Abstract
The escalating burden of antibiotic drug resistance necessitates research into novel classes of antibiotics and their mechanism of action. Pyrrolomycins are a family of potent natural product antibiotics with nanomolar activity against Gram-positive bacteria, yet with an elusive mechanism of action. In this work, we dissect the apparent Gram-positive specific activity of pyrrolomycins and show that Gram-negative bacteria are equally sensitive to pyrrolomycins when drug efflux transporters are removed and that albumin in medium plays a large role in pyrrolomycin activity. The selection of resistant mutants allowed for the characterization and validation of a number of mechanisms of resistance to pyrrolomycins in both Staphylococcus aureus and an Escherichia coli ΔtolC mutant, all of which appear to affect compound penetration rather than being target associated. Imaging of the impact of pyrrolomycin on the E. coli ΔtolC mutant using scanning electron microscopy showed blebbing of the bacterial cell wall often at the site of bacterial division. Using potentiometric probes and an electrophysiological technique with an artificial bilayer lipid membrane, it was demonstrated that pyrrolomycins C and D are very potent membrane-depolarizing agents, an order of magnitude more active than conventional carbonyl cyanide m-chlorophenylhydrazone (CCCP), specifically disturbing the proton gradient and uncoupling oxidative phosphorylation via protonophoric action. This work clearly unveils the until-now-elusive mechanism of action of pyrrolomycins and explains their antibiotic activity as well as mechanisms of innate and acquired drug resistance in bacteria.
https://pubmed.ncbi.nlm.nih.gov/31405863
Protonophore
A protonophore, also known as a proton translocator, is an ionophore that moves protons across lipid bilayers or other type of membranes. This would otherwise not occur as protons cations (H+) have positive charge and hydrophilic properties, making them unable to cross without a channel or transporter in the form of a protonophore. Protonophores are generally aromatic compounds with a negative charge, that are both hydrophobic and capable of distributing the negative charge over a number of atoms by π-orbitals which delocalize a proton's charge when it attaches to the molecule.Both the neutral and the charged protonophore can diffuse across the lipid bilayer by passive diffusion and simultaneously facilitate proton transport. Protonophores uncouple oxidative phosphorylation via a decrease in the membrane potential of the inner membrane of mitochondria. They stimulate mitochondria respiration and heat production. Protonophores (uncouplers) are often used in biochemistry research to help explore the bioenergetics of chemiosmotic and other membrane transport processes. It has been reported that the protonophore has antibacterial activity by perturbing bacterial proton motive force.
Representative anionic protonophores include:
- 2,4-dinitrophenol (DNP)
- Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP)
- Carbonyl cyanide m-chlorophenyl hydrazone (CCCP)
https://en.wikipedia.org/wiki/Protonophore
Hyperpolarization (biology)
Hyperpolarization is a change in a cell's membrane potential that makes it more negative. It is the opposite of a depolarization. It inhibits action potentials by increasing the stimulus required to move the membrane potential to the action potential threshold.
Hyperpolarization is often caused by efflux of K+ (a cation) through K+ channels, or influx of Cl– (an anion) through Cl– channels. On the other hand, influx of cations, e.g. Na+ through Na+ channels or Ca2+ through Ca2+ channels, inhibits hyperpolarization. If a cell has Na+ or Ca2+ currents at rest, then inhibition of those currents will also result in a hyperpolarization. This voltage-gated ion channel response is how the hyperpolarization state is achieved. In neurons, the cell enters a state of hyperpolarization immediately following the generation of an action potential. While hyperpolarized, the neuron is in a refractory period that lasts roughly 2 milliseconds, during which the neuron is unable to generate subsequent action potentials. Sodium-potassium ATPases redistribute K+ and Na+ ions until the membrane potential is back to its resting potential of around –70 millivolts, at which point the neuron is once again ready to transmit another action potential.
https://en.wikipedia.org/wiki/Hyperpolarization_(biology)
Potassium channel-driven bioelectric signalling regulates metastasis in triple-negative breast cancer
Our study demonstrates that hyperpolarization of breast tumour cells increases invasion and metastasis and that depolarization of cells can be used to reduce metastatic burden.
https://ncbi.nlm.nih.gov/pmc/articles/PMC8688589
Why cancer cells have a more hyperpolarised mitochondrial membrane potential and emergent prospects for therapy
https://biorxiv.org/content/10.1101/025197v1.full
Cancer cells have distinct electrical properties that predict a susceptibility to lipophilic anions; a new cancer drug paradigm
Hyperpolarization is one of the characteristics of cancer cells, so depolarization of cells by protonophores is one of the options for cancer treatment.
Thanks. Are you aware of available and accessible Protonophores?
Note: the same can be achieved with e.g. potassium ionophors, that are indirectly creating the move of protons across membranes (which I think is one of the fundamental mechanism related to its anticancer activity, as this has impact on so many functions inside cells).
Kind regards,
Daniel
Thanks @daniel, it seems that drugs containing salicylic acid and its derivatives, such as salicylamide and salicylanilide, act as protonophores. However, other salicylate derivatives such as mesalazine and sulfasalazine have not yet been identified as protonophores!
Protonophore Compounds:
Niclosamide / salicylic acid derivative
https://doi.org/10.1371/journal.ppat.1002976Diflunisal / salicylic acid derivative
https://doi.org/10.1016/0024-3205(85)90180-8Nitazoxanide / salicylic acid derivative
https://doi.org/10.1016/j.apsb.2021.09.009Aspirin, Sodium Benzoate and Sodium Salicylate
https://doi.org/10.1093/jac/dkaa371Methylene Blue
https://doi.org/10.1016/j.freeradbiomed.2020.04.015Tamoxifen
https://doi.org/10.1016%2Fj.jcmgh.2020.04.012Carvedilol
https://doi.org/10.1016/S0024-3205(01)01109-2Clofazimine and Bedaquiline
https://doi.org/10.1073/pnas.1521988112Hyperforin / St. John's wort
https://doi.org/10.1038/srep07500Monensin / veterinary drug
https://doi.org/10.2527/1991.6952196xClosantel / veterinary drug, salicylanilide derivative
https://doi.org/10.1021/jm5006435
Hyperpolarization is often caused by efflux of K+ through potassium channels, or influx of Cl- through chloride channels, so as you said, potassium ionophores as well as chloride channel blockers cause depolarization of cancer cells. Interestingly, a chloride channel blocking chemical compound acts as an effective protonophore.
https://doi.org/10.1016/0014-5793(91)80992-C
Most protonophores are mitochondrial uncouplers, so they increase mitochondria respiration and heat production, and excessive heat or hyperthermia also causes tumor regression. It should be noted that aspirin (at a concentration of 5mM or 500mg) increases mitochondrial respiration by 300%, while DNP (the strongest protophore) increases it by 400%.
https://doi.org/10.1046/j.1365-2036.2000.00723.x
Other drugs such as retinoids, artemisinin, indomethacin, ketoprofen, meloxicam, pitavastatin, rosiglitazone, resveratrol, temsirolimus, etc., which have been identified in screening as mitochondrial uncouplers, may also be a protonophore!
Uncoupling of oxidative phosphorylation by curcumin
Curcumin is a phytochemical isolated from the rhizome of turmeric. Recent reports have shown curcumin to have antioxidant, anti-inflammatory and anti-tumor properties as well as affecting the 5′-AMP activated protein kinase (AMPK), mTOR and STAT-3 signaling pathways. We provide evidence that curcumin acts as an uncoupler. Well-established biochemical techniques were performed on isolated rat liver mitochondria in measuring oxygen consumption, F0F1-ATPase activity and ATP biosynthesis. Curcumin displays all the characteristics typical of classical uncouplers like fccP and 2,4-dinitrophenol (DNP). In addition, at concentrations higher than 50 μM (or 100 mg intravenous), curcumin was found to inhibit mitochondrial respiration which is a characteristic feature of inhibitory uncouplers. As a protonophoric uncoupler and as an activator of F0F1-ATPase, curcumin causes a decrease in ATP biosynthesis in rat liver mitochondria. The resulting change in ATP:AMP could disrupt the phosphorylation status of the cell; this provides a possible mechanism for its activation of AMPK and its downstream mTOR and STAT-3 signaling.
https://doi.org/10.1016/j.bbrc.2009.08.121
So Curcumin can be added to the list of protonophore compounds. Interestingly, Ccurcumin as well as EGCG, Honokiol and Myricetin cause cell membrane depolarization like 2,4-dinitrophenol (the strongest protonophore).
https://doi.org/10.1089/ars.2017.7404
Let me add that all these compounds are actually a member of phenols and their derivatives (polyphenol, nitrophenol, etc.).
Would you consider phenylbutyrate as such a compound?
Short-chain fatty acids such as Butyrate and medium-chain fatty acids are not protonophore, only long-chain acids such as Stearate are protonophore and mitochondrial uncouplers.
However, being a protonophore is not a very important chemical compound, what is important is the depolarization of cancer cells that phenyl butyrate does (like DCA and 3BP).
Phenyl butyrate inhibits pyruvate dehydrogenase kinase 1 and contributes to its anti-cancer effect
Phenyl butyrate (PB) has been proved to decrease pyruvate dehydrogenase (PDH) phosphorylation level and increase PDH activity by inhibiting pyruvate dehydrogenase kinase 1 (PDK1) in fibroblast cells, PDH deficiency zebrafish and wild type mice. PB has also shown efficacy in many cancers and so far, all of its anti-tumor activity has been attributed to the histone deacetylase (HDAC) inhibition. As PDK1/PDH controls the critical switch between oxidative phosphorylation and glycolysis in cancer cells, PDK1 is a key target in tumor metabolism for anti-cancer treatment. We hypothesize that the therapeutic effects of PB in cancers might also depend on suppressing PDK1 and promoting PDH activity, in addition to its proposed role as HDAC inhibitor. We showed that PB directly inhibited the kinase activity of PDK1 and increased the activity of PDH in an enzyme assay. In several different cancer cell lines, PB reduced the phosphorylation level of PDH, increased the mitochondrial respiration, decreased glycolysis in cytoplasm, reversed mitochondrial hyperpolarization, activated several proteins in apoptotic signaling pathway and then induced the apoptosis of cells. In summary, this is the first study indicated that PB could exert its anti-cancer effects through inhibiting PDK1, altering the mitochondrial bioenergetics and inducing apoptosis.
Would you consider phenylbutyrate as such a compound?
Short-chain fatty acids such as Butyrate and medium-chain fatty acids are not protonophore, only long-chain acids such as Stearate are protonophore and mitochondrial uncouplers.
However, being a protonophore is not a very important chemical compound, what is important is the depolarization of cancer cells that phenyl butyrate does (like DCA and 3BP).
Phenyl butyrate inhibits pyruvate dehydrogenase kinase 1 and contributes to its anti-cancer effect
Phenyl butyrate (PB) has been proved to decrease pyruvate dehydrogenase (PDH) phosphorylation level and increase PDH activity by inhibiting pyruvate dehydrogenase kinase 1 (PDK1) in fibroblast cells, PDH deficiency zebrafish and wild type mice. PB has also shown efficacy in many cancers and so far, all of its anti-tumor activity has been attributed to the histone deacetylase (HDAC) inhibition. As PDK1/PDH controls the critical switch between oxidative phosphorylation and glycolysis in cancer cells, PDK1 is a key target in tumor metabolism for anti-cancer treatment. We hypothesize that the therapeutic effects of PB in cancers might also depend on suppressing PDK1 and promoting PDH activity, in addition to its proposed role as HDAC inhibitor. We showed that PB directly inhibited the kinase activity of PDK1 and increased the activity of PDH in an enzyme assay. In several different cancer cell lines, PB reduced the phosphorylation level of PDH, increased the mitochondrial respiration, decreased glycolysis in cytoplasm, reversed mitochondrial hyperpolarization, activated several proteins in apoptotic signaling pathway and then induced the apoptosis of cells. In summary, this is the first study indicated that PB could exert its anti-cancer effects through inhibiting PDK1, altering the mitochondrial bioenergetics and inducing apoptosis.
Fascinating!
Potassium channel openers are uncoupling protonophores
Excessive build-up of mitochondrial protonic potential is harmful to cellular homeostasis, and modulation of inner membrane permeability a proposed countermeasure. Here, we demonstrate that structurally distinct potassium channel openers, diazoxide and pinacidil, facilitated transmembrane proton translocation generating H+-selective current through planar phospholipid membrane. Both openers depolarized mitochondria, activated state 4 respiration and reduced oxidative phosphorylation, recapitulating the signature of mitochondrial uncoupling. This effect was maintained in K+-free conditions and shared with the prototypic protonophore 2,4-dinitrophenol. Diazoxide, pinacidil and 2,4-dinitrophenol, but not 2,4-dinitrotoluene lacking protonophoric properties, preserved functional recovery of ischemic heart. The identified protonophoric property of potassium channel openers, thus, implicates a previously unrecognized component in their mechanism of cardioprotection.
https://doi.org/10.1016/j.febslet.2004.05.031
Minoxidil is also a potassium channel opener, but it causes hyperpolarization of the cell membrane by decreasing intracellular potassium level, so it is not a good choice for cancer treatment. Of course, maybe I'm wrong!
Anti-invasive effects of minoxidil on breast cancer cells
A plethora of ion channels have been shown to be involved systemically in the pathophysiology of cancer and ion channel blockers can produce anti-metastatic effects. However, although ion channels are known to frequently function in concerted action, little is known about possible combined effects of ion channel modulators on metastatic cell behaviour. Here, we investigated functional consequences of pharmacologically modulating ATP-gated potassium (KATP) channel and voltage-gated sodium channel (VGSC) activities individually and in combination. Two triple-negative human breast cancer cell lines were used: MDA-MB-231 and MDA-MB-468, the latter mainly for comparison. Most experiments were carried out on hypoxic cells. Electrophysiological effects were studied by whole-cell patch clamp recording. Minoxidil (a KATP channel opener) and ranolazine (a blocker of the VGSC persistent current) had no effect on cell viability and proliferation, alone or in combination. In contrast, invasion was significantly reduced in a dose-dependent manner by clinical concentrations of minoxidil and ranolazine. Combining the two drugs produced significant additive effects at concentrations as low as 0.625 μM ranolazine and 2.5 μM minoxidil. Electrophysiologically, acute application of minoxidil shifted VGSC steady-state inactivation to more hyperpolarised potentials and slowed recovery from inactivation, consistent with inhibition of VGSC activation. We concluded (i) that clinically relevant doses of minoxidil and ranolazine individually could inhibit cellular invasiveness dose dependently and (ii) that their combination was additionally effective. Accordingly, ranolazine, minoxidil and their combination may be repurposed as novel anti-metastatic agents.
Fifty Years of Research on Protonophores: Mitochondrial Uncoupling As a Basis for Therapeutic Action
Abstract
Protonophores are compounds capable of electrogenic transport of protons across membranes. Protonophores have been intensively studied over the past 50 years owing to their ability to uncouple oxidation and phosphorylation in mitochondria and chloroplasts. The action mechanism of classical uncouplers, such as DNP and CCCP, in mitochondria is believed to be related to their protonophoric activity; i.e., their ability to transfer protons across the lipid part of the mitochondrial membrane. Given the recently revealed deviations in the correlation between the protonophoric activity of some uncouplers and their ability to stimulate mitochondrial respiration, this review addresses the involvement of some proteins of the inner mitochondrial membrane, such as the ATP/ADP antiporter, dicarboxylate carrier, and ATPase, in the uncoupling process. However, these deviations do not contradict the Mitchell theory but point to a more complex nature of the interaction of DNP, CCCP, and other uncouplers with mitochondrial membranes. Therefore, a detailed investigation of the action mechanism of uncouplers is required for a more successful pharmacological use, including their antibacterial, antiviral, anticancer, as well as cardio-, neuro-, and nephroprotective effects.
INTRODUCTION
The term protonophore was first used in a review by Skulachev published in 1970 [1], but protonophores were discovered several years earlier in the laboratories of Lehninger (1966 [2]), Skulachev [3], and Lieberman [4]. Those studies showed that some compounds previously identified as uncouplers of oxidative phosphorylation in mitochondria increase the proton conductivity of lipid membranes. This observation was in agreement with the Mitchell theory on the coupling of oxidation and phosphorylation in mitochondria through the electrochemical potential difference between protons [5]. In 1967, Mitchell observed proton transfer by some uncouplers in mitochondrial membranes [6]. As already mentioned, the term protonophore was coined in 1970 [1]; before that, uncouplers were called proton conductors, or H+ carriers [2]. It is worth noting a study in 1967 [7] on an uncoupler-mediated increase in the proton conductivity of liposomes, but that study did not attract as much research attention as the publication in Nature [3]. Skulachev’s group's priority in the discovery of protonophores was also confirmed by a publication in Nature in 1969 [8], which reported a quantitative correlation between protonophore activity in lipid membranes (planar bilayers, BLM) and stimulation of mitochondrial respiration in state 4 (P-vs-U-correlation) for many uncouplers of various chemical structures. This publication in 1969 [8] is now considered classic. It should be noted that the term ionophore, which denotes a compound that transports ions through membranes, had appeared earlier and was actively used in Pressman’s works in the mid-1960s [9]. However, Pressman focused on the transport of metal ions and did not use the term protonophore. At that time, Russian-language articles often used the term membrane-active complexone [10], which was later replaced by the term ionophore.
Apart from phenols (dinitrophenol, pentachlorophenol, etc.), various hydrazones (CCCP, FCCP), benzimidazoles (TTFB and DTFB), dicoumarol, and salicylic acid were studied among the first protonophores. These compounds, which are weak aromatic acids, correspond well to the general protonophore structure.
https://doi.org/10.32607/actanaturae.11610
Benzimidazoles
Compounds with a BENZENE fused to IMIDAZOLES.
