High-throughput amenable fluorescence-assays to screen for calmodulin-inhibitors

Manoharan, Ganesh babu; Kopra, Kari; Eskonen, Ville; Härmä, Harri; Abankwa, Daniel

doi:10.1016/j.ab.2019.02.015

Cited by 15 publications

(31 citation statements)

References 47 publications

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“…Assay principle: Förster resonance energy transfer (FRET) assays can be applied in various configurations in cells [50] and in reporter systems [51] to detect molecular proximity and with the proper biosensor also activity. Here we employed an in vitro Assays involving principles of time-resolved FRET may be suitable for future follow-up screening of compounds with background fluorescence [53,54].…”

Section: In Vitro Assay For Assessing the 3clpro Inhibitory Activity mentioning

confidence: 99%

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A Pharmacophore Model for SARS-CoV-2 3CLpro Small Molecule Inhibitors and in Vitro Experimental Validation of Computationally Screened Inhibitors

Glaab

Manoharan

Abankwa

2021

Preprint

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Among the biomedical efforts in response to the current coronavirus (COVID-19) pandemic, pharmacological strategies to reduce viral load in patients with severe forms of the disease are being studied intensively. One of the main drug target proteins proposed so far is the SARS-CoV-2 viral protease 3CLpro (also called Mpro), an essential component for viral replication. Ongoing ligand- and receptor-based computational screening efforts would be facilitated by an improved understanding of the electrostatic, hydrophobic and steric features that characterize small molecule inhibitors binding stably to 3CLpro, as well as by an extended collection of known binders.Here, we present combined virtual screening, molecular dynamics simulation, machine learning and in vitro experimental validation analyses which have led to the identification of small molecule inhibitors of 3CLpro with micromolar activity, and to a pharmacophore model that describes functional chemical groups associated with the molecular recognition of ligands by the 3CLpro binding pocket. Experimentally validated inhibitors using a ligand activity assay include natural compounds with available prior knowledge on safety and bioavailability properties, such as the natural compound rottlerin (IC50 = 37 μM), and synthetic compounds previously not characterized (e.g. compound CID 46897844, IC50 = 31 μM). In combination with the developed pharmacophore model, these and other confirmed 3CLpro inhibitors may provide a basis for further similarity-based screening in independent compound databases and structural design optimization efforts, to identify 3CLpro ligands with improved potency and selectivity.Overall, this study suggests that the integration of virtual screening, molecular dynamics simulations and machine learning can facilitate 3CLpro-targeted small molecule screening investigations. Different receptor-, ligand- and machine learning-based screening strategies provided complementary information, helping to increase the number and diversity of identified active compounds. Finally, the resulting pharmacophore model and experimentally validated small molecule inhibitors for 3CLpro provide resources to support follow-up computational screening efforts for this drug target.

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Section: In Vitro Assay For Assessing the 3clpro Inhibitory Activity mentioning

confidence: 99%

“…Therefore, out of 96 compounds tested in the 3CLpro activity assay, 12 compounds were filtered out from further analysis (highlighted in blue color in the lists of tested compounds in the Supporting Information). Assays involving principles of time-resolved FRET may be suitable for future follow-up screening of compounds with background fluorescence[64,65].…”

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confidence: 99%

A Pharmacophore Model for SARS-CoV-2 3CLpro Small Molecule Inhibitors and in Vitro Experimental Validation of Computationally Screened Inhibitors

Glaab

Manoharan

Abankwa

2021

Preprint

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“…The fluorescence polarization assay was performed as previously described (22,23). IC50 value was converted into Kd as described previously (59).…”

Section: Fluorescence Polarization Assaymentioning

confidence: 99%

Potential of phenothiazines to synergistically block calmodulin and reactivate PP2A in cancer cells

Okutachi

Manoharan

Abankwa

2021

Preprint

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Phenothiazines (PTZ) are well known as inhibitors of monoamine neurotransmitter receptors, notably dopamine receptors. Because of this activity they are used for decades as antipsychotic drugs. In addition, significant anti-cancer properties have been ascribed to them. Several attempts for their repurposing were made, however, their incompletely understood polypharmacology is challenging. Here we examined the potential of PTZ to synergistically act on two cancer associated targets, calmodulin (CaM) and the tumor suppressor protein phosphatase 2A (PP2A). Both proteins are known to modulate the Ras-MAPK pathway activity. Consistently, combinations of a CaM inhibitor and a PP2A activator synergistically inhibited cancer cells with KRAS or BRAF mutations. We identified the covalently reactive PTZ derivative fluphenazine mustard as an inhibitor of Ras driven proliferation and Ras membrane organization. We confirmed its anti-CaM activity in vitro and through a cellular CaM target engagement bioluminescence resonance energy transfer (BRET) assay. Our results suggest that improved PTZ derivatives retaining their synergistic CaM inhibitory and PP2A activating properties, but without neurological side-effects, may be interesting to pursue further as anti-cancer agents.

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“…As in fluorescence polarization (FP), also applicable for homogeneous monitoring of cGMP forming and consumption activity 29 , 30 , only one label conjugation is needed for QRET, which simplifies assay optimization. However, unlike in FP performed with traditional organic fluorophores, QRET utilizes millisecond scale fluorescence lifetime of the Ln(III)-chelate, which is shown to improved sensitivity compared to FP in competitive assay 31 , 32 . In addition, wash-free homogeneous protocol ensures short assay time, while time-resolved fluorescence (TRF) detection provided a low background fluorescence even in biological milieu, as shown with e.g.…”

Section: Introductionmentioning

confidence: 99%

Homogeneous single-label cGMP detection platform for the functional study of nitric oxide-sensitive (soluble) guanylyl cyclases and cGMP-specific phosphodiesterases

Kopra

Sharina²,

Martin³

et al. 2020

Sci Rep

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Cardiovascular diseases are the number one death worldwide. Nitric oxide (NO)—NO-sensitive (soluble) guanylyl cyclase (sGC)—cyclic guanosine monophosphate (cGMP) pathway regulates diverse set of important physiological functions, including maintenance of cardiovascular homeostasis. Resting and activated sGC enzyme converts guanosine triphosphate to an important second messenger cGMP. In addition to traditional NO generators, a number of sGC activators and stimulators are currently in clinical trials aiming to support or increase sGC activity in various pathological conditions. cGMP-specific phosphodiesterases (PDEs), which degrade cGMP to guanosine monophosphate, play key role in controlling the cGMP level and the strength or length of the cGMP-dependent cellular signaling. Thus, PDE inhibitors also have clear clinical applications. Here, we introduce a homogeneous quenching resonance energy transfer (QRET) for cGMP to monitor both sGC and PDE activities using high throughput screening adoptable method. We demonstrate that using cGMP-specific antibody, sGC or PDE activity and the effect of small molecules modulating their function can be studied with sub-picomole cGMP sensitivity. The results further indicate that the method is suitable for monitoring enzyme reactions also in complex biological cellular homogenates and mixture.

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High-throughput amenable fluorescence-assays to screen for calmodulin-inhibitors

Cited by 15 publications

References 47 publications

A Pharmacophore Model for SARS-CoV-2 3CLpro Small Molecule Inhibitors and in Vitro Experimental Validation of Computationally Screened Inhibitors

A Pharmacophore Model for SARS-CoV-2 3CLpro Small Molecule Inhibitors and in Vitro Experimental Validation of Computationally Screened Inhibitors

Potential of phenothiazines to synergistically block calmodulin and reactivate PP2A in cancer cells

Homogeneous single-label cGMP detection platform for the functional study of nitric oxide-sensitive (soluble) guanylyl cyclases and cGMP-specific phosphodiesterases

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