Anticancer efficacy triggered by synergistically modulating the homeostasis of anions and iron: Design, synthesis and biological evaluation of dual-functional squaramide-hydroxamic acid conjugates.

Targeting the homeostasis of anions and iron has emerged as a promising therapeutic approach for the treatment of cancers. However, single-targeted agents often fall short of achieving optimal treatment efficacy. Herein we designed and synthesized a series of novel dual-functional squaramide-hydroxamic acid conjugates that are capable of synergistically modulating the homeostasis of anions and iron. Among them, compound 16 exhibited the most potent antiproliferative activity against a panel of selected cancer cell lines, and strong in vivo anti-tumor efficacy. This compound effectively elevated lysosomal pH through anion transport, and reduced the levels of intracellular iron. Compound 16 could disturb autophagy in A549 cells and trigger robust apoptosis. This compound caused cell cycle arrest at the G1/S phase, altered the mitochondrial function and elevated ROS levels. The present findings clearly demonstrated that synergistic modulation of anion and iron homeostasis has high potentials in the development of promising chemotherapeutic agents with dual action against cancers.

Cloning, expression, and purification of an α-carbonic anhydrase from Toxoplasma gondii to unveil its kinetic parameters and anion inhibition profile.

Toxoplasmosis, induced by the intracellular parasite Toxoplasma gondii, holds considerable implications for global health. While treatment options primarily focusing on folate pathway enzymes have notable limitations, current research endeavours concentrate on pinpointing specific metabolic pathways vital for parasite survival. Carbonic anhydrases (CAs, EC 4.2.1.1) have emerged as potential drug targets due to their role in fundamental reactions critical for various protozoan metabolic processes. Within T. gondii, the Carbonic Anhydrase-Related Protein (TgCA_RP) plays a pivotal role in rhoptry biogenesis. Notably, α-CA (TcCA) from another protozoan, Trypanosoma cruzi, exhibited considerable susceptibility to classical CA inhibitors (CAIs) such as anions, sulphonamides, thiols, and hydroxamates. Here, the recombinant DNA technology was employed to synthesise and clone the identified gene in the T. gondii genome, which encodes an α-CA protein (Tg_CA), with the purpose of heterologously overexpressing its corresponding protein. Tg_CA kinetic constants were determined, and its inhibition patterns explored with inorganic metal-complexing compounds, which are relevant for rational compound design. The significance of this study lies in the potential development of innovative therapeutic strategies that disrupt the vital metabolic pathways crucial for T. gondii survival and virulence. This research may lead to the development of targeted treatments, offering new approaches to manage toxoplasmosis.

Aerobic exercises regulate the epididymal anion homeostasis of high-fat diet-induced obese rats through TRPA1-mediated Cl- and HCO3- secretion†.

Aerobic exercises could improve the sperm motility of obese individuals. However, the underlying mechanism has not been fully elucidated, especially the possible involvement of the epididymis in which sperm acquire their fertilizing capacity. This study aims to investigate the benefit effect of aerobic exercises on the epididymal luminal milieu of obese rats. Sprague-Dawley male rats were fed on a normal or high-fat diet (HFD) for 10 weeks and then subjected to aerobic exercises for 12 weeks. We verified that TRPA1 was located in the epididymal epithelium. Notably, aerobic exercises reversed the downregulated TRPA1 in the epididymis of HFD-induced obese rats, thus improving sperm fertilizing capacity and Cl- concentration in epididymal milieu. Ussing chamber experiments showed that cinnamaldehyd (CIN), agonist of TRPA1, stimulated an increase of the short-circuit current (ISC) in rat cauda epididymal epithelium, which was subsequently abolished by removing the ambient Cl- and HCO3-. In vivo data revealed that aerobic exercises increased the CIN-stimulated Cl- secretion rate of epididymal epithelium in obese rats. Pharmacological experiments revealed that blocking cystic fibrosis transmembrane regulator (CFTR) and Ca2+-activated Cl- channel (CaCC) suppressed the CIN-stimulated anion secretion. Moreover, CIN application in rat cauda epididymal epithelial cells elevated intracellular Ca2+ level, and thus activate CACC. Interfering with the PGHS2-PGE2-EP2/EP4-cAMP pathway suppressed CFTR-mediated anion secretion. This study demonstrates that TRPA1 activation can stimulate anion secretion via CFTR and CaCC, which potentially forming an appropriate microenvironment essential for sperm maturation, and aerobic exercises can reverse the downregulation of TRPA1 in the epididymal epithelium of obese rats.

Inhibition studies with simple and complex (in)organic anions of the γ-carbonic anhydrase from Mammaliicoccus (Staphylococcus) sciuri, MscCAγ.

The γ-carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic bacterium, Mammaliicoccus (Staphylococcus) sciuri (MscCAγ) was recently cloned and purified by our groups. Here we investigated inhibition of this enzyme with (in)organic simple and complex anions, in the search of inhibitors with potential applications. The most effective inhibitors (KIs in the micromolar range) were peroxydisulfate and trithiocarbonate, whereas submillimolar inhibition was observed with N,N-diethyldithiocarbamate and phenylboronic acid (KIs of 0.5-0.9 mM). Thiocyanate, hydrogensulfide, bisulphite, stannate, divanadate, tetraborate, perrhenate, perruthenate, hexafluorophosphate, triflate and iminodisulfonate showed KIs of 1.0-13.7 mM. Cyanate, cyanide, azide, carbonate, nitrate, tellurate, selenocyanide, tetrafluoroborate, sulfamide, sulphamic acid and phenylarsonic acid were weaker inhibitors, with KIs in the range of 25.2-95.5 mM, whereas halides, bicarbonate, nitrite, sulphate, perchlorate and fluorosulfonate did not show inhibitory action up until 100 mM concentrations in the assay system. Finding more effective MscCAγ inhibitors may be helpful to fight drug resistance to antibiotics.

Cloning, purification, kinetic and anion inhibition studies of a recombinant ß-carbonic anhydrase from the Atlantic salmon parasite platyhelminth Gyrodactylus salaris.

A ß-class carbonic anhydrase (CA, EC 4.2.1.1) was cloned from the genome of the Monogenean platyhelminth Gyrodactylus salaris, a parasite of Atlantic salmon. The new enzyme, GsaCAß has a significant catalytic activity for the physiological reaction, CO2 + H2O ⇋ HCO3- + H+ with a kcat of 1.1 × 105 s-1 and a kcat/Km of 7.58 × 106 M-1 × s-1. This activity was inhibited by acetazolamide (KI of 0.46 µM), a sulphonamide in clinical use, as well as by selected inorganic anions and small molecules. Most tested anions inhibited GsaCAß at millimolar concentrations, but sulfamide (KI of 81 µM), N,N-diethyldithiocarbamate (KI of 67 µM) and sulphamic acid (KI of 6.2 µM) showed a rather efficient inhibitory action. There are currently very few non-toxic agents effective in combating this parasite. GsaCAß is subsequently proposed as a new drug target for which effective inhibitors can be designed.

Compensation mechanism for membrane potential against hypoosmotic stress in the Onchidium neuron.

The effect of acute hypoosmotic stress on the neural response was investigated using the neurons identified in the abdominal ganglion of the amphibious mollusk Onchidium. The membrane potential of an identified neuron (Ip-1/2) was not significantly altered in 50% hypoosmotic artificial sea water. In isotonic 50% artificial seawater (ASW) with osmolarity that was compensated for using glycerol or urea, the membrane potentials of Ip-1/2 were also not altered compared to those in 50% hypoosmotic ASW. However, hyperpolarization was induced in isotonic 50% ASW when osmolarity was compensated for using sucrose or mannose. In the presence of volume-regulated anion channel (VRAC) inhibitors (niflumic acid and glibenclamide), the Ip-1/2 membrane potentials were hyperpolarized in 50% hypoosmotic ASW. These results suggest that there is a compensatory mechanism involving aquaglyceroporin and VRAC-like channels that maintains membrane potential under hypoosmotic conditions. Here, we detected the expression of aquaglyceroporin mRNA in neural tissues of Onchidium.

Cationic N,N-Dimethylglycine Ester Prodrug of 2R-α-Tocotrienol Promotes Intestinal Absorption via Efficient Self-Micellization with Intrinsic Bile Acid Anion.

The intestinal absorption of hydrophobic compounds is severely influenced by their transportation rate through the unstirred water layer in the intestinal lumen. A member of the vitamin E family, α-Tocotrienol (α-T3) has remarkable pharmacological effects, but its intestinal absorption is hampered due to its hydrophobicity. Here, we prepared three ester derivatives of 2R-α-T3, and we selected a suitable prodrug compound using rat plasma and liver microsomes. The micellization profile of the selected compound in the presence of taurocholic acid (TCA) was evaluated. After gastrostomy administration of the prodrug candidate or α-T3 solution containing TCA, AUC values were determined for α-T3 in plasma obtained from bile duct-ligated rats. Among the three types in the efficiency of the reconversion to the parent drug, α-T3 N,N-dimethylglycinate (α-T3DMG) was the best prodrug; α-T3DMG formed mixed micelles via ion pairs with anionic TCA. The solubility of α-T3DMG in n-octanol/water depended on its ratio to TCA. The AUC after α-T3DMG administration to ligated rats was 2-fold higher than that after α-T3 administration, suggesting a smooth interaction with intrinsic bile acids. In conclusion, utilization of the prodrug synthesized using N,N-dimethylglycine ester may be a beneficial approach to promote intestinal absorption of α-T3 via self-micellization with intrinsic bile acid.

Effects of air discharge on surface charges and cell walls of Fusarium oxysporum.

Air discharge showed significant inhibition on mycelial growth and spore germination of Fusarium oxysporum, one of the main spoilage fungi in post-harvest lotus roots which is an important economic aquatic vegetable in China. However, the antimicrobial mechanism of air discharge is not clear yet. In the present study, the effects of air discharge on F. oxysporum separated from post-harvest rotten lotus roots were characterized by analyzing surface charges, cell wall permeability, and changes in chitin and chitosan including surface morphology, functional groups, degree of deacetylation, crystallinity, and C/N ratio. After air discharge treatments, alkaline phosphatase leak assay revealed that cell wall permeability of F. oxysporum was magnified. What's more, zeta potentials of F. oxysporum increased and negative charges on cell surfaces decreased. The ordered and compact molecular arrangements of chitin and chitosan in cell walls of F. oxysporum were reduced. The deacetylation degree of chitin and chitosan increased, and the C/N ratios of chitin and chitosan decreased. It was concluded from these results that air discharge caused the transformation in structures of chitin and chitosan, resulting in the exposure of positively charged amino groups and decrease of negative charges on cell surfaces which brought damage to the structure and function of F. oxysporum's cell walls.

Crystal structures of a double-barrelled fluoride ion channel.

To contend with hazards posed by environmental fluoride, microorganisms export this anion through F(-)-specific ion channels of the Fluc family. Since the recent discovery of Fluc channels, numerous idiosyncratic features of these proteins have been unearthed, including strong selectivity for F(-) over Cl(-) and dual-topology dimeric assembly. To understand the chemical basis for F(-) permeation and how the antiparallel subunits convene to form a F(-)-selective pore, here we solve the crystal structures of two bacterial Fluc homologues in complex with three different monobody inhibitors, with and without F(-) present, to a maximum resolution of 2.1 Å. The structures reveal a surprising 'double-barrelled' channel architecture in which two F(-) ion pathways span the membrane, and the dual-topology arrangement includes a centrally coordinated cation, most likely Na(+). F(-) selectivity is proposed to arise from the very narrow pores and an unusual anion coordination that exploits the quadrupolar edges of conserved phenylalanine rings.

Reconsidering anion inhibitors in the general context of drug design studies of modulators of activity of the classical enzyme carbonic anhydrase.

Inorganic anions inhibit the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) generally by coordinating to the active site metal ion. Cyanate was reported as a non-coordinating CA inhibitor but those erroneous results were subsequently corrected by another group. We review the anion CA inhibitors (CAIs) in the more general context of drug design studies and the discovery of a large number of inhibitor classes and inhibition mechanisms, including zinc binders (sulphonamides and isosteres, dithiocabamates and isosteres, thiols, selenols, benzoxaboroles, ninhydrins, etc.); inhibitors anchoring to the zinc-coordinated water molecule (phenols, polyamines, sulfocoumarins, thioxocoumarins, catechols); CAIs occluding the entrance to the active site (coumarins and derivatives, lacosamide), as well as compounds that bind outside the active site. All these new chemotypes integrated with a general procedure for obtaining isoform-selective compounds (the tail approach) has resulted, through the guidance of rigorous X-ray crystallography experiments, in the development of highly selective CAIs for all human CA isoforms with many pharmacological applications.

Anion inhibition studies of the Zn(II)-bound ι-carbonic anhydrase from the Gram-negative bacterium Burkholderia territorii.

Burkholderia territorii, a Gram-negative bacterium, encodes for the ι-class carbonic anhydrase (CA, EC 4.2.1.1) BteCAι, which was recently characterised. It acts as a good catalyst for the hydration of CO2 to bicarbonate and protons, with a kcat value of 3.0 × 105 s-1 and kcat/KM value of 3.9 × 107 M-1 s-1. No inhibition data on this new class of enzymes are available to date. We report here an anion and small molecules inhibition study of BteCAι, which we prove to be a zinc(II)- and not manganese(II)-containing enzyme, as reported for diatom ι-CAs. The best inhibitors were sulphamic acid, stannate, phenylarsonic acid, phenylboronic acid and sulfamide (KI values of 6.2-94 µM), whereas diethyldithiocarbamate, tellurate, selenate, bicarbonate and cyanate were submillimolar inhibitors (KI values of 0.71-0.94 mM). The halides (except iodide), thiocyanate, nitrite, nitrate, carbonate, bisulphite, sulphate, hydrogensulfide, peroxydisulfate, selenocyanate, fluorosulfonate and trithiocarbonate showed KI values in the range of 3.1-9.3 mM.

An anion and small molecule inhibition study of the ß-carbonic anhydrase from Staphylococcus aureus.

Pathogenic bacteria resistant to most antibiotics, including the methicillin-resistant Staphylococcus aureus (MRSA) represent a serious medical problem. The search for new antiinfectives, possessing a diverse mechanism of action compared to the clinically used antibiotics, has become an attractive research field. S. aureus DNA encodes a ß-class carbonic anhydrase, SauBCA. It is a druggable target that can be inhibited by certain aromatic and heterocyclic sulphonamides. Here we investigated inorganic anions and some other small molecules for their inhibition of SauBCA. The halides, nitrite, nitrate, bicarbonate, carbonate, bisulphite, sulphate, stannate, and N,N-diethyldithiocarbamate were submillimolar SauBCA inhibitors with KIs in the range of 0.26 - 0.91 mM. The most effective inhibitors were sulfamide, sulfamate, phenylboronic acid, and phenylarsonic acid with KIs of 7 - 43 µM. Several interesting inhibitors detected here may be considered lead compounds for the development of even more effective derivatives, which should be investigated for their bacteriostatic effects.

Anion inhibition studies of the α-carbonic anhydrases from Neisseria gonorrhoeae.

The bacterial pathogen Neisseria gonorrhoeae encodes for an α-class carbonic anhydrase (CA, EC 4.2.1.1), NgCA, which was investigated for its inhibition with a series of inorganic and organic anions. Perchlorate and hexafluorophosphate did not significantly inhibit NgCA CO2 hydrase activity, whereas the halides, azide, bicarbonate, carbonate, stannate, perosmate, diphosphate, divanadate, perruthenate, and trifluoromethanesulfonate showed inhibition constants in the range of 1.3-9.6 mM. Anions/small molecules such as cyanate, thiocyanate, nitrite, nitrate, bisulphite, sulphate, hydrogensulfide, phenylboronic acid, phenylarsonic acid, selenate, tellurate, tetraborate, perrhenate, peroxydisulfate, selenocyanate, iminodisulfonate, and fluorosulfonate showed KIs in the range of 0.15-1.0 mM. The most effective inhibitors detected in this study were sulfamide, sulfamate, trithiocarbonate and N,N-diethyldithiocarbamate, which had KIs in the range of 5.1-88 µM. These last compounds incorporating the CS2- zinc-binding group may be used as leads for developing even more effective NgCA inhibitors in addition to the aromatic/heterocyclic sulphonamides, as this enzyme was recently validated as an antibacterial drug target for obtaining novel antigonococcal agents.

The Role of the Anion in Salt (NaCl) Detection by Mouse Taste Buds.

How taste buds detect NaCl remains poorly understood. Among other problems, applying taste-relevant concentrations of NaCl (50-500 mm) onto isolated taste buds or cells exposes them to unphysiological (hypo/hypertonic) conditions. To overcome these limitations, we used the anterior tongue of male and female mice to implement a slice preparation in which fungiform taste buds are in a relatively intact tissue environment and stimuli are limited to the taste pore. Taste-evoked responses were monitored using confocal Ca2+ imaging via GCaMP3 expressed in Type 2 and Type 3 taste bud cells. NaCl evoked intracellular mobilization of Ca2+ in the apical tips of a subset of taste cells. The concentration dependence and rapid adaptation of NaCl-evoked cellular responses closely resembled behavioral and afferent nerve responses to NaCl. Importantly, taste cell responses were not inhibited by the diuretic, amiloride. Post hoc immunostaining revealed that >80% of NaCl-responsive taste bud cells were of Type 2. Many NaCl-responsive cells were also sensitive to stimuli that activate Type 2 cells but never to stimuli for Type 3 cells. Ion substitutions revealed that amiloride-insensitive NaCl responses depended on Cl- rather than Na+ Moreover, choline chloride, an established salt taste enhancer, was equally effective a stimulus as sodium chloride. Although the apical transducer for Cl- remains unknown, blocking known chloride channels and cotransporters had little effect on NaCl responses. Together, our data suggest that chloride, an essential nutrient, is a key determinant of taste transduction for amiloride-insensitive salt taste.SIGNIFICANCE STATEMENT Sodium and chloride are essential nutrients and must be regularly consumed to replace excreted NaCl. Thus, understanding salt taste, which informs salt appetite, is important from a fundamental sensory perspective and forms the basis for interventions to replace/reduce excess Na+ consumption. This study examines responses to NaCl in a semi-intact preparation of mouse taste buds. We identify taste cells that respond to NaCl in the presence of amiloride, which is significant because much of human salt taste also is amiloride-insensitive. Further, we demonstrate that Cl-, not Na+, generates these amiloride-insensitive salt taste responses. Intriguingly, choline chloride, a commercial salt taste enhancer, is also a highly effective stimulus for these cells.

Electron Attachment Studies with the Potential Radiosensitizer 2-Nitrofuran.

Nitrofurans belong to the class of drugs typically used as antibiotics or antimicrobials. The defining structural component is a furan ring with a nitro group attached. In the present investigation, electron attachment to 2-nitrofuran (C4H3NO3), which is considered as a potential radiosensitizer candidate for application in radiotherapy, has been studied in a crossed electron-molecular beams experiment. The present results indicate that low-energy electrons with kinetic energies of about 0-12 eV effectively decompose the molecule. In total, twelve fragment anions were detected within the detection limit of the apparatus, as well as the parent anion of 2-nitrofuran. One major resonance region of ≈0-5 eV is observed in which the most abundant anions NO2-, C4H3O-, and C4H3NO3- are detected. The experimental results are supported by ab initio calculations of electronic states in the resulting anion, thermochemical thresholds, connectivity between electronic states of the anion, and reactivity analysis in the hot ground state.

Negatively Charged Carbon Nanodots with Bacteria Resistance Ability for High-Performance Antibiofilm Formation and Anticorrosion Coating Design.

Multifunctional coatings, especially those with simultaneous antibiofilm formation and anticorrosion properties are of great significance for the marine industry. Inspired by the function of fish mucus of blackhead fish, a biological epidermal secretion with negative surface potential that protects blackhead fish from colonization of microorganisms, a concept is introduced to use negatively charged carbon nanodots (CDs) as a secure and economical dual-functional additive to prepare protective coatings. The prepared CDs with strong negative surface potential initiate robust antibiofilm formation (antiadhesion and antibacteria) and anticorrosion properties (about 60 days' durability in seawater) of polymeric coatings. The incorporated CDs with negative surface potential take effect in the following ways: 1) suppressing bacterial adhesion by virtue of strong electrostatic repulsion; 2) sterilizing anchored bacteria via destroying bacterial cell walls; 3) impeding electron ejection from the metallic surface; and 4) blocking aggressive species (H2 O and O2 ) by narrowing the microchannels. This work provides a new train of thought propelling the development of potential materials for industrial and engineering applications.

Anionic Carbosilane Dendrimers Destabilize the GP120-CD4 Complex Blocking HIV-1 Entry and Cell to Cell Fusion.

Cell-to-cell transmission is the most effective pathway for the spread of human immunodeficiency virus (HIV-1). Infected cells expose virus-encoded fusion proteins on their surface as a consequence of HIV-1 replicative cycle that interacts with noninfected cells through CD4 receptor and CXCR4 coreceptor leading to the formation of giant multinucleated cells known as syncytia. Our group previously described the potent activity of dendrimers against CCR5-tropic viruses. Nevertheless, the study of G1-S4, G2-S16, and G3-S16 dendrimers in the context of X4-HIV-1 tropic cell-cell fusion referred to syncytium formation remains still unknown. These dendrimers showed a suitable biocompatibility in all cell lines studied and our results demonstrated that anionic carbosilane dendrimers G1-S4, G2-S16, and G3-S16 significantly inhibit the X4-HIV-1 infection, as well as syncytia formation, in a dose dependent manner. We also demonstrated that G2-S16 and G1-S4 significantly reduced syncytia formation in HIV-1 Env-mediated cell-to-cell fusion model. Molecular modeling and in silico models showed that G2-S16 dendrimer interfered with gp120-CD4 complex and demonstrated its potential use for a treatment.

Synthetic Access to the Mandelalide Family of Macrolides: Development of an Anion Relay Chemistry Strategy.

The mandelalides comprise a family of structurally complex marine macrolides that display significant cytotoxicity against several human cancer cell lines. Presented here is a full account on the development of an Anion Relay Chemistry (ARC) strategy for the total synthesis of (-)-mandelalides A and L, the two most potent members of the mandelalide family. The design and implementation of a three-component type II ARC/cross-coupling protocol and a four-component type I ARC union permits rapid access respectively to the key tetrahydrofuran and tetrahydropyran structural motifs of these natural products. Other highlights of the synthesis include an osmium-catalyzed oxidative cyclization of an allylic 1,3-diol, a mild Yamaguchi esterification to unite the northern and southern hemispheres, and a late-stage Heck macrocyclization. Synthetic mandelalides A and L displayed potent cytotoxicity against human HeLa cervical cancer cells (EC50, 1.3 and 3.1 nM, respectively). This synthetic approach also provides access to several highly potent non-natural mandelalide analogs, including a biotin-tagged mandelalide probe for future biological investigation.

Anion inhibition studies of a beta carbonic anhydrase from the malaria mosquito Anopheles gambiae.

An anion inhibition study of the ß-class carbonic anhydrase, AgaCA, from the malaria mosquito Anopheles gambiae is reported. A series of simple as well as complex inorganic anions, and small molecules known to interact with CAs were included in the study. Bromide, iodide, bisulphite, perchlorate, perrhenate, perruthenate, and peroxydisulphate were ineffective AgaCA inhibitors, with KIs > 200 mM. Fluoride, chloride, cyanate, thiocyanate, cyanide, bicarbonate, carbonate, nitrite, nitrate, sulphate, stannate, selenate, tellurate, diphosphate, divanadate, tetraborate, selenocyanide, and trithiocarbonate showed KIs in the range of 1.80-9.46 mM, whereas N,N-diethyldithiocarbamate was a submillimolar AgaCA inhibitor (KI of 0.65 mM). The most effective AgaCA inhibitors were sulphamide, sulphamic acid, phenylboronic acid and phenylarsonic acid, with inhibition constants in the range of 21-84 µM. The control of insect vectors responsible of the transmission of many protozoan diseases is rather difficult nowadays, and finding agents which can interfere with these processes, as the enzyme inhibitors investigated here, may arrest the spread of these diseases worldwide.

Abnormal Anionic Porphyrin Sensing Effect for HER2 Gene Related DNA Detection via Impedance Difference between MWCNTs and Single-Stranded DNA or Double-Stranded DNA.

Human epidermal growth factor receptor 2 (HER2) is a key tumor marker for several common and deadly cancers. It is of great importance to develop efficient detection methods for its over-expression. In this work, an electrochemical impedance spectroscopy (EIS) method adjustable by anionic porphyrin for HER2 gene detection has been proposed, based on the impedance difference between multi-walled carbon nanotubes (MWCNTs) and DNA. The interesting finding herein is that with the addition of anionic porphyrin, i.e., meso-tetra(4-sulfophenyl)-porphyrin (TSPP), the impedance value obtained at a glass carbon electrode (GCE) modified with MWCNTs and a single stranded DNA (ssDNA), the probe DNA that might be assembled tightly onto MWCNTs through π-π stacking interaction, gets a slight decrease; however, the impedance value from a GCE modified with MWCNTs and a double stranded DNA (dsDNA), the hybrid of the probe DNA with a target DNA, which might be assembled loosely onto MWCNTs for the screening effect of phosphate backbones in dsDNA, gets an obvious decrease. The reason may be that on the one hand, being rich in negative sulfonate groups, TSPP will try to push DNA far away from CNTs surface due to its strong electrostatic repulsion towards DNA; on the other hand, rich in planar phenyl or pyrrole rings, TSPP will compete with DNA for the surface of CNTs since it can also be assembled onto CNTs through conjugative interactions. In this way, the "loosely assembled" dsDNA will be repelled by this anionic porphyrin and released off CNTs surface much more than the "tightly assembled" ssDNA, leading to a bigger difference in the impedance value between dsDNA and ssDNA. Thus, through the amplification effect of TSPP on the impedance difference, the perfectly matched target DNA could be easily determined by EIS without any label. Under the optimized experimental conditions, this electrochemical sensor shows an excellent linear response to target DNA in a concentration range of 2.0 × 10-11⁻2.0 × 10-6 M with a limit of detection (LOD) of 6.34 × 10-11 M (S/N = 3). This abnormally sensitive electrochemical sensing performance resulting from anionic porphyrin for DNA sequences specific to HER2 gene will offer considerable promise for tumor diagnosis and treatment.