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Small molecule induction of MSH2-dependent cell death suggests a vital role of mismatch repair proteins in cell death - PubMed

  • ️Thu Jan 01 2009

Small molecule induction of MSH2-dependent cell death suggests a vital role of mismatch repair proteins in cell death

Aksana Vasilyeva et al. DNA Repair (Amst). 2009.

Abstract

Avoidance of apoptosis is one of the hallmarks of cancer development and progression. Chemotherapeutic agents aim to initiate an apoptotic response, but often fail due to dysregulation. MSH proteins are capable of recognizing cisplatin damage in DNA and participate in the initiation of cell death. We have exploited this recognition and computationally simulated a MutS homolog (MSH) "death conformation". Screening and docking experiments based on this model determined that the MSH2-dependent cell-death pathway can be induced by a small molecule without DNA damage, reserpine. Reserpine was identified via virtual screening on structures obtained from molecular dynamics as a small molecule that selectively binds a protein "death" conformation. The virtual screening predicts that this small molecule binds in the absence of DNA. Cell biology confirmed that reserpine triggers the MSH2-dependent cell-death pathway. This result supports the hypothesis that the MSH2-dependent pathway is initiated by specific protein conformational changes triggered by binding to either DNA damage or small compound molecules. These findings have multiple implications for drug discovery and cell biology. Computational modeling may be used to identify and eventually design small molecules that selectively activate particular pathways through conformational control. Molecular dynamics simulations can be used to model the biologically relevant conformations and virtual screening can then be used to select for small molecules that bind specific conformations. The ability of a small molecule to induce the cell-death pathway suggests a broader role for MMR proteins in cellular events, such as cell-death pathways, than previously suspected.

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Figures

Fig. 1
Fig. 1

Structures of reserpic acid and reserpine. Standard two-dimensional chemical drawings of reserpine (left) and reserpic acid (right).

Fig. 2
Fig. 2

Small-Molecule binding mode is in the DNA binding pocket. The predicted binding poise is depicted in two different representations. On the left, the entire protein complex, taken from the MutS/cisplatinated DNA molecular dynamics simulation [7], is shown in a cartoon representation, F36, M33 and E38 are in a gray vdW representation, and reserpine is in a blue vdW representation. On the right is a rotated close-up of the binding pocket with the same color scheme.

Fig. 3
Fig. 3

MTS cell survival assay shows MSH2-dependent cell viability after concentration-dependent (a) reserpine and (b) cisplatin treatment. A representative MTS cell survival assay of MSH2-proficient (triangle) and –deficient (square) cells in the presence of increasing concentrations of reserpine (as indicated) and cisplatin is shown. Percent cell survival is plotted in reference to untreated cells. Mean values and standard deviation of three independent experiments are shown. Results are shown 24h ((a) and (b) left panel) and 72h ((b) right panel) after exposure.

Fig. 4
Fig. 4

MTS cell survival assay with two reserpine analogs: (A) Structure and MTS assay of MSH2-proficient (triangle) and –deficient (square) cells after exposure to increasing concentrations (as indicated) of rescinnamine. (B) Structure and MTS assay of MSH2-proficient (triangle) and –deficient (square) cells after exposure to increasing concentrations (as indicated) of evodiamine. Shown is cell viability in percent compared to untreated cells. Mean values and standard deviation of three independent experiments are shown.

Fig. 5
Fig. 5

Western blot analysis of caspase-3 cleavage in MSH2-proficient (Hec59(2)) and – deficient (Hec59) cells after exposure to reserpine and staurosporine (STS, control), as indicated. The 17 and 19 kDa cleavage products of caspase-3 are shown. Actin was added as a loading control.

Fig. 6
Fig. 6

(A) Van der Waals image of DNA containing a single 1,2GpG cisplatin adduct (red) as predicted to bind by MutS in the structural model and its overlap with reserpine in its predicted binding mode with MutS (F36, M33 and E38 are in a gray vdW representation, and Reserpine is in a blue vdW representation). Predicted binding of reserpine (blue) and rescinnamine (gold) as it overlaps with (B) mismatched DNA, and (C) cisplatinated DNA, respectively. (D, E) Gel shift assay with yeast Msh2/Msh6 protein in the presence of reserpine and evodiamine, as indicated. 100 nM protein were incubated with 10 nM TAMRA-labeled oligonucleotides (mismatched DNA) in the absence or presence of 200 μM reserpine and evodiamine, respectively (see Materials and Methods).

Fig. 7
Fig. 7

(A) Silver-stained SDS polyacrylamide gel of trypsin proteolysis of MSH2/MSH6 in the presence of mismatched (lane 1), cisplatinated DNA (lane 2) and reserpine (lane 3), respectively. Diamonds indicate fragments detected by the imaging software (B). ImageQuant TL analysis of pixel intensity of individual fragments. Banding was detected automatically after setting the slope to 300. Fragments present with mismatched DNA are labeled 1-7. Additional fragments observed with DNA damage or small molecule are labeled by letters. See Materials and Methods for details.

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