High-Throughput Turbidimetric Screening for Heparin-Neutralizing Agents and Low-Molecular-Weight Heparin Mimetics

Abstract

Safer heparin-neutralizing agents are currently required to replace protamine, the use of which causes adverse effects such as anaphylaxia. Low-molecular-weight (LMW) heparin mimetics that potentiate antithrombin III (AT) action are also valuable as anti-thrombotics. This paper describes a high-throughput assay for both heparin-neutralizing agents and LMW heparin mimetics without the use of blood preparations. The assay is based on turbidimetric measurement of a solution of collagen, heparin, and a test compound. Native collagen molecules spontaneously form insoluble fibrils when transferred to a physiological buffer, and this process is inhibited by heparin. In the presence of a heparin-neutralizing agent or an LMW heparin mimetic, the inhibitory effect of heparin is canceled and turbidity increase is retrieved. We demonstrated that this assay is effective in detecting potential agents with high reliability (Z′ factor 0.9). The screening of a chemical library (34400 compounds) was further performed in a 384-well format, and led to the identification of a novel heparin-neutralizing agent. Since this assay protocol is feasible for an automated highthroughput screening (HTS) system, it could enhance the lead seeking process for drugs related to heparin/ heparan sulfate (HS) functions.

A simple new competition assay for heparin binding in serum applied to multivalent PAMAM dendrimers

“…6 Previous approaches to screening heparin binders have used radioisotopes, affinity co-electrophoresis, 7 competitive inhibition assays 8 and recently, turbidimetric screening. 9 Having previously demonstrated that MalB could detect heparin in competitive conditions, we designed a simple dyedisplacement competition assay (Fig. 2) to probe synthetic heparin binders.…”

A Transparent and Injectable Biomaterial Prepared by Mixing Collagen and Anti‐Cancer Platinum Derivatives

“…Our group has previously investigated inhibitors of collagen fibril formation using a high-throughput multi-well screening system that is based on monitoring turbidity increase as an indicator of collagen fibril formation. [12][13][14] In a chemical library containing 13 780 low-molecular-weight compounds, our screening identified only one hit compound, cisplatin, a well-known anti-cancer drug. We also clarified that the active compound was not cisplatin itself but rather a cisplatin derivative formed during storage in dimethyl sulfoxide (DMSO), a solvent commonly used in the preparation of chemical libraries.…”