Examination of sulfonamide-based inhibitors of MMP3 using the conditioned media of invasive glioma cells

Abstract

Glioblastoma multiforme (GBM) is the deadliest and the most common primary malignant brain tumour. The median survival for patients with GBM is around one year due to the nature of glioma cells to diffusely invade that make the complete surgical resection of tumours difficult. Based upon the connexin43 (Cx43) model of glioma migration we have developed a computational framework to evaluate MMP inhibition in materials relevant to GBM. Using the ilomastat Leu-Trp backbone, we have synthesised novel sulphonamides and monitored the performance of these compounds in conditioned media expressing MMP3. From the results discussed herein we demonstrate the performance of sulfonamide based MMPIs included AP-3, AP-6, and AP-7.

Keywords: Matrix metalloproteinase; glioblastoma multiforme; ilomastat; inhibition.

Figure 1.

General synthesis scheme for derivatives: (1) A (1.0 eq.), CH₃NH₂/CH₃OH (33% CH₃NH₂ by wt., 10 eq.); (2) B (1.0 eq.), FMOC-Leu-OH (2.0 eq.), DIC (2.0 eq.), Oxyma Pure (2.0 eq.), N,N-Diisopropylethylamine (2.0 eq.); (3) C (1.0 eq.), 20% piperidine in DMF; (4) D (1.0 eq.), chloromethane sulphonyl chloride (7.0 eq.), N,N-Diisopropylethylamine (7.0 eq.).

Figure 2.

(A) Analysis of MMP3 in C6-Cx43 (C6-13) and low motility C6 parental line confirm MMP3 expression in high motility cells^,. (B) Relative to untreated control, zymographic assays of C6-13 conditioned media demonstrate dose-dependent loss of MMP3 activity due to ilomastat (N = 3, significance level *p ≤ 0.05, ***p ≤ 0.001) (C). (D) Structures of the Leu-Trp backbone, ilomastat and sulphonamide derivatives computationally (Leu-Trp to AP-7) and experimentally (Leu-Trp, ilomastat and AP-1) examined in this study.

Figure 3.

The placement of AP-3, AP-6, AP-7 and ilomastat in MMP3. The colour scheme is green=AP-3, blue=AP-6, yellow=AP-7, and red = ilomastat. Regarding the molecular surface green indicates lipophilic regions and purple indicates low lipophilic regions. The Zn²⁺ ion is represented by the large light blue coloured sphere.

Figure 4.

Inhibition of MMP-3 activity detected by NFF-3 assay. Assays were monitored over six hours, relative to untreated controls, 50 μM and 100 μM concentration for each of the listed compounds were compared for: (A) Ilomastat, (B) Leu-Trp, and (C) AP-1. Value reported here represent average, control normalised values performed in triplicate (N = 3), R² values >0.99. Error bars representing standard error of the mean.

Figure 5.

Comparison of MMP3 activity (NFF-3 fluoresence, RFU/min) in the presence of Leu-Trp, AP-1, ilomastat at (A) 50 μM and (B) 100 μM and negative control as a function of calculated binding affinity. Anticipated inhibitory performance of (AP-2, AP-3, AP-4, AP-5, AP-6 and AP-7) are based on linear regression models.