Zika virus NS2B-NS3 protease (ZIKVpro). The compound showed >50% inhibition of ZIKVpro activity at 20 µM. It was confirmed to directly bind to ZIKVpro at 50 µM in an Octet binding assay [1]

Merbromin inhibited the enzymatic activity of Zika virus NS2B-NS3 protease (ZIKVpro) in a dose-dependent manner in a fluorescence-based assay. It was identified as a hit from a primary screen of a natural compound library, showing >50% inhibition at 20 µM. A subsequent Octet binding assay using biotinylated ZIKVpro confirmed that Merbromin directly binds to the protease at a concentration of 50 µM. However, in a cell-based cytopathic effect (CPE) protection assay using ZIKV-infected Vero E6 cells, Merbromin did not show appreciable anti-ZIKV replication activity (e.g., less than 30% CPE protection at 10 µM) [1]

1) Fluorescence-based Protease Activity Assay: The Zika virus NS2B-NS3 protease (ZIKVpro) was expressed and purified. For screening and confirmation, the protease (150 nM) was incubated with compounds (e.g., at 20 µM for screening) in assay buffer for 1 hour at 37°C. The proteolytic reaction was then initiated by adding the fluorogenic substrate Bz-nKKR-AMC (50 µM). The fluorescence increase was monitored over time. The inhibition activity was calculated relative to a DMSO control [1]
2) Bio-Layer Interferometry (BLI) Binding Assay: Biotinylated ZIKVpro was immobilized on super streptavidin biosensors. The biosensors were then dipped into wells containing the test compound (e.g., 50 µM for initial binding check). Binding interactions were measured in real-time, and sensorgrams were analyzed to confirm direct binding [1]

Cytopathic Effect (CPE) Protection Assay: African green monkey kidney cells (Vero E6) were seeded and grown. The cells were treated with the compound and then infected with Zika virus (ZIKV) at a low multiplicity of infection (MOI). After incubation, cell viability was measured using a cell counting kit to assess the compound's ability to protect cells from virus-induced CPE. Ribavirin and DMSO were used as positive and negative controls, respectively [1]

Metabolism / Metabolites
Organic mercury is primarily absorbed through the gastrointestinal tract and then distributed throughout the body via the bloodstream. Organic mercury forms complexes with free cysteine and cysteine and sulfhydryl groups on proteins such as hemoglobin. These complexes mimic methionine, allowing them to be transported throughout the body, including across the blood-brain barrier and placenta. Organic mercury is metabolized into inorganic mercury, which is ultimately excreted in urine and feces. Bromine is primarily absorbed through inhalation, but can also enter the body through skin contact. Bromine salts can be ingested. Due to its reactivity, bromine rapidly forms bromides and may deposit in tissues, replacing other halogens. (L626, T11)

Toxicity Summary
The high affinity of divalent mercury ions for thiols or sulfhydryl groups in proteins is considered the primary mechanism by which mercury exerts its effects. Mercury promotes oxidative stress, lipid peroxidation, mitochondrial dysfunction, and altered heme metabolism by altering intracellular thiol states. Mercury is known to bind to microsomes and mitochondrial enzymes, leading to cell damage and death. For example, mercury is known to inhibit aquaporins, blocking the flow of water molecules across cell membranes. It also inhibits LCK proteins, leading to weakened T-cell signaling and immunosuppression. Mercury is also thought to inhibit neuronal excitability by acting on postsynaptic neuronal membranes. Furthermore, it affects the nervous system by inhibiting protein kinase C and alkaline phosphatase, thereby impairing the formation and function of brain microvessels and altering the blood-brain barrier. Organomercury influences development by binding to tubulin, preventing microtubule assembly and inhibiting mitosis. Mercury can also trigger autoimmune responses, possibly through modification of major histocompatibility complex (MHC) class II molecules, autopeptides, T-cell receptors, or cell surface adhesion molecules. Bromine is a strong oxidizing agent that can release oxygen free radicals from water in mucous membranes. These free radicals are also strong oxidizing agents and can cause tissue damage. Furthermore, the formation of hydrobromic acid and bromic acid can lead to secondary irritation. Bromine ions are also known to affect the central nervous system, leading to bromine poisoning. This is thought to be due to bromide ions replacing chloride ions in the action of neurotransmitters and transport systems, thereby affecting various synaptic processes. (L626, L627, A543, L7, A8, A25, A26)

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Toxicity Data>
LD50: 50 mg/kg (intravenous injection, mice) (L330)

[1]. Identification of Theaflavin-3,3'-Digallate as a Novel Zika Virus Protease Inhibitor. Front Pharmacol. 2020 Oct 21;11:514313.

Merbromin is an organosodium salt with the chemical formula 2,7-dibromo-4-hydroxyfluorescein, in which the carboxyl and phenolic hydroxyl groups are deprotonated, and the resulting charge is neutralized by two sodium ions. It is used as a disinfectant, fluorescent dye, and histological staining agent. It contains 2,7-dibromo-4-hydroxyfluorescein (2-). Merbromin is the trade name for Merbromin and is commonly used as a topical first-aid disinfectant. It is an organomercury disodium salt compound and fluorescein, sold in many countries except Switzerland, France, Germany, and the United States, where it has been withdrawn from the market due to potential mercury poisoning. Merbromin is an organomercury disodium salt compound and fluorescein. It is used as a topical disinfectant for minor cuts and abrasions. Merbromin is also used as a biological dye to mark tissue edges and as a metal dye in industrial penetrant testing to detect metal cracks. Due to its mercury content, it is unusable in some countries (such as the United States). Mercury is a heavy metal, silvery-white in color, belonging to the d-block element, and is one of the six elements that are liquid at or near room temperature and atmospheric pressure. It is a naturally occurring substance that can combine with other elements (such as chlorine, sulfur, or oxygen) to form inorganic mercury compounds (salts). Mercury can also combine with carbon to form organomercury compounds. Bromine is a halogen element with the symbol Br and atomic number 35. Diatomic bromine does not exist naturally, but bromine salts are found in Earth's crustal rocks. (L625, L1, L264) A once-popular mercury-containing external disinfectant. Merbromin (also referred to as ZP1 in this study) was one of the first active compounds discovered from a high-throughput screening of a library of natural compounds targeting the Zika virus NS2B-NS3 protease. It, along with four other compounds (including tannic acid, 1,2,3,4,6-O-pentagalloglucoside, and theaflavin-3,3'-digallate), was found to both inhibit protease activity and bind directly to ZIKVpro, suggesting their potential for further development as ZIKVpro inhibitors [1].

C20H8BR2HGNA2O6

750.65

752.824

129-16-8

441373

Light brown to black solid powder

≥300 °C

3.592

1

6

2

31

801

0

KQSRXDXJGUJRKE-UHFFFAOYSA-L

InChI=1S/C20H9Br2O5.Hg.2Na.H2O/c21-13-5-11-17(7-15(13)23)27-18-8-16(24)14(22)6-12(18)19(11)9-3-1-2-4-10(9)20(25)26;;;;/h1-7,24H,(H,25,26);;;;1H2/q;;2*+1;/p-2

disodium;[2,7-dibromo-9-(2-carboxylatophenyl)-3-oxido-6-oxoxanthen-4-yl]mercury;hydrate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~6.67 mg/mL (~8.86 mM)

Solubility in Formulation 1: ≥ 0.67 mg/mL (0.89 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 6.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 0.67 mg/mL (0.89 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 6.7 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)