Inhibition of the cellular function of perforin by 1-amino-2,4-dicyanopyrido[1,2-a]benzimidazoles

doi:10.1016/j.bmc.2011.05.013

Bioorganic & Medicinal Chemistry

Volume 19, Issue 13, 1 July 2011, Pages 4091-4100

https://doi.org/10.1016/j.bmc.2011.05.013 Get rights and content

Abstract

A high throughput screen showed the ability of a 1-amino-2,4-dicyanopyrido[1,2-a]benzimidazole analogue to directly inhibit the lytic activity of the pore-forming protein perforin. A series of analogues were prepared to study structure–activity relationships (SAR) for the this activity, either directly added to cells or released in situ by KHYG-1 NK cells, at non-toxic concentrations. These studies showed that the pyridobenzimidazole moiety was required for effective activity, with strongly basic centres disfavoured. This class of compounds was relatively unaffected by the addition of serum, which was not the case for a previous class of direct inhibitors.

Graphical abstract

Introduction

Perforin (PRF) is a pore-forming member of the membrane-attack-complex/PRF (MACPF) protein family¹, and is secreted, along with a range of serine proteases (granzymes), by both cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells.² Together, perforin and granzymes comprise a principal mechanism of CTL/NK cytotoxicity by which the immune systems of higher organisms protect themselves against pathogens and transformed cells. The critical role of perforin in this process is its ability, upon binding calcium, to assemble into aggregates of 12–18 molecules that form trans-membrane pores of 10–20 nm in diameter in the plasma membrane of the target cell, facilitating the entry of granzymes, which can trigger apoptosis.3, 4 Initially, the calcium-dependent membrane binding of perforin monomers is mediated by their C terminal C2 domains.⁵ Successive perforin monomers then assemble into a cylindrical pore via a series of salt bridges linking Arg213 on the ‘front’ of one monomer with Glu343 on the ‘back’ of the next.⁶ The structure of mouse perforin monomer at a resolution of 2.75 Å has recently been reported as a bent and twisted four-stranded β-sheet MACPF domain flanked by two clusters of α-helices that insert into membranes as β-strands upon pore formation.⁷ In the same report, cryo-electron microscopy also confirmed the distribution of individual perforin sub-domains within an assembled pore structure.

Apart from well-recognised disease syndromes due to mutation and/or down-regulation of perforin expression, there is also much evidence linking inappropriate perforin activity to several human pathologies, including cerebral malaria, type 1 diabetes, juvenile idiopathic arthritis and postviral myocarditis,⁷ as well as therapy-induced conditions such as allograft rejection and graft-versus-host disease.8, 9 Perforin is therefore a potentially druggable target, especially as it is encoded by a single-copy gene in both mice and humans.

However, there have been few reports of small-molecule inhibitors of perforin function, as distinguished from compounds that affect it indirectly by inhibiting cell respiration or perforin processing.¹⁰ Enzastaurin (1) (Fig. 1), a bisindolylmaleimide structurally related to staurosporine and currently in Phase II trial for cancer, has recently been reported¹¹ to inhibit perforin release from NK cells, but is thought to act via the PKC/PI3K/AKT pathway. We recently reported¹⁰ a series of dihydrofuro[3,4-c]pyridinones (e.g., 2) (Fig. 1) as direct inhibitors of the cytolytic effects of perforin in cells, and demonstrated limited structure–activity effects across the series. The most active compound in the series (2) had a IC₅₀ of 1 μM for inhibition of the lysis of Jurkat cells by added perforin, and at a concentration of 20 μM showed a 60% inhibition in the killing of K562 cells by perforin released from KHYG-1 NK cells, with little toxicity to the latter. However, the cellular activity of these compounds was later shown to be adversely affected by increasing concentrations of serum, limiting their further development. In the present paper we discuss the discovery of this new class of direct perforin inhibitors, the 1-amino-2,4-dicyanopyrido[1,2-a]benzimidazoles, and initial structure–activity relationship (SAR) studies on this new class.

Section snippets

Chemistry

The lead compound (5) was selected from a high throughput screen¹² of 100,000 compounds sourced from commercial libraries, using a 384-well plate format. Compounds were screened (at 20 μM) by incubation with sheep red blood cells, which in the absence of an inhibitor are lysed by perforin, generating turbidity that can be measured by the change in absorbance at 650 nm. Compound 5 was one of a few hits that were confirmed in an assay measuring its potency for inhibiting the lysis of Jurkat human

Conclusions

Molecules with the ability to directly inhibit the function of perforin are of potential use in the treatment of some autoimmune diseases and therapy-induced conditions characterised by undesired perforin secretion, but to date only one class, the dihydrofuro[3,4-c]pyridinones have been reported.¹⁰ Compound 5 in the present paper was one of a small number of compounds from a high throughput screen to show confirmatory inhibition of the lytic activity of perforin in a cell-based assay, and as a

Chemistry

Analyses were performed by the Microchemical Laboratory, University of Otago, Dunedin, NZ. Many samples tenaciously held water or DMF (verified by NMR). Melting points were determined using an Electrothermal Model 9200 and are as read. NMR spectra were measured in CD₃SOCD₃ (unless otherwise specified) on a Bruker Advance 400 spectrometer and referenced to Me₄Si. Mass spectra were recorded either on a Varian VG 7070 spectrometer at nominal 5000 resolution or a Finnigan MAT 900Q spectrometer. All

Acknowledgments

The authors thank the Wellcome Trust for partial support. K.M.H. thanks the Academy of Finland (Grant no. 135439), the Finnish Cultural Foundation, and Jenny and Antti Wihuri Foundation for financial support.

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Perforin is a pore-forming protein whose normal function enables cytotoxic T and natural killer (NK) cells to kill virus-infected and transformed cells. Conversely, unwanted perforin activity can also result in auto-immune attack, graft rejection and aberrant responses to pathogens. Perforin is critical for the function of the granule exocytosis cell death pathway and is therefore a target for drug development. In this study, by screening a fragment library using NMR and surface plasmon resonance, we identified 4,4-diaminodiphenyl sulfone (dapsone) as a perforin ligand. We also found that dapsone has modest (mM) inhibitory activity of perforin lytic activity in a red blood cell lysis assay in vitro. Sequential modification of this lead fragment, guided by structural knowledge of the ligand binding site and binding pose, and supported by SPR and ligand-detected ¹⁹F NMR, enabled the design of nanomolar inhibitors of the cytolytic activity of intact NK cells against various tumour cell targets. Interestingly, the ligands we developed were largely inert with respect to direct perforin-mediated red blood cell lysis but were very potent in the context of perforin's action on delivering granzymes in the immune synapse, the context in which it functions physiologically. Our work indicates that a fragment-based, structure-guided drug discovery strategy can be used to identify novel ligands that bind perforin. Moreover, these molecules have superior physicochemical properties and solubility compared to previous generations of perforin ligands.
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The discovery of a novel class of 1-amino-2,4-dicyanopyrido [1,2-a]benzimidazoles as direct inhibitors of perforin overcame these pitfalls later. Analogs with O-linked side chains 3 and amide-linked side chains including its NMe analogs 4,and 5 exhibited an optimized solubility considering steric factors also in the frame [121]. Compared to dihydro[3,4-c]pyridinone (IC50 = 0.97 μM), aryl substituted isobenzofuran-1(3H)-one produced consistent inhibitory activity (C; IC50 = 6.20 μM).
Perforin is a granular effector pore-forming protein formed in NK cells and Cytotoxic T lymphocytes. These cytotoxic proteins are part of the first-line immune defense in the human body. They ensure apoptosis of pathogen-infected cells or tumor cells in the human body. Activation of receptors on NK cell or T cell triggers secondary proteins in these cells. Further, it leads to Ca²⁺ dependent perforin egress towards the target cell, ensued by PI3K signaling pathway. Perforin undergoes oligomerization over the target cell membrane and forms transmembrane pores with the membrane-spanning domain-MACPF domain. Granzymes, proapoptotic serine proteases are released through these pores and initiate the target cell apoptotic pathway leading to the cell death. Although perforin is a savior for humans from tumor and viral infections, uncontrolled expression of the perforins leads to the autoimmune conditions, including Familial Hemophagocytic lymphohistiocytosis, insulin-dependent diabetes, and cerebral myocarditis.
The present review is the concerted effort to highlight the mechanistic pathways concerning perforin secretion, NK cell and T cell-mediated cytotoxicity towards virus-infected and transformed cells. This is followed by the discussion on synthetic derivatives tested so far to inhibit the perforin in pre and clinical arena for certain unusual conditions.
Microwave-assisted annulation for the construction of pyrido-fused heterocycles and their application as photoluminescent chemosensors
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A catalyst-free microwave-assisted annulation protocol for the preparation of biologically interesting pyrido-fused quinazolinones and pyrido[1,2-a]benzimidazoles is developed. This reaction involves the [3 + 3] annulation of various quinazolinones or benzimidazoles with 3-formylchromones to yield functionalized 11H-pyrido[2,1-b]quinazolin-11-one and pyrido[1,2-a] benzimidazole derivatives. This approach is successfully extended to the construction of various pyrazolo[4,3-d]pyrido[1,2-a]pyrimidin-10(1H)-ones. The present approach is complementary to the existing synthetic methodologies and offers a rapid and facile approach with a broad substrate scope, good yields, catalyst-free conditions, and a high functional group tolerance. The optimal synthesized compound is also employed as an “on–off” photoluminescent probe for the selective detection of Fe³⁺ and Ag⁺ metal ions.
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The one unexpected outlier was the 3-cyano compound 49 which, although it had poor activity against isolated recombinant perforin, had excellent activity against perforin produced by whole NK cells. The NK cells also retained excellent viability across all examples, consistent with our previous findings for this series [32] and in contrast to earlier reported classes [26–28]. Preliminary physicochemical data was collected on the same (active) subset of compounds in order to assess their potential for progression to in vivo pharmacokinetic (PK) studies (Table 5).
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Inhibition of the cellular function of perforin by 1-amino-2,4-dicyanopyrido[1,2-a]benzimidazoles

Abstract

Graphical abstract

Introduction

Section snippets

Chemistry

Conclusions

Chemistry

Acknowledgments

Nature

J. Exp. Med.

Science

Nature

J. Biol. Chem.

Immunity

Nature

Cell Death Differ.