Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Jun 1:65:128713.
doi: 10.1016/j.bmcl.2022.128713. Epub 2022 Apr 1.

Dimers of isatin derived α-methylene-γ-butyrolactone as potent anti-cancer agents

Sandeep Rana  1 , Smit Kour  2 , Smitha Kizhake  2 , Hannah M King  2 , Jayapal Reddy Mallareddy  2 , Adam J Case  3 , Tom Huxford  4 , Amarnath Natarajan  5
Affiliations

Dimers of isatin derived α-methylene-γ-butyrolactone as potent anti-cancer agents

Sandeep Rana et al. Bioorg Med Chem Lett. .

Abstract

The IKK-NFκB complex is a key signaling node that facilitates activation of gene expression in response to extracellular signals. The kinase IKKβ and the transcription factor RELA have been targeted by covalent modifiers that bind to surface exposed cysteine residues. A common feature in well characterized covalent modifiers of RELA and IKKβ is the Michael acceptor containing α-methylene-γ-butyrolactone functionality. Through synthesis and evaluation of a focused set of α-methylene-γ-butyrolactone containing spirocyclic dimers (SpiDs) we identified SpiD3 as an anticancer agent with low nanomolar potency. Using cell-free and cell-based studies we show that SpiD3 is a covalent modifier that generates stable RELA containing high molecular weight complexes. SpiD3 inhibits TNFα-induced IκBα phosphorylation resulting in the blockade of RELA nuclear translocation. SpiD3 induces apoptosis, inhibits colony formation and migration of cancer cells. The NCI-60 cell line screen revealed that SpiD3 potently inhibits growth of leukemia cell lines, making it a suitable pre-therapeutic lead for hematological malignancies.

Keywords: Cancer; Irreversible inhibitor; NFκB pathway; RELA inhibitor; Spirocyclic compounds; α-methylene-γ-butyrolactone.

PubMed Disclaimer

Figures

Figure 1:
Figure 1:
Previously reported anticancer sesquiterpene lactones and simplified helenalin derived NFκB Inhibitors.
Figure 2.
Figure 2.
(A) Equimolar concentrations (7.72 μM) of the reactants were mixed and incubated for 24h at 4 °C. The mixture was subjected to SDS-PAGE and the gel subjected to silver staining silver staining of the in vitro RELA and IKKβ reaction with SpiD3. Black arrows indicate the HMW protein cross-link bands. (B) OVCAR5 cells were treated with and without SpiD3 and SpiD7 for 2 h. The lysates were subjected to WB analyses with antibodies against the indicated proteins. (C) WB analysis of panel of cell lines treated with and without 10 μM concentration of dimer SpiD3 for 2h. Black arrows indicate the HMW protein cross-link bands. (D) OVCAR5 cells were treated with 10μM dimers SpiD3, SpiD7 or SpiD12 for 2h and stimulated with and without 40ng/ml TNFα for 10 min post inhibitor treatment. The cytoplasmic and nuclear fractions were isolated, and lysates were subjected to western blotting. (E) Dose-response study with SpiD3 to assess inhibition of TNFα-induced RELA activation in a dual luciferase reporter assay (n=3, ±SD). (F) Proposed docking pose and (F) zoom in image showing crosslinking of SpiD3 to cys105 of RELA (pdb code 1IKN) and cys412 of IKKβ (pdb code 4KIK) respectively.
Figure 3:
Figure 3:
(A) SpiD3 inhibited the colony formation in A2780 cells. (B) A2780 cells were treated with SpiD3 for 6h at difference concentrations. Cell growth medium containing SpiD3 was then removed (washout) and cells were allowed to grow over three days. (C) SpiD3 disrupts migration of A2780 cells. Wound gap images taken during the 24h and 48h incubation of HCT116 cells with DMSO or 500 nM SpiD3. (D) Quantification of % wound closure after treatment of HCT116 cells with DMSO of 500 nM SpiD3.
Figure 4:
Figure 4:
Growth Inhibition (GI50) for SpiD3 and monomeric analog 19 against a panel of 60 Human Cancer Cell Lines. GI50 is the drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in control cells during the drug incubation.
Scheme 1.
Scheme 1.. Synthesis of dimeric spirocyclic based analogs.
(i). Dibromoalkane, NaH, DMF, RT, O/N (60–90%); (ii). Methyl 2-(bromomethyl)acrylate, In, THF: water, RT, 24h (40–70%); (iii). TsOH, DCM, RT, 24h (60–80%).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Oeckinghaus A; Ghosh S The NF-kappaB family of transcription factors and its regulation. Cold Spring Harb Perspect Biol 2009, 1, a000034. - PMC - PubMed
    1. Liu F; Xia Y; Parker AS; Verma IM IKK biology. Immunol Rev 2012, 246, 239–53. - PMC - PubMed
    1. DiDonato JA; Hayakawa M; Rothwarf DM; Zandi E; Karin M A cytokine-responsive IkB kinase that activates the transcription factor NF-kB. Nature 1997, 388, 549–554. - PubMed
    1. DiDonato J; Mercurio F; ROSETTE C; WU-LI J; SUYANG H; GHOSH S; KARIN M Mapping of the Inducible IkB Phosphorylation Sites That Signal Its Ubiquitination and Degradation. Mol Cell Biol 1996, 16, 1295–1304. - PMC - PubMed
    1. Prabhu L; Mundade R; Korc M; Loehrer PJ; Lu T Critical role of NF-kappaB in pancreatic cancer. Oncotarget 2014, 5, 10969–75. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources