Thiacalix[4]arenes Counteract Zinc‑Induced Inhibition of Uterine Myosin ATPase Activity

Background

Environmental exposure to heavy metals, especially zinc, poses significant health risks. While zinc is a vital cofactor for over 300 metalloenzymes, chronic high intake can disrupt physiological processes, including uterine smooth‑muscle contraction, leading to reproductive complications such as infertility and preterm labor. Zinc’s impact on myosin ATPase—a key driver of smooth‑muscle contractility—has been documented, yet effective countermeasures are limited. Calixarenes, particularly thiacalix[4]arenes, have emerged as promising chelating agents due to their low cytotoxicity and strong metal‑binding capabilities. Prior work demonstrated that C‑798 protects myosin S1 from Pb²⁺, Cd²⁺, and Ni²⁺ inhibition. This study extends those insights to zinc toxicity in uterine myosin.

This collaborative effort between the Palladin Institute of Biochemistry and the Institute of Organic Chemistry, NAS of Ukraine, explores the interaction between uterine myosin ATPase and thiacalix[4]arenes.

Results

Effect of Zn²⁺ on Myosin S1 ATPase

Incremental Zn²⁺ (0.5–5 mM) reduced ATPase activity, with the maximum inhibition (43 ± 8 %) observed at 5 mM. Subsequent kinetic analysis (Lineweaver–Burk) confirmed a significant 1.6‑fold decrease in V_max,ATP without a statistically meaningful change in K_m,ATP.

ATP Concentration Dependence under 5 mM Zn²⁺

ATPase activity peaked at 3 mM ATP in both control and zinc‑treated samples. Under zinc, the peak activity was 30 % lower, corroborating the inhibitory effect on catalytic turnover.

Mg²⁺ Dependence in Presence of 5 mM Zn²⁺

Unlike the control, varying Mg²⁺ (0.5–5 mM) did not alter ATPase activity when 5 mM Zn²⁺ was present, indicating that zinc occupies the Mg²⁺‑binding sites and renders the enzyme insensitive to Mg²⁺ concentration.

Zinc Binding Sites on Myosin S1

Docking studies identified multiple Zn‑binding loci within the ATPase domain—particularly near the P‑loop and switch 1/2 regions—stabilized by residues Glu, Asp, and Arg. These sites are integral to ATP binding and hydrolysis.

Thiacalix[4]arenes Restore ATPase Activity

Adding 100 µM C‑798 or C‑800 to the 5 mM Zn medium fully restored ATPase activity to control levels. C‑798 exhibited a modest, reversible inhibition (likely due to Mg²⁺ sequestration), whereas C‑800 had no effect on the enzyme in the absence of zinc.

Mechanistic Insights from Docking

Simulations revealed that C‑798 and C‑800 bind Zn²⁺ via their lower‑rim oxygen and sulfur atoms, displacing the metal from key catalytic residues. This chelation reduces Zn–myosin interactions, thereby relieving the inhibitory effect. Energy minimization indicated favorable host‑guest interactions (total energies: 64.5 kcal/mol for C‑798, 83 kcal/mol for C‑798‑Zn complex).

Discussion

The 5 mM zinc concentration employed mimics pathological exposure and demonstrates a pronounced, reversible suppression of uterine myosin ATPase. The lack of Mg²⁺ responsiveness in zinc‑treated samples supports the hypothesis that Zn²⁺ competitively occupies Mg²⁺ sites within the ATPase domain. Docking results align with this, pinpointing Zn coordination to residues critical for nucleotide binding and hydrolysis.

Thiacalix[4]arenes, with their anionic sulfonate or phosphonate groups, provide robust chelation of Zn²⁺ without disrupting Mg²⁺ homeostasis at physiological levels. Their ability to restore ATPase activity suggests a viable therapeutic strategy for zinc‑induced uterine dysfunction and potentially for other zinc‑related toxicities.

Conclusions

High‑level zinc (5 mM) impairs uterine myosin S1 ATPase by reducing V_max without altering K_m. Water‑soluble thiacalix[4]arenes C‑798 and C‑800 effectively neutralize this inhibition by chelating zinc at the ATPase active site, restoring enzymatic activity to baseline. These findings warrant further investigation into C‑798 and C‑800 as antidotes for zinc toxicity.

Methods

Reagents

All reagents were analytical grade (Sigma, Merck, Acros, or domestic suppliers). Solutions were prepared in ultrapure water (conductivity < 0.1 µS). Metal ion concentrations were verified by the Mohr titration method.

Actomyosin and Myosin S1 Isolation

Actomyosin was extracted from porcine uterine smooth muscle via a modified Barany protocol, and myosin S1 was purified using the Suzuki method. Protein purity was confirmed by PAAG‑SDS electrophoresis.

ATPase Activity Assay

ATPase activity was measured in 96‑well plates at 37 °C in a 0.1 mL medium (20 mM Tris‑HCl pH 7.2, 100 mM KCl, 0.01 mM CaCl₂, 5 mM MgCl₂, 3 mM ATP). Myosin S1 concentration was 20 µg mL⁻¹. After 5 min, inorganic phosphate release was quantified by the Chen method (absorbance 820 nm). Activity was expressed as nmol Pi min⁻¹ mg⁻¹ protein. Zinc and thiacalixarene effects were assessed by adding appropriate concentrations to the incubation medium; control activity (no Zn or ligand) was set to 100 %.

Kinetic and Statistical Analysis

Michaelis–Menten parameters were derived from Lineweaver–Burk plots. Statistical significance was evaluated using two‑way ANOVA (p < 0.05). Data analysis employed Microsoft Excel and Statistica 4.5.

Thiacalix[4]Arene Synthesis and Characterization

Compounds C‑798 and C‑800 were synthesized in the Phosphoranes Chemistry Department (Institute of Organic Chemistry, NAS of Ukraine) and confirmed by NMR and IR spectroscopy.

Computer Modeling

Ligand–protein docking used AutoDock 4.2 with the 1b7t PDB structure. 100 Lamarckian genetic algorithm runs (population = 100, 10⁶ energy evaluations) identified the lowest‑energy poses. Visualization employed Chimera and Yassara. Binding energies incorporated van der Waals, electrostatic, and hydrogen‑bond contributions.

Abbreviations

C-798:

Tetrahydroxythiacalix[4]arene‑tetrasulfonate

C-800:

Tetrahydroxythiacalix[4]arene‑tetraphosphonate

CNDO:

Complete Neglect of Differential Overlap (method)

K_m:

Michaelis constant

L50:

Lower 50‑kDa domain of myosin

LD₅₀:

Lethal dose for 50 % mortality

MM2:

Force field class

Myosin S1:

Myosin subfragment‑1

NASU:

National Academy of Science of Ukraine

PDB:

Protein Data Bank

P-loop:

Phosphate‑binding loop of myosin

RCSB:

Research Collaboratory for Structural Bioinformatics

U50:

Upper 50‑kDa domain of myosin

V_max:

Maximum velocity of the enzyme

V_max,ATP:

Maximum velocity for ATP hydrolysis