Development of a colorimetric assay for heparanase activity suitable for kinetic analysis and inhibitor screening

The role that heparanase plays during metastasis and angiogenesis in tumors makes it an attractive target for cancer therapeutics. Despite this enzyme’s significance, most of the assays developed to measure its activity are complex. Moreover, they usually rely on labeling variable preparations of the natural substrate heparan sulfate, making comparisons across studies precarious. To overcome these problems, we have developed a convenient assay based on the cleavage of the synthetic heparin oligosaccharide fondaparinux. The assay measures the appearance of the disaccharide product of heparanase-catalyzed fondaparinux cleavage colorimetrically using the tetrazolium salt WST-1. Because this assay has a homogeneous substrate with a single point of cleavage, the kinetics of the enzyme can be reliably characterized, giving a K_m of 46 μM and a k_cat of 3.5 s⁻¹ with fondaparinux as substrate. The inhibition of heparanase by the published inhibitor, PI-88, was also studied, and a K_i of 7.9 nM was determined. The simplicity and robustness of this method, should, not only greatly assist routine assay of heparanase activity but also could be adapted for high-throughput screening of compound libraries, with the data generated being directly comparable across studies.     

A rapid two-step method for isolation of functional primary mouse hepatocytes: cell characterization and asialoglycoprotein receptor based assay development

Primary mouse hepatocytes are an important tool in the biomedical research field for the assessment of hepatocyte function. Several methods for hepatocyte isolation have been published; however, many of these methods require extensive handling and can therefore compromise the viability and function of the isolated cells. Since one advantage of utilizing freshly isolated cells is to maintain an environment in which the cells are more comparable to their in vivo state, it is important to have robust methods that produce cells with high viability, good purity and that function in a similar manner to that in their in vivo state. Here we describe a modified two-step method for the rapid isolation and characterization of mouse primary hepatocytes that results in high yields of viable cells. The asialoglycoprotein receptor (ASGPR), which is one of the most abundant cell surface receptors on hepatocytes, was used to monitor the function of the isolated hepatocytes by demonstrating specific binding of its ligand using a newly developed flow cytometry based ligand-receptor binding assay. Also, an in vitro screening method for siRNA drug candidates was successfully developed utilizing freshly isolated hepatocytes with minimum culture time.     

Virtual screening of small molecules databases for discovery of novel PARP-1 inhibitors: combination of in silico and in vitro studies

Poly(ADP-ribose) polymerase-1 (PARP-1) enzyme has critical roles in DNA replication repair and recombination. Thus, PARP-1 inhibitors play an important role in the cancer therapy. In the current study, we have performed combination of in silico and in vitro studies in order to discover novel inhibitors against PARP-1 target. Structure-based virtual screening was carried out for an available small molecules database. A total of 257,951 ligands from Otava database were screened at the binding pocket of PARP-1 using high-throughput virtual screening techniques. Filtered structures based on predicted binding energy results were then used in more sophisticated molecular docking simulations (i.e. Glide/standard precision, Glide/XP, induced fit docking – IFD, and quantum mechanics polarized ligand docking – QPLD). Potential high binding affinity compounds that are predicted by molecular simulations were then tested by in vitro methods. Computationally proposed compounds as PARP-1 inhibitors (Otava Compound Codes: 7111620047 and 7119980926) were confirmed by in vitro studies. In vitro results showed that compounds 7111620047 and 7119980926 have IC₅₀ values of 0.56 and 63 μM against PARP-1 target, respectively. The molecular mechanism analysis, free energy perturbation calculations using long multiple molecular dynamics simulations for the discovered compounds which showed high binding affinity against PARP-1 enzyme, as well as structure-based pharmacophore development (E-pharmacophore) studies were also studied.