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  • Microscale Thermophoresis as a Screening Tool to Predict Melanin Binding of Drugs.

Microscale Thermophoresis as a Screening Tool to Predict Melanin Binding of Drugs.

Pharmaceutics (2020-06-21)
Laura Hellinen, Sina Bahrpeyma, Anna-Kaisa Rimpelä, Marja Hagström, Mika Reinisalo, Arto Urtti
ABSTRACT

Interactions between drugs and melanin pigment may have major impacts on pharmacokinetics. Therefore, melanin binding can modify the efficacy and toxicity of medications in ophthalmic and other disease of pigmented tissues, such as melanoma. As melanin is present in many pigmented tissues in the human body, investigation of pigment binding is relevant in drug discovery and development. Conventionally, melanin binding assays have been performed using an equilibrium binding study followed by chemical analytics, such as LC/MS. This approach is laborious, relatively slow, and limited to facilities with high performance quantitation instrumentation. We present here a screening of melanin binding with label-free microscale thermophoresis (MST) that utilizes the natural autofluorescence of melanin. We determined equilibrium dissociation constants (Kd) of 11 model compounds with melanin nanoparticles. MST categorized the compounds into extreme (chloroquine, penicillin G), high (papaverine, levofloxacin, terazosin), intermediate (timolol, nadolol, quinidine, propranolol), and low melanin binders (atropine, methotrexate, diclofenac) and displayed good correlation with binding parameter values obtained with the conventional binding study and LC/MS analytics. Further, correlation was seen between predicted melanin binding in human retinal pigment epithelium and choroid (RPE-choroid) and Kd values obtained with MST. This method represents a useful and fast approach for classification of compounds regarding melanin binding. Thus, the method can be utilized in various fields, including drug discovery, pharmacokinetics, and toxicology.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Quinidine, crystallized, ≥98.0% (dried material, NT)
Sigma-Aldrich
Levofloxacin, 98.0-102.0% anhydrous basis (HPLC)
Supelco
Nadolol, analytical standard
Sigma-Aldrich
Quinidine, anhydrous
Sigma-Aldrich
(±)-Propranolol hydrochloride, ≥99% (TLC), powder
Sigma-Aldrich
Timolol maleate salt, ≥98% (TLC), powder
Sigma-Aldrich
Penicillin G potassium salt, powder, BioReagent, suitable for cell culture
Supelco
Methotrexate, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Terazosin hydrochloride, ≥98% (TLC), powder
Sigma-Aldrich
Diclofenac sodium salt
Sigma-Aldrich
Chloroquine diphosphate salt, powder or crystals, 98.5-101.0% (EP)
Sigma-Aldrich
Pazopanib