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Novel peptidomimetic MMP-9 inhibitors as potential anti-epileptogenic therapeutics

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Khomiak D., Bertran A., Rejmak E., Kaczmarek L.

Acta Neurobiologiae Experimentalis

2017

tom 77

nr Suppl.1

INTRODUCTION: Epilepsy is the most widespread neurological disorder (prevalence – 50 million). The recent discoveries suggest that remodelling of the brain extracellular matrix, executed by extracellularly operating proteases, may play a fundamental role in the pathogenesis of epilepsy. One of them, MMP-9 has been particularly linked to epileptogenesis and the functional involvement of MMP-9 in kainic acid and pentylenetetrazole-kindling models of temporal lobe epilepsy demonstrated. AIM(S): Considering the inhibition MMP-9 as a promising therapeutic strategy, MMP-9 Inhibitors of peptidomimetic nature IPR-X, IPR-Y and IPR-Z were developed at Iproteos and initial conjoint testing of these compounds has been performed. METHOD(S): Compounds ability to penetrate blood‑brain barrier was analyzed by PAMPA (Parallel artificial membrane permeability assay. Stability to degradation in liver and cytotoxicity were tested by exposing to rat liver microsomes and by MTT assay, respectively. Selectivity and potency of IPR‑X‑Z were pre‑estimated by fluorescent assay using DQ‑gelatin and recombinant MMP‑9. Finally, the inhibitors’ ability to inhibit cleavage of of MMP-9 substrate – Nectin-3 was tested in primary hippocampal cell culture upon 50 μM glutamate stimulation. RESULTS: PAMPA assay studies yielded in 7.7–13.3% penetration percentage for artificial blood-brain barrier. The values of intrinsic clearance for IPR-X-Z determined by stability in liver microsomes assay ranged from 33 to 46 µL/min/mg protein, indicating moderate degradation. Compounds proved to be non-toxic in MTT cytotoxicity assay, except of IPR-Z at the highest concentration tested (200 µM). IC50 values from DQ‑gelatinase assay were shown to be 4–10 µM. In hippocampal cell cultures compounds inhibited MMP-9 dependent nectin-3 cleavage with 60% of total band intensity remaining on western blot. CONCLUSIONS: The obtained results confirm that IPR‑X, IPR‑Y and IPR‑Z are specific and potent towards MMP-9 and could be further tested in animal models of epileptogenesis. FINANCIAL SUPPORT: This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 642881.

MMP-9 inhibitors as a new drug that blocks the development of epilepsy

100%

Rejmak-Kozicka E., Khomiak D., Kaczmarek L.

Acta Neurobiologiae Experimentalis

2017

tom 77

nr Suppl.1

INTRODUCTION: Matrix metalloproteinase-9 (MMP-9) is a member of matrix metalloproteinase family that remodels the extracellular matrix. Recently, cumulative evidence indicates that MMP-9 is upregulated in experimental epilepsy models. Increased MMP-9 is also implicated in clinical epilepsy studies. AIM(S): Thus, we hypothesize that MMP-9 may be a novel therapeutic target for epilepsy and some agents, such as OAT1 or OAT2, may be potential antiepileptogenic drugs. METHOD(S): First we estimated IC50 of selected inhibitors using recombinant MMP‑9 and DQ gelatin, fluorescent substrate of MMP-9. Next, we estimated the level of cleavage of MMP-9 substrate – Nectin-3. To determine whether Nectin-3 cleavage might be blocked by MMP-9 inhibitors, we added to hippocampal neurons different concentrations of inhibitors upon 50 μM glutamate stimulation. Extracts from whole cell lysates were analyzed on Western blots. We also adapted gel zymography, method for estimating the level of MMP-9. Due to the MMP-9 low brain expression level and its secretion on the synapse upon neuronal stimulation we decided to inject mice with PTZ (50 mg/kg, 15 min). Next we added inhibitors of MMP-9 directly to developing buffer. RESULTS: IC50 value for OAT1 is 0,5 nM and for OAT2 is 13 nM. Glutamate-dependent stimulation of Nectin-3 cleavage was abolished only in the presence of OAT1 in 5 μM concentration in culture. Further, the gel zymography analysis showed inhibition of MMP-9 activity in the gel with OAT1 in the buffer. The inhibitor’s specificity towards MMP-9 was supported by the absence of MMP-2 activity in this probe, which is another abundant in the brain metalloproteinase showing gelatinase activity. CONCLUSIONS: This results strongly confirm that OAT1 is specific towards MMP‑9 and could be used in animal models of epileptogenesis. FINANCIAL SUPPORT: PBS3/A8/36/2015 from The National Centre for Research and Development.

Ograniczanie wyników

2 Acta Neurobiologiae Experimentalis

2 Kaczmarek L.

2 Khomiak D.

1 Bertran A.

1 Rejmak E.

1 Rejmak-Kozicka E.

2 2017

Wyniki wyszukiwania

Novel peptidomimetic MMP-9 inhibitors as potential anti-epileptogenic therapeutics

MMP-9 inhibitors as a new drug that blocks the development of epilepsy