – KC (SDH) and master regulator of energy metabolism (AMPKα) than patients who were satisfied with the results of TKA. At the same time, no differences were found in the expression of PDH and other KC enzyme genes (IDH, MDH) and ATP synthase in patients with and without CPP.Conclusion. The development of CPP is associated with a higher rate of glycolysis and energy deficiency, presumably due to the higher uncoupling activity of oxidation and phosphorylation that can be observed before TKA.

Proton transfer and regulation across chemical interfaces by small-molecule assemblies.

We report on measurements and control of proton gradient across interfaces of water and dichloroethane. Such interfaces are interesting as mimics of biological membranes. We use impedance spectroscopy to quantify interfacial proton gradient and identify proton transfer modes. We quantify proton movement using reciprocal of time constant (τ-1 ) acquired from electrochemical impedance modeling. We show that proton gradient across interfaces of water/dichloroethane and τ-1 correlate with the aqueous phase pH, changing from ca. 1 s-1 at pH 1 to 0.2 s-1 at pH 7. τ-1 changes in the presence of proton shuttling fat-soluble molecules. Dinitrophenol acts as a pH activated proton coupler which is active at around neutral pH and inert at pH <4. However, quinone type cofactors change the interfacial proton transport when activated by redox reactions with ferrocene type molecules, such as decamethyl ferrocence (DMFc). Quinone type cofactors show distinct features in their impedance response assigned to a proton coupled electron transfer (PCET) process, different from the uncoupled proton transfer activity of dinitrophenol. The observed PCET reaction significantly changes τ-1 . We use τ-1 as a proton transport descriptor. In particular, CoQ10 -DMFc shows a τ-1 of 3.5 s-1 at pH 7, indicating how small-molecule assemblies change proton availability.

Investigation of barium iron oxides for CO2 capture and chemical looping oxygen uncoupling

The performance of two underexploited ternary oxides – Ba3Fe2O6 and Ba5Fe2O8 are investigated for carbon dioxide capture and chemical looping oxygen uncoupling. The ternary compound Ba3Fe2O6 was found to have a structure characterised by space group Pa3¯. Experimental results have shown that both Ba3Fe2O6 and Ba5Fe2O8 are capable of cyclically capturing CO2 at temperatures above 800 °C. Ba5Fe2O8 demonstrated superior CO2 capture performance compared to Ba3Fe2O6, with consistent gravimetric CO2 uptake capacities of 4.35 wt% and 13.39 wt% at 900 °C and 1000 °C, respectively, over 20 cycles. In comparison, Ba3Fe2O6 demonstrated high initial CO2 uptake capacities which deteriorated cyclically, with 20 cycle average capacities of 7.73 wt% and 11.99 wt% at 900 °C and 1000 °C, respectively. Ba3Fe2O6 also exhibits excellent recyclability and satisfactory chemical looping oxygen uncoupling (CLOU) activity over temperature swing cycles between 550 °C and 950 °C. In contrast, the strong affinity with CO2 makes Ba5Fe2O8 unsuitable for application in chemical looping oxygen uncoupling or chemical looping air separation, especially in the presence of substantial partial pressures of CO2.

Mitochondria-targeted Uncouplers Decrease Inflammatory Reactions in Endothelial Cells by Enhancing Methylation of the ICAM1 Gene Promoter.

The study aimed to investigate the effects of low concentrations of mitochondrial uncouplers in endothelial cells on the CpG dinucleotide methylation of the ICAM1 gene promoter. The excessive inflammatory response in the endothelium is responsible for the development of many cardiovascular diseases. Mitochondria are important regulators of endothelial cell functions. Mild uncoupling of oxidative phosphorylation and respiration in endothelial mitochondria exerts a long lasting anti-inflammatory effect. However, the detailed mechanism of the anti-inflammatory activity of mitochondrial uncouplers remains unclear.We hypothesized that mild mitochondrial uncoupling leads to epigenetic changes in genomic DNA contributing to the anti-inflammatory response. We studied the long-term effects of mitochondria-targeted compounds with the uncoupler's activities: the antioxidant plastoquinonyl-decyltriphenylphosphonium (SkQ1), dodecyl-triphenylphosphonium (C12TPP), and 2,4-dinitrophenol (DNP). The mRNA expression of the intercellular adhesion molecule 1 (ICAM1), a marker of inflammatory activation of endothelial cells, was measured by RT-qPCR. Cytosine methylation in the CpG sites of the ICAM1 gene promoter was estimated by bisulfite sequencing of individual clones. It was found that downregulation of ICAM1 expression caused by DNP and C12TPP was accompanied by an increase in the methylation of CpG sites in the ICAM1 gene promoter. None of the compounds affected intracellular or intramitochondrial ATP levels. Low concentrations of mitochondrial oxidative phosphorylation uncouplers are able to increase methylation of ICAM1 gene promoter, which corresponds to the observed decrease in the levels of mRNA of this gene. Thus, the change in methylation of the ICAM1 gene promoter may underlie the mechanism of decreased ICAM1 expression caused by mild mitochondrial depolarization. Mitochondrial uncouplers may be exploited as possible therapeutic candidates to treat excessive inflammation in endothelium, by changing the methylation status of genomic DNA.

The transient character of mitochondrial uncoupling by the popular fungicide fluazinam is specific for liver

The popular fungicide fluazinam is known to exhibit an unusual cyclic pattern of the protonophoric uncoupling activity in isolated rat liver mitochondria (RLM), with membrane deenergization followed by spontaneous recoupling in the minute scale, which is associated with glutathione conjugation of fluazinam catalyzed by glutathione-S-transferase (GST). Here, we compare the fluazinam effect on RLM with that on rat kidney (RKM) and heart (RHM) mitochondria by monitoring three bioenergetic parameters: oxygen consumption rate, mitochondrial membrane potential and reduction of nucleotides. Only in RLM, the uncoupling activity of fluazinam was transient, i.e. disappeared in a few minutes, whereas in RKM and RHM it was stable in this time scale. We attribute this difference to the increased activity of mitochondrial GST in liver. We report data on the detection of glutathione-fluazinam conjugates by mass-spectrometry, thin layer chromatography and capillary electrophoresis after incubation of fluazinam with RLM but not with RKM, which supports the assumption of the tissue specificity of the conjugation.

Acanthamoeba castellanii Uncoupling Protein: A Complete Sequence, Activity, and Role in Response to Oxidative Stress.

Uncoupling proteins (UCPs) are mitochondrial inner membrane transporters that mediate free-fatty-acid-induced, purine-nucleotide-inhibited proton leak into the mitochondrial matrix, thereby uncoupling respiratory substrate oxidation from ATP synthesis. The aim of this study was to provide functional evidence that the putative Acucp gene of the free-living protozoan amoeba, A. castellanii, encodes the mitochondrial protein with uncoupling activity characteristic of UCPs and to investigate its role during oxidative stress. We report the sequencing and cloning of a complete Acucp coding sequence, its phylogenetic analysis, and the heterologous expression of AcUCP in the S. cerevisiae strain InvSc1. Measurements of mitochondrial respiratory activity and membrane potential indicate that the heterologous expression of AcUCP causes AcUCP-mediated uncoupling activity. In addition, in a model of oxidative stress with increased reactive oxygen species levels (superoxide dismutase 1 knockout yeasts), AcUCP expression strongly promotes cell survival and growth. The level of superoxide anion radicals is greatly reduced in the ΔSOD1 strain expressing AcUCP. These results suggest that AcUCP targeted to yeast mitochondria causes uncoupling and may act as an antioxidant system. Phylogenetic analysis shows that the A. castellanii UCP diverges very early from other UCPs, but clearly locates within the UCP subfamily rather than among other mitochondrial anion carrier proteins.

Efficiency of mitochondrial uncoupling by modified butyltriphenylphosphonium cations and fatty acids correlates with lipophilicity of cations: Protonophoric vs leakage mechanisms

In order to determine the share of protonophoric activity in the uncoupling action of lipophilic cations a number of analogues of butyltriphenylphosphonium with substitutions in phenyl rings (C4TPP-X) were studied on isolated rat liver mitochondria and model lipid membranes. An increase in the rate of respiration and a decrease in the membrane potential of isolated mitochondria were observed for all the studied cations, the efficiency of these processes was significantly enhanced in the presence of fatty acids and correlated with the octanol-water partition coefficient of the cations. The ability of C4TPP-X cations to induce proton transport across the lipid membrane of liposomes loaded with a pH-sensitive fluorescent dye increased also with their lipophilicity and depended on the presence of palmitic acid in the liposome membrane. Of all the cations, only butyl[tri(3,5-dimethylphenyl)]phosphonium (C4TPP-diMe) was able to induce proton transport by the mechanism of formation of a cation-fatty acid ion pair on planar bilayer lipid membranes and liposomes. The rate of oxygen consumption by mitochondria in the presence of C4TPP-diMe increased to the maximum values corresponding to conventional uncouplers; for all other cations the maximum uncoupling rates were significantly lower. We assume that the studied cations of the C4TPP-X series, with the exception of C4TPP-diMe at low concentrations, cause nonspecific leak of ions through lipid model and biological membranes which is significantly enhanced in the presence of fatty acids.

Investigation into the Mechanism of Action of the Tuberculosis Drug Candidate SQ109 and Its Metabolites and Analogues in Mycobacteria.

We tested a series of SQ109 analogues against Mycobacterium tuberculosis and M. smegmatis, in addition to determining their uncoupling activity. We then investigated potential protein targets, involved in quinone and cell wall biosynthesis, using "rescue" experiments. There was little effect of menaquinone on growth inhibition by SQ109, but there were large increases in the IC50 of SQ109 and its analogues (up to 20×) on addition of undecaprenyl phosphate (Up), a homologue of the mycobacterial decaprenyl (C50) diphosphate. Inhibition of an undecaprenyl diphosphate phosphatase, an ortholog of the mycobacterial phosphatase, correlated with cell growth inhibition, and we found that M. smegmatis cell growth inhibition could be well predicted by using uncoupler and Up-rescue results. We also investigated whether SQ109 was metabolized inside Mycobacterium tuberculosis, finding only a single metabolite, previously shown to be inactive. The results are of general interest since they help explain the mechanism of SQ109 in mycobacteria.

Base-excision restriction enzymes: expanding the world of epigenetic immune systems.

The restriction enzymes examined so far are phosphodiesterases, which cleave DNA strands by hydrolyzing phosphodiester bonds. Based on the mobility of restriction-modification systems, recent studies have identified a family of restriction enzymes that excise a base in their recognition sequence to generate an abasic (AP) site unless the base is properly methylated. These restriction glycosylases also show intrinsic but uncoupled AP lyase activity at the AP site, generating an atypical strand break. Action of an AP endonuclease at the AP site may generate another atypical break, rejoining/repairing of which is difficult. The PabI family of restriction enzymes contain a novel fold (HALFPIPE) and show unusual properties, such as non-requirement of divalent cations for cleavage. These enzymes are present in Helicobacteraceae/Campylobacteraceae and in few hyperthermophilic archaeal species. In Helicobacter genomes, their recognition sites are strongly avoided, and the encoding genes are often inactivated by mutations or replacement, indicating that their expression is toxic for the cells. The discovery of restriction glycosylases generalizes the concept of restriction-modification systems to epigenetic immune systems, which may use any mode of damage to DNA that are considered "non-self" based on epigenetic modifications. This concept will add to our understanding of immunity and epigenetics.

Molecular determinants of inhibition of UCP1-mediated respiratory uncoupling

Brown adipose tissue expresses uncoupling protein 1 (UCP1), which dissipates energy as heat, making it a target for treating metabolic disorders. Here, we investigate how purine nucleotides inhibit respiration uncoupling by UCP1. Our molecular simulations predict that GDP and GTP bind UCP1 in the common substrate binding site in an upright orientation, where the base moiety interacts with conserved residues R92 and E191. We identify a triplet of uncharged residues, F88/I187/W281, forming hydrophobic contacts with nucleotides. In yeast spheroplast respiration assays, both I187A and W281A mutants increase the fatty acid-induced uncoupling activity of UCP1 and partially suppress the inhibition of UCP1 activity by nucleotides. The F88A/I187A/W281A triple mutant is overactivated by fatty acids even at high concentrations of purine nucleotides. In simulations, E191 and W281 interact with purine but not pyrimidine bases. These results provide a molecular understanding of the selective inhibition of UCP1 by purine nucleotides.