Synthesis and evaluation of novel radioiodinated PSMA targeting ligands for potential radiotherapy of prostate cancer

Radioligand therapy (RLT) using prostate-specific membrane antigen (PSMA) targeting ligands is an attractive option for the treatment of Prostate cancer (PCa) and its metastases. We report herein a series of radioiodinated glutamate-urea-lysine-phenylalanine derivatives as new PSMA ligands in which l-tyrosine and l-glutamic acid moieties were added to increase hydrophilicity concomitant with improvement of in vivo targeting properties. Compounds 8, 15, 19a/19b and 23a/23b were synthesized and radiolabeled with ¹²⁵I by iododestannylation. All iodinated compounds displayed high binding affinities toward PSMA (IC₅₀ = 1–13 nM). In vitro cell uptake studies demonstrated that compounds containing an l-tyrosine linker moiety (8, 15 and 19a/19b) showed higher internalization than MIP-1095 and 23a/23b, both without the l-tyrosine linker moiety. Biodistribution studies in mice bearing PC3-PIP and PC3 xenografts showed that [¹²⁵I]8 and [¹²⁵I]15 with higher lipophilicity exhibited higher nonspecific accumulations in the liver and intestinal tract, whereas [¹²⁵I]19a/19b and [¹²⁵I]23a/23b containing additional glutamic acid moieties showed higher accumulations in the kidney and implanted PC3-PIP (PSMA+) tumors. [¹²⁵I]23b displayed a promising biodistribution profile with favorable tumor retention, fast clearance from the kidney, and 2–3-fold lower uptake in the liver and blood than that observed for [¹²⁵I]MIP-1095. [^125/131I]23b may serve as an optimal PSMA ligand for radiotherapy treatment of prostate cancer over-expressing PSMA.     

In vitro cytotoxic potential of extracts from Aristolochia foetida Kunth against MCF-7 and bMECs cell lines

The aim of this study was to evaluate the cytotoxic potential of Aristolochia foetida Kunth. Stems and leaves of A. foetida Kunth (Aristolochiaceae) have never been investigated pharmacologically. Recent studies of species of the Aristolochiaceae family found significant cytotoxic activities. Hexane, dichloromethane, ethyl acetate and methanol extracts were analyzed by ¹H NMR and GC–MS to know the metabolites in each extract. In GC–MS analysis, the main compounds were methyl hexadecanoate (3); hexadecanoic acid (4); 2-butoxyethyl dodecanoate (9); ethyl hexadecanoate (20); methyl octadeca-9,12,15-trienoate (28) and (9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid (40). The results showed a significant reduction in cell viability of the MCF-7 (breast cancer) cell line caused by organic extracts in a dose-dependent manner. The cytotoxicity activity of the dichloromethane extract from the stems (DSE) showed IC₅₀ values of 45.9 μg/mL and the dichloromethane extract of the leaves (DLE) showed IC₅₀ values of 47.3 μg/mL. DSE and DLE had the highest cytotoxic potential in an in vitro study against the MCF-7 cell line and non-tumor cells obtained from the bovine mammary epithelial (bMECs). DSE and DLE induced a loss in mitochondrial membrane potential (ΔΨm) and can cause cell death by apoptosis through the intrinsic pathway in the MCF-7 cell line. DSE and DLE are cytotoxic in cancer cells and cause late apoptosis. Higher concentrations of DSE and DLE are required to induce a cytotoxic effect in healthy mammary epithelial cells. This is the first report of the dichloromethane extract of A. foetida Kunth that induces late apoptosis in MCF-7 cancer cells and may be a candidate for pharmacological study against breast cancer.     

Rational design,synthesis and biological evaluation of novel multitargeting anti-AD iron chelators with potent MAO-B inhibitory and antioxidant activity

A series of (3-hydroxypyridin-4-one)-coumarin hybrids were developed and investigated as potential multitargeting candidates for the treatment of Alzheimer's disease (AD) through the incorporation of iron-chelating and monoamine oxidase B (MAO-B) inhibition. This combination endowed the hybrids with good capacity to inhibit MAO-B as well as excellent iron-chelating effects. The pFe³⁺ values of the compounds were ranging from 16.91 to 20.16, comparable to more potent than the reference drug deferiprone (DFP). Among them, compound 18d exhibited the most promising activity against MAO-B, with an IC₅₀ value of 87.9 nM. Moreover, compound 18d exerted favorable antioxidant activity, significantly reversed the amyloid-β_1-42 (Aβ_1-42) induced PC12 cell damage. More importantly, 18d remarkably ameliorated the cognitive dysfunction in a scopolamine-induced mice AD model. In brief, a series of hybrids with potential anti-AD effect were successfully obtained, indicating that the design of iron chelators with MAO-B inhibitory and antioxidant activities is an attractive strategy against AD progression.     

Analogs of nitrofuran antibiotics are potent GroEL/ES inhibitor pro-drugs

In two previous studies, we identified compound 1 as a moderate GroEL/ES inhibitor with weak to moderate antibacterial activity against Gram-positive and Gram-negative bacteria including Bacillus subtilis, methicillin-resistant Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, and SM101 Escherichia coli (which has a compromised lipopolysaccharide biosynthetic pathway making bacteria more permeable to drugs). Extending from those studies, we developed two series of analogs with key substructures resembling those of known antibacterials, nitroxoline (hydroxyquinoline moiety) and nifuroxazide/nitrofurantoin (bis-cyclic-N-acylhydrazone scaffolds). Through biochemical and cell-based assays, we identified potent GroEL/ES inhibitors that selectively blocked E. faecium, S. aureus, and E. coli proliferation with low cytotoxicity to human colon and intestine cells in vitro. Initially, only the hydroxyquinoline-bearing analogs were found to be potent inhibitors in our GroEL/ES-mediated substrate refolding assays; however, subsequent testing in the presence of an E. coli nitroreductase (NfsB) in situ indicated that metabolites of the nitrofuran-bearing analogs were potent GroEL/ES inhibitor pro-drugs. Consequently, this study has identified a new target of nitrofuran-containing drugs, and is the first reported instance of such a unique class of GroEL/ES chaperonin inhibitors. The intriguing results presented herein provide impetus for expanded studies to validate inhibitor mechanisms and optimize this antibacterial class using the respective GroEL/ES chaperonin systems and nitroreductases from E. coli and the ESKAPE bacteria.     

Optimisation of estrogen receptor subtype-selectivity of a 4-Aryl-4H-chromene scaffold previously identified by virtual screening

4-Aryl-4H-Chromene derivatives have been previously shown to exhibit anti-proliferative, apoptotic and anti-angiogenic activity in a variety of tumor models in vitro and in vivo generally via activation of caspases through inhibition of tubulin polymerisation. We have previously identified by Virtual Screening (VS) a 4-aryl-4H-chromene scaffold, of which two examples were shown to bind Estrogen Receptor α and β with low nanomolar affinity and <20-fold selectivity for α over β and low micromolar anti-proliferative activity in the MCF-7 cell line. Thus, using the 4-aryl-4H-chromene scaffold as a starting point, a series of compounds with a range of basic arylethers at C-4 and modifications at the C3-ester substituent of the benzopyran ring were synthesised, producing some potent ER antagonists in the MCF-7 cell line which were highly selective for ERα (compound 35; 350-fold selectivity) or ERβ (compound 42; 170-fold selectivity).     

Rhodopirellula aestuarii sp. nov., a novel member of the genus Rhodopirellula isolated from brackish sediments collected in the Tagus River estuary,Portugal

Bacteria within the phylum Planctomycetota are biologically relevant due to unique characteristics among prokaryotes. Members of the genus Rhodopirellula can be abundant in marine habitats, however, only six species are currently validly described. In this study, we expand the explored genus diversity by formally describing a novel species. The pink-coloured strain ICT_H3.1^T was isolated from brackish sediments collected in the Tagus estuary (Portugal) and a 16S rRNA gene sequence-based analysis placed this strain into the genus Rhodopirellula (family Pirellulaceae). The closest type strain is Rhodopirellula rubra LF2^T, suggested by a similarity of 98.4% of the 16S rRNA gene sequence. Strain ICT_H3.1^T is heterotrophic, aerobic and able to grow under microaerobic conditions. The strain grows between 15 and 37 °C, over a range of pH 6.5 to 11.0 and from 1 to 8% (w/v) NaCl. Several nitrogen and carbon sources were utilized by the novel isolate. Cells have an elongated pear-shape with 2.0 ± 0.3 × 0.9 ± 0.2 µm in size. Cells of strain ICT_H3.1^T cluster in rosettes through a holdfast structure and divide by budding. Younger cells are motile. Ultrathin cell sections show cytoplasmic membrane invaginations and polar fimbriae. The genome size is 9,072,081 base pairs with a DNA G + C content of 56.1 mol%. Genomic, physiological and morphological comparison of strain ICT_H3.1^T with its relatives suggest that it belongs to a novel species within the genus Rhodopirellula. Hence, we propose the name Rhodopirellula aestuarii sp. nov., represented by ICT_H3.1^T (=CECT30431^T = LMG32464^T) as the type strain of this novel species.16S rRNA gene accession number: GenBank = OK001858.Genome accession number: The Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession JAMQBK000000000. The version described in this paper is version JAMQBK010000000.     

Anti-aging and antioxidant of four traditional malaysian plants using simplex centroid mixture design approach

Aging is a naturally biological process with adverse effects. The continuous accumulation of reactive oxygen species (ROS) trigger cellular and tissue damage by activating several aging enzymes. The antioxidant properties of traditional medicinal plants used by Jakun aborigine’s community are a promising approach to alleviate aging process and prevent Alzheimer. The aim of the current investigation was to optimize a novel anti-aging formulation from traditional plants (Cnestis palala stem, Urceola micrantha stem, Marantodes pumilum stem and Microporus xanthopus fruiting bodies) using simplex centroid mixture design (SCMD). After selecting the optimal formulations based on desirability function of antioxidant activity (DPPḢ, ABTS^̇+ and FRAP), they were further examined against the activity of aging-related-enzymes (collagenase, tyrosinase, acetyl- and butyrylcholinesterase). The single extracts of C. palala, U. micrantha and the binary mixture of C. palala and U. micrantha were the optimal formulations with high antioxidant activities. Single extract of U. micrantha showed the highest inhibition towards matrix metalloproteinase-1 (49.44 ± 4.11 %), while C. palala water extract showed highest inhibitions towards tyrosinase (14.06 ± 0.31%), acetylcholinesterase (32.92 ± 2.13%) and butyrylcholinesterase (34.89 ± 2.84%) enzymes. The single extracts of C. palala and U. micrantha displayed better activity as compared to the binary mixture formulation. In conclusion, these findings could be a baseline for further exploration of novel anti-aging agents from natural resources.     

Discovery of ORN0829, a potent dual orexin 1/2 receptor antagonist for the treatment of insomnia

Here, we present the design, synthesis, and SAR of dual orexin 1 and 2 receptor antagonists, which were optimized by balancing the antagonistic activity for orexin receptors and lipophilicity. Based on the prototype compound 1, ring construction and the insertion of an additional heteroatom into the resulting ring led to the discovery of orexin 1 and 2 receptor antagonists, which were 3-benzoyl-1,3-oxazinane derivatives. Within these derivatives, (−)-3h enabled a high dual orexin receptor antagonistic activity and a low lipophilicity. Compound (−)-3h exhibited potent sleep-promoting effects at a po dose of 1 mg/kg in a rat polysomnogram study, and optimal PK properties with a rapid T_max and short half-lives in rats and dogs were observed, indicating a predicted human half-life of 0.9–2.0 h. Thus, (−)-3h (ORN0829; investigation code name, TS-142) was selected as a viable candidate and is currently in clinical development for the treatment of insomnia.     

Immunomodulation-mediated anticancer activity of a novel compound from Brugmansia suaveolens leaves

Immunomodulation activity-guided fractionation of ethanol extract of Brugmansia suaveolens leaves was carried out to isolate a novel compound SUPH036-022A (1) by co-culturing the test fraction/compound activated PBMC with MCF7 and A549 cancer cell lines. Assessment of immune markers in PBMC, and analysis of apoptosis markers and cell cycle was carried out for cancer cells. The structure of the isolated compound was elucidated by spectral analysis. Compound 1 enhanced the secretion of immune markers, IL-2 and IFN-γ, from PBMC. Further, compound 1 treated PBMC increased cell death in MCF7 and A549 cell lines and induced ROS production and mitochondrial membrane perturbation, leading to apoptosis. Flow cytometry analysis revealed; compound 1 stimulated PBMC to cause a five-fold increase in cell cycle perturbations in the sub-G1 stage of cancer cells as compared to the negative control. The compound, in the absence of PBMC, only had a weak cytotoxic activity against these cell lines. Thus, compound 1 is a novel lead for immunomodulation-mediated anticancer activity.     

Development of an efficient semisynthetic modification of FR901459 via a novel regioselective N,O-acyl migration

HCV utilizes cellular protein cyclophilins in the virus replication cycle and cyclophilin inhibitors have become a new class of anti-HCV agents. In our screening of natural products, we identified a unique cyclosporin analogue, FR901459, as a cyclophilin inhibitor with potent anti-HCV activity. In this work, we developed an efficient synthetic methodology to prepare FR901459 derivatives via an N, O-acyl migration reaction. This method allows us to efficiently manipulate the amino acid residues at the 3 position while avoiding lengthy total synthesis for each compound. By using this methodology, we discovered 4, which has superior anti-HCV activity and decreased immunosuppressive activity compared to FR901459.     

Isolation of diverse bioactive compounds from Euphorbia balsamifera: Cytotoxicity and antibacterial activity studies

Antibacterial and cytotoxic activities of Euphorbia balsamifera, fractions and pure compounds were evaluated. The cytotoxic assays for HCT116, HePG2 and MCF7 showed a significant IC₅₀: 54.7 and 76.2 µg/mL of non-polar fraction “n-hexane” against HCT116 and HePG2, respectively. Antibacterial results revealed that plant fractions exhibited significant potential against the tested pathogens than the total extract where n-butanol and ethyl acetate fractions showed significant antibacterial activity (P < 0.05) against tested bacterial strains. Isolation and structure determination of compounds from n-hexane and n-butanol fractions were performed. From n-hexane fraction, 29-nor-cycloartanol (1), lanost-8-en-3-ol (2a), cycloartanol (2b) and kampferol-3,4'-dimethyl ether (3) were isolated and structurally identified, along with 24 compounds were tentatively identified by GC–MS. From the polar n-butanol fraction, 4-O-β-D-glucopyranosyl-2-hydroxy-6-methoxyacetophenone (4), 4-O-α-L-rhamnosyl-(1 → 6)-β-D-glucopyranosyl-2-hydroxy-6methoxy-acetophenone (5), quercetin-3-O-glucopyranoside (6) and isoorientin (7) were assigned. Structures of the obtained compounds were determined by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. Except compounds 1 and 5, all reported compounds announced antibacterial efficiency. Compound 2 showed selectively the highest activity against Enterococcus faecalis (22 ± 0.13 mm), meanwhile 4-O-β-D-glucopyranosyl-2-hydroxy-6-methoxyacetophenone (4) showed broadly the highest antibacterial activity with MIC of 1.15–1.88 mg/mL against the test Gram-positive and Gram-negative bacteria. Cytotoxic assays indicated that kampferol-3,4'-dimethyl ether (3) exhibited the highest activity with matching IC₅₀ values to doxorubicin; 111.46, 42.67 and 44.90 µM against HCT116, HePG2 and MCF7, respectively, however, it is toxic on retina normal cell line RPE1.     

Design,synthesis, and biological activity of new endomorphin analogs with multi-site modifications

Endomorphin (EM)-1 and EM-2 are the most effective endogenous analgesics with efficient separation of analgesia from the risk of adverse effects. Poor metabolic stability and ineffective analgesia after peripheral administration were detrimental for the use of EMs as novel clinical analgesics. Therefore, here, we aimed to establish new EM analogs via introducing different bifunctional d-amino acids at position 2 of [(2-furyl)Map⁴]EMs. The combination of [(2-furyl)Map⁴]EMs with D-Arg² or D-Cit² yielded analogs with enhanced binding affinity to the μ-opioid receptor (MOR) and increased stability against enzymatic degradation (t_1/2 > 300 min). However, the agonistic activities of these analogs toward MOR were slightly reduced. Similar to morphine, peripheral administration of the analog [D-Cit², (2-furyl)Map⁴]EM-1 (10) significantly inhibited the pain behavior of mice in multiple pain models. In addition, this EM-1 analog was associated with reduced tolerance, less effect on gastrointestinal mobility, and no significant motor impairment. Compared to natural EMs, the EM analogs synthesized herein had enhanced metabolic stability, bioavailability, and analgesic properties.     

Inhibition of Mycobacterium tuberculosis InhA: Design,synthesis and evaluation of new di-triclosan derivatives

Multi-drug resistant tuberculosis (MDR-TB) represents a growing problem for global healthcare systems. In addition to 1.3 million deaths in 2018, the World Health Organisation reported 484,000 new cases of MDR-TB. Isoniazid is a key anti-TB drug that inhibits InhA, a crucial enzyme in the cell wall biosynthesis pathway and identical in Mycobacterium tuberculosis and M. bovis. Isoniazid is a pro-drug which requires activation by the enzyme KatG, mutations in KatG prevent activation and confer INH-resistance. ‘Direct inhibitors’ of InhA are attractive as they would circumvent the main clinically observed resistance mechanisms. A library of new 1,5-triazoles, designed to mimic the structures of both triclosan molecules uniquely bound to InhA have been synthesised. The inhibitory activity of these compounds was evaluated using isolated enzyme assays with 2 (5-chloro-2-(4-(5-(((4-(4-chloro-2-hydroxyphenoxy)benzyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)phenoxy)phenol) exhibiting an IC₅₀ of 5.6 µM. Whole-cell evaluation was also performed, with 11 (5-chloro-2-(4-(5-(((4-(cyclopropylmethoxy)benzyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)phenoxy)phenol) showing the greatest potency, with an MIC₉₉ of 12.9 µM against M. bovis.     

Synthesis of novel 2-pyrrolidinone and pyrrolidine derivatives and study of their inhibitory activity against autotaxin enzyme

Autotaxin (ATX), a glycoprotein (~125 kDa) isolated as an autocrine motility factor from melanoma cells, belongs to a seven-membered family of ectonucleotide pyrophosphatase/phosphodiesterase (ENPP), and exhibits lysophospholipase D activity. ATX is responsible for the hydrolysis of lysophosphatidylcholine (LPC) to produce the bioactive lipid lysophosphatidic acid (LPA), which is upregulated in a variety of pathological inflammatory conditions, including fibrosis, cancer, liver toxicity and thrombosis. Given its role in human disease, the ATX-LPA axis is an interesting target for therapy, and the development of novel potent ATX inhibitors is of great importance. In the present work a novel class of ATX inhibitors, optically active derivatives of 2-pyrrolidinone and pyrrolidine heterocycles were synthesized. Some of them exhibited interesting in vitro activity, namely the hydroxamic acid 16 (IC₅₀ 700 nM) and the carboxylic acid 40b (IC₅₀ 800 nM), while the boronic acid derivatives 3k (IC₅₀ 50 nM), 3l (IC₅₀ 120 nM), 3 m (IC₅₀ 180 nM) and 21 (IC₅₀ 35 nM) were found to be potent inhibitors of ATX.     

Discovery of isoquinolinone and naphthyridinone-based inhibitors of poly(ADP-ribose) polymerase-1 (PARP1) as anticancer agents: Structure activity relationship and preclinical characterization

The exploitation of GLU988 and LYS903 residues in PARP1 as targets to design isoquinolinone (I & II) and naphthyridinone (III) analogues is described. Compounds of structure I have good biochemical and cellular potency but suffered from inferior PK. Constraining the linear propylene linker of structure I into a cyclopentene ring (II) offered improved PK parameters, while maintaining potency for PARP1. Finally, to avoid potential issues that may arise from the presence of an anilinic moiety, the nitrogen substituent on the isoquinolinone ring was incorporated as part of the bicyclic ring. This afforded a naphthyridinone scaffold, as shown in structure III. Further optimization of naphthyridinone series led to identification of a novel and highly potent PARP1 inhibitor 34, which was further characterized as preclinical candidate molecule. Compound 34 is orally bioavailable and displayed favorable pharmacokinetic (PK) properties. Compound 34 demonstrated remarkable antitumor efficacy both as a single-agent as well as in combination with chemotherapeutic agents in the BRCA1 mutant MDA-MB-436 breast cancer xenograft model. Additionally, compound 34 also potentiated the effect of agents such as temozolomide in breast cancer, pancreatic cancer and Ewing’s sarcoma models.     

Scaffold-hopping identifies furano[2,3-d]pyrimidine amides as potent Notum inhibitors

The carboxylesterase Notum is a key negative regulator of the Wnt signaling pathway by mediating the depalmitoleoylation of Wnt proteins. Our objective was to discover potent small molecule inhibitors of Notum suitable for exploring the regulation of Wnt signaling in the central nervous system. Scaffold-hopping from thienopyrimidine acids 1 and 2, supported by X-ray structure determination, identified 3-methylimidazolin-4-one amides 20–24 as potent inhibitors of Notum with activity across three orthogonal assay formats (biochemical, extra-cellular, occupancy). A preferred example 24 demonstrated good stability in mouse microsomes and plasma, and cell permeability in the MDCK-MDR1 assay albeit with modest P-gp mediated efflux. Pharmacokinetic studies with 24 were performed in vivo in mouse with single oral administration of 24 showing good plasma exposure and reasonable CNS penetration. We propose that 24 is a new chemical tool suitable for cellular studies to explore the fundamental biology of Notum.     

Pseudomonas petrae sp. nov. isolated from regolith samples in Antarctica

A polyphasic taxonomic approach was used to characterize the four strains P2653^T, P2652, P2498, and P2647, isolated from Antarctic regolith samples. Initial genotype screening performed by PCR fingerprinting based on repetitive sequences showed that the isolates studied formed a coherent cluster separated from the other Pseudomonas species. Identification results based on 16S rRNA gene sequences showed the highest sequence similarity with Pseudomonas graminis (99.7%), which was confirmed by multilocus sequence analysis using the rpoB, rpoD, and gyrB genes. Genome sequence comparison of P2653^T with the most related P. graminis type strain DSM 11363^T revealed an average nucleotide identity of 92.1% and a digital DNA-DNA hybridization value of 46.6%. The major fatty acids for all Antarctic strains were C_16:0, Summed Feature 3 (C_16:1 ω7c/C_16:1 ω6c) and Summed Feature 8 (C_18:1 ω7c/C_18:1 ω6c). The predominant respiratory quinone was Q-9, and the major polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, and phosphatidylglycerol. The regolith strains could be differentiated from related species by the absence of arginine dihydrolase, ornithine and lysine decarboxylase and by negative tyrosine hydrolysis. The results of this polyphasic study allowed the genotypic and phenotypic differentiation of four analysed strains from the closest related species, which confirmed that the strains represent a novel species within the genus Pseudomonas, for which the name Pseudomonas petrae sp. nov. is proposed with P2653^T (CCM 8850^T = DSM 112068^T = LMG 30619^T) as the type strain.     

Conformation-specific Synthetic Antibodies Discriminate Multiple Functional States of the Ion Channel CorA

CorA, the primary magnesium ion channel in prokaryotes and archaea, is a prototypical homopentameric ion channel that undergoes ion-dependent conformational transitions. CorA adopts five-fold symmetric non-conductive states in the presence of high concentrations of Mg²⁺, and highly asymmetric flexible states in its complete absence. However, the latter were of insufficient resolution to be thoroughly characterized. In order to gain additional insights into the relationship between asymmetry and channel activation, we exploited phage display selection strategies to generate conformation-specific synthetic antibodies (sABs) against CorA in the absence of Mg²⁺. Two sABs from these selections, C12 and C18, showed different degrees of Mg²⁺-sensitivity. Through structural, biochemical, and biophysical characterization, we found the sABs are both conformation-specific but probe different features of the channel under open-like conditions. C18 is highly specific to the Mg²⁺-depleted state of CorA and through negative-stain electron microscopy (ns-EM), we show sAB binding reflects the asymmetric arrangement of CorA protomers in Mg²⁺-depleted conditions. We used X-ray crystallography to determine a structure at 2.0 Å resolution of sAB C12 bound to the soluble N-terminal regulatory domain of CorA. The structure shows C12 is a competitive inhibitor of regulatory magnesium binding through its interaction with the divalent cation sensing site. We subsequently exploited this relationship to capture and visualize asymmetric CorA states in different [Mg²⁺] using ns-EM. We additionally utilized these sABs to provide insights into the energy landscape that governs the ion-dependent conformational transitions of CorA.