Dataset Information

A microscopy-based small molecule screen in primary neurons reveals neuroprotective properties of the FDA-approved anti-viral drug Elvitegravir.

ABSTRACT: Glutamate toxicity is a pathomechanism that contributes to neuronal cell death in a wide range of acute and chronic neurodegenerative and neuroinflammatory diseases. Activation of the N-methyl-D-aspartate (NMDA)-type glutamate receptor and breakdown of the mitochondrial membrane potential are key events during glutamate toxicity. Due to its manifold functions in nervous system physiology, however, the NMDA receptor is not well suited as a drug target. To identify novel compounds that act downstream of toxic NMDA receptor signaling and can protect mitochondria from glutamate toxicity, we developed a cell viability screening assay in primary mouse cortical neurons. In a proof-of-principle screen we tested 146 natural products and 424 FDA-approved drugs for their ability to protect neurons against NMDA-induced cell death. We confirmed several known neuroprotective drugs that include Dutasteride, Enalapril, Finasteride, Haloperidol, and Oxybutynin, and we identified neuroprotective properties of Elvitegravir. Using live imaging of tetramethylrhodamine ethyl ester-labelled primary cortical neurons, we found that Elvitegravir, Dutasteride, and Oxybutynin attenuated the NMDA-induced breakdown of the mitochondrial membrane potential. Patch clamp electrophysiological recordings in NMDA receptor-expressing HEK293 cell lines and primary mouse hippocampal neurons revealed that Elvitegravir does not act at the NMDA receptor and does not affect the function of glutamatergic synapses. In summary, we have developed a cost-effective and easy-to-implement screening assay in primary neurons and identified Elvitegravir as a neuro- and mitoprotective drug that acts downstream of the NMDA receptor.

SUBMITTER: Merz SF

PROVIDER: S-EPMC7489219 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A microscopy-based small molecule screen in primary neurons reveals neuroprotective properties of the FDA-approved anti-viral drug Elvitegravir.

Merz Simon F SF Bengtson C Peter CP Tepohl Clara C Hagenston Anna M AM Bading Hilmar H Bas-Orth Carlos C

Molecular brain 20200914 1

Glutamate toxicity is a pathomechanism that contributes to neuronal cell death in a wide range of acute and chronic neurodegenerative and neuroinflammatory diseases. Activation of the N-methyl-D-aspartate (NMDA)-type glutamate receptor and breakdown of the mitochondrial membrane potential are key events during glutamate toxicity. Due to its manifold functions in nervous system physiology, however, the NMDA receptor is not well suited as a drug target. To identify novel compounds that act downstr ...[more]

PMID: 32928261

Similar Datasets

Project description:PurposeTo assess the neuroprotective effects of flibanserin (formerly BIMT-17), a dual 5-HT1A agonist and 5-HT2A antagonist, in a light-induced retinopathy model.MethodsAlbino BALB/c mice were injected intraperitoneally with either vehicle or increasing doses of flibanserin ranging from 0.75 to 15 mg/kg flibanserin. To assess 5-HT1A-mediated effects, BALB/c mice were injected with 10 mg/kg WAY 100635, a 5-HT1A antagonist, prior to 6 mg/kg flibanserin and 5-HT1A knockout mice were injected with 6 mg/kg flibanserin. Injections were administered once immediately prior to light exposure or over the course of five days. Light exposure lasted for one hour at an intensity of 10,000 lux. Retinal structure was assessed using spectral domain optical coherence tomography and retinal function was assessed using electroretinography. To investigate the mechanisms of flibanserin-mediated neuroprotection, gene expression, measured by RT-qPCR, was assessed following five days of daily 15 mg/kg flibanserin injections.ResultsA five-day treatment regimen of 3 to 15 mg/kg of flibanserin significantly preserved outer retinal structure and function in a dose-dependent manner. Additionally, a single-day treatment regimen of 6 to 15 mg/kg of flibanserin still provided significant protection. The action of flibanserin was hindered by the 5-HT1A antagonist, WAY 100635, and was not effective in 5-HT1A knockout mice. Creb, c-Jun, c-Fos, Bcl-2, Cast1, Nqo1, Sod1, and Cat were significantly increased in flibanserin-injected mice versus vehicle-injected mice.ConclusionsIntraperitoneal delivery of flibanserin in a light-induced retinopathy mouse model provides retinal neuroprotection. Mechanistic data suggests that this effect is mediated through 5-HT1A receptors and that flibanserin augments the expression of genes capable of reducing mitochondrial dysfunction and oxidative stress. Since flibanserin is already FDA-approved for other indications, the potential to repurpose this drug for treating retinal degenerations merits further investigation.

Project description:Protein kinases are key enzymes that catalyze the covalent phosphorylation of substrates via the transfer of the γ-phosphate of ATP, playing a crucial role in cellular proliferation, differentiation, and various cell regulatory processes. Due to their pivotal cellular role, the aberrant function of kinases has been associated with cancers and many other diseases. Consequently, competitive inhibition of the ATP binding site of protein kinases has emerged as an effective means of curing these diseases. Decades of intense development of protein kinase inhibitors (PKIs) resulted in 71 FDA-approved PKI drugs that target dozens of protein kinases for the treatment of various diseases. How do FDA-approved protein kinase inhibitor PKI drugs compete with ATP in their own binding pocket? This is the central question we attempt to address in this work. Based on modes of non-bonded interactions and their calculated interaction strengths by means of the advanced double hybrid DFT method B2PLYP, the molecular recognition of PKI drugs in the ATP-binding pockets was systematically analyzed. It was found that (1) all the FDA-approved PKI drugs studied here form one or more hydrogen bond(s) with the backbone amide N, O atoms in the hinge region of the ATP binding site, mimicking the adenine base; (2) all the FDA-approved PKI drugs feature two or more aromatic rings. The latter reach far and deep into the hydrophobic regions I and II, forming multiple CH-π interactions with aliphatic residues L(3), V(11), A(15), V(36), G(51), L(77) and π-π stacking interactions with aromatic residues F(47) and F(82), but ATP itself does not utilize these regions extensively; (3) all FDA-approved PKI drugs studied here have one thing in common, i.e., they frequently formed non-bonded interactions with a total of 12 residues L(3),V(11), A(15), K(17), E(24),V(36),T(45), F(47), G(51), L(77), D(81) and F(82) in the ATP binding. Many of those 12 commonly involved residues are highly conserved residues with important structural and catalytic functional roles. K(17) and E(24) are the two highly conserved residues crucial for the catalytic function of kinases. D(81) and F(82) belong to the DFG motif; T(45) was dubbed the gate keeper residue. F(47) is located on the hinge region and G(51) sits on the linker that connects the hinge to the αD-helix. It is this targeting of highly conserved residues in protein kinases that led to promiscuous PKI drugs that lack selectivity. Although the formation of hydrogen bond(s) with the backbone of the hinge gives PKI drugs the added binding affinity and the much-needed directionality, selectivity is sacrificed. That is why so many FDA-approved PKI drugs are known to have multiple targets. Moreover, off-target-mediated toxicity caused by a lack of selectivity was one of the major challenges facing the PKI drug discovery community. This work suggests a road map for future PKI drug design, i.e., targeting non-conserved residues in the ATP binding pocket to gain better selectivity so as to avoid off-target-mediated toxicity.

Project description:The FDA has approved 31 small-molecule kinase inhibitors (KIs) for human use as of November 2016, with six having black box warnings for hepatotoxicity (BBW-H) in product labeling. The precise mechanisms and risk factors for KI-induced hepatotoxicity are poorly understood. Here, the 31 KIs were tested in isolated rat liver mitochondria, an in vitro system recently proposed to be a useful tool to predict drug-induced hepatotoxicity in humans. The KIs were incubated with mitochondria or submitochondrial particles at concentrations ranging from therapeutic maximal blood concentrations (Cmax) levels to 100-fold Cmax levels. Ten endpoints were measured, including oxygen consumption rate, inner membrane potential, cytochrome c release, swelling, reactive oxygen species, and individual respiratory chain complex (I-V) activities. Of the 31 KIs examined only three including sorafenib, regorafenib and pazopanib, all of which are hepatotoxic, caused significant mitochondrial toxicity at concentrations equal to the Cmax, indicating that mitochondrial toxicity likely contributes to the pathogenesis of hepatotoxicity associated with these KIs. At concentrations equal to 100-fold Cmax, 18 KIs were found to be toxic to mitochondria, and among six KIs with BBW-H, mitochondrial injury was induced by regorafenib, lapatinib, idelalisib, and pazopanib, but not ponatinib, or sunitinib. Mitochondrial liability at 100-fold Cmax had a positive predictive power (PPV) of 72% and negative predictive power (NPV) of 33% in predicting human KI hepatotoxicity as defined by product labeling, with the sensitivity and specificity being 62% and 44%, respectively. Similar predictive power was obtained using the criterion of Cmax ≥1.1 µM or daily dose ≥100 mg. Mitochondrial liability at 1-2.5-fold Cmax showed a 100% PPV and specificity, though the NPV and sensitivity were 32% and 14%, respectively. These data provide novel mechanistic insights into KI hepatotoxicity and indicate that mitochondrial toxicity at therapeutic levels can help identify hepatotoxic KIs.

Dataset Information

A microscopy-based small molecule screen in primary neurons reveals neuroprotective properties of the FDA-approved anti-viral drug Elvitegravir.

Publications

A microscopy-based small molecule screen in primary neurons reveals neuroprotective properties of the FDA-approved anti-viral drug Elvitegravir.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets