Discovery of Potent Multikinase Type-II Inhibitors Targeting CDK5 in the DFG-out Inactive State with Promising Potential against Glioblastoma.

Kinases have proven valuable targets in successful cancer drug discovery projects, but not yet for malignant brain tumors where type-II inhibition of cyclin-dependent kinase 5 (CDK5) stabilizing the DFG-out inactive state has potential for design of selective and clinically efficient drug candidates. In the absence of crystallographic evidence for a CDK5 DFG-out inactive state protein-ligand complex, for the first time, a model was designed using metadynamics/molecular dynamics simulations. Glide docking of the ZINC15 biogenic database identified [pyrimidin-2-yl]amino-furo[3,2-b]-furyl-urea/amide hit chemical scaffolds. For four selected analogues (4, 27, 36, and 42), potent effects on glioblastoma cell viability in U87-MG, T98G, and U251-MG cell lines and patient-derived cultures were generally observed (IC50s ∼ 10-40 µM at 72 h). Selectivity profiling against 11 homologous kinases revealed multikinase inhibition (CDK2, CDK5, CDK9, and GSK-3α/ß), most potent for GSK-3α in the nanomolar range (IC50s ∼ 0.23-0.98 µM). These compounds may therefore have diverse anticancer mechanisms of action and are of considerable interest for lead optimization.

Multidisciplinary docking, kinetics and X-ray crystallography studies of baicalein acting as a glycogen phosphorylase inhibitor and determination of its' potential against glioblastoma in cellular models.

Glycogen phosphorylase (GP) is the rate-determining enzyme in the glycogenolysis pathway. Glioblastoma (GBM) is amongst the most aggressive cancers of the central nervous system. The role of GP and glycogen metabolism in the context of cancer cell metabolic reprogramming is recognised, so that GP inhibitors may have potential treatment benefits. Here, baicalein (5,6,7-trihydroxyflavone) is studied as a GP inhibitor, and for its effects on glycogenolysis and GBM at the cellular level. The compound is revealed as a potent GP inhibitor against human brain GPa (Ki = 32.54 µM), human liver GPa (Ki = 8.77 µM) and rabbit muscle GPb (Ki = 5.66 µM) isoforms. It is also an effective inhibitor of glycogenolysis (IC50 = 119.6 µM), measured in HepG2 cells. Most significantly, baicalein demonstrated anti-cancer potential through concentration- and time-dependent decrease in cell viability for three GBM cell-lines (U-251 MG, U-87 MG, T98-G) with IC50 values of ∼20-55 µM (48- and 72-h). Its effectiveness against T98-G suggests potential against GBM with resistance to temozolomide (the first-line therapy) due to a positive O6-methylguanine-DNA methyltransferase (MGMT) status. The solved X-ray structure of rabbit muscle GP-baicalein complex will facilitate structure-based design of GP inhibitors. Further exploration of baicalein and other GP inhibitors with different isoform specificities against GBM is suggested.

The Effect of Cryoprotectants and Storage Conditions on the Transfection Efficiency, Stability, and Safety of Lipid-Based Nanoparticles for mRNA and DNA Delivery.

Lipid-based nanoparticles have recently shown great promise, establishing themselves as the gold standard in delivering novel RNA therapeutics. However, research on the effects of storage on their efficacy, safety, and stability is still lacking. Herein, the impact of storage temperature on two types of lipid-based nanocarriers, lipid nanoparticles (LNPs) and receptor-targeted nanoparticles (RTNs), loaded with either DNA or messenger RNA (mRNA), is explored and the effects of different cryoprotectants on the stability and efficacy of the formulations are investigated. The medium-term stability of the nanoparticles was evaluated by monitoring their physicochemical characteristics, entrapment and transfection efficiency, every two weeks over one month. It is demonstrated, that the use of cryoprotectants protects nanoparticles against loss of function and degradation in all storage conditions. Moreover, it is shown that the addition of sucrose enables all nanoparticles to remain stable and maintain their efficacy for up to a month when stored at -80 °C, regardless of cargo or type of nanoparticle. DNA-loaded nanoparticles also remain stable in a wider variety of storage conditions than mRNA-loaded ones. Importantly, these novel LNPs show increased GFP expression that can signify their future use in gene therapies, beyond the established role of LNPs in RNA therapeutics.

Intracellular mechanisms underlying the nicotinic enhancement of LTP in the rat dentate gyrus.

We have previously shown that activation of nicotinic acetylcholine receptors (nAChRs) enhanced long-term potentiation (LTP) in the rat dentate gyrus in vitro via activation of alpha7 nAChR. In the present studies, mechanisms underlying the acute and chronic nicotinic enhancement of LTP were examined. In particular, the involvement of activation of intracellular kinases was examined using selective kinase antagonists, and the effects of enhancing cholinergic function with positive allosteric modulators of the alpha7 nAChR and with acetylcholinesterase (AChE) inhibitors were also investigated. Activation of extracellular signal-regulated kinase (ERK) and cAMP-dependent protein kinase (PKA) was found to be involved in the induction of the acute nicotinic enhancement of LTP, although not control LTP. In contrast, activation of the tyrosine kinase Src, Ca(2+)-calmodulin-dependent protein kinase II, Janus kinase 2 and p38 mitogen-activated protein kinase was not involved in the acute nicotinic enhancement of LTP, although Src activation was necessary for control LTP. Moreover, activation of phosphoinositide 3-kinase was involved in the acute nicotinic enhancement of LTP to a much lesser extent than in control LTP. Chronic nicotine enhancement of LTP was found to be dependent on PKA, ERK and Src kinases. Acute nicotinic enhancement of LTP was occluded by chronic nicotine treatment. The positive allosteric modulator PNU-120596 was found to strongly reduce the threshold for nicotinic enhancement of LTP, an affect mediated via the alpha7 nAChR as it was blocked by the selective antagonist methyllycaconitine. The AChE inhibitors tacrine and physostigmine enhanced control LTP.

Molecular basis for the modulation of native T-type Ca2+ channels in vivo by Ca2+/calmodulin-dependent protein kinase II.

Ang II receptor activation increases cytosolic Ca2+ levels to enhance the synthesis and secretion of aldosterone, a recently identified early pathogenic stimulus that adversely influences cardiovascular homeostasis. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a downstream effector of the Ang II-elicited signaling cascade that serves as a key intracellular Ca2+ sensor to feedback-regulate Ca2+ entry through voltage-gated Ca2+ channels. However, the molecular mechanism(s) by which CaMKII regulates these important physiological targets to increase Ca2+ entry remain unresolved. We show here that CaMKII forms a signaling complex with alpha1H T-type Ca2+ channels, directly interacting with the intracellular loop connecting domains II and III of the channel pore (II-III loop). Activation of the kinase mediated the phosphorylation of Ser1198 in the II-III loop and the positive feedback regulation of channel gating both in intact cells in situ and in cells of the native adrenal zona glomerulosa stimulated by Ang II in vivo. These data define the molecular basis for the in vivo modulation of native T-type Ca2+ channels by CaMKII and suggest that the disruption of this signaling complex in the zona glomerulosa may provide a new therapeutic approach to limit aldosterone production and cardiovascular disease progression.

Beta-amyloid blocks high frequency stimulation induced LTP but not nicotine enhanced LTP.

Nicotine has been postulated to be a possible neuroprotective agent in Alzheimer's Disease (AD). In the present studies, the effect of beta-amyloid (Abeta) was investigated on the nicotine enhancement of high-frequency-induced LTP. Perfusion of nicotine substantially enhanced HFS-induced LTP in both rat and mouse dentate gyrus. The enhancing action of nicotine was mediated via alpha7 nAChRs as it was absent in mice null for alpha7 nAChR. Abeta strongly inhibited the induction of LTP in control animals, with LTP being completely inhibited at 1h post-HFS. Although Abeta also inhibited LTP in the presence of nicotine, the extent of the inhibition of LTP in nicotine perfused slices was similar to that in control, resulting in substantial LTP remaining in the presence of Abeta in the nicotine perfused slices. The nicotine enhanced LTP and the LTP remaining in the presence of Abeta and nicotine, although not the control LTP was dependent on activation of PKA. Chronic nicotine treatment also enhanced HFS-LTP recorded in acute slices taken from the nicotine-treated animals, and such LTP was only partially inhibited by Abeta. We postulate that nicotine-enhanced LTP has certain different mechanisms to that of control LTP which results in a resistance to inhibition by Abeta.

A mechanism for the direct regulation of T-type calcium channels by Ca2+/calmodulin-dependent kinase II.

Low-voltage-activated (LVA) Ca2+ channels are widely distributed throughout the CNS and are important determinants of neuronal excitability, initiating dendritic and somatic Ca2+ spikes that trigger and shape the pattern of action potential firing. Here, we define a molecular mechanism underlying the dynamic regulation of alpha1H channels (Cav3.2), by Ca2+/CaM-dependent protein kinase II (CaMKII). We show that channel regulation is selective for the LVA alpha1H Ca2+ channel subtype, depends on determinants in the alpha1H II-III intracellular loop, and requires the phosphorylation of a serine residue absent from unregulated alpha1G (Cav3.1) channels. These studies identify the alpha1H channel as a new substrate for CaMKII and provide the first molecular mechanism for the direct regulation of T-type Ca2+ channels by a protein kinase. Our data suggest a novel mechanism for modulating the integrative properties of neurons.

Regulation of the avidity of ternary complexes containing the human 5-HT(1A) receptor by mutation of a receptor contact site on the interacting G protein alpha subunit.

1 Fusion proteins were constructed between the human 5-HT(1A) receptor and pertussis toxin-resistant forms of both G(i1)alpha and G(o1)alpha mutated at residue(351) from cysteine to either glycine or isoleucine. Each of these was expressed stably in HEK293 cells. 2 Increasing concentrations of GDP inhibited binding of the agonist [(3)H]-8-OH-DPAT but not the antagonist [(3)H]-MPPF to each construct. 3 The IC(50) for GDP was greater for constructs containing isoleucine at residue(351) of the G proteins compared to those with glycine at this position. 4 The G protein antagonist suramin had similar effects to GDP on the binding of [(3)H]-8-OH-DPAT. 5 The proportion of 5-HT(1A) receptor binding sites detected by [(3)H]-MPPF that displayed high affinity for 8-OH-DPAT was significantly greater when the interacting G protein contained isoleucine rather than glycine at residue(351). 6 The 5-HT(1A) receptor displayed similar avidity of interaction with G(i1)alpha and G(o1)alpha. 7 These results indicate that a higher avidity ternary complex is formed between 8-OH-DPAT, the 5-HT(1A) receptor and G proteins when isoleucine rather than glycine is located at residue(351) of the interacting G protein.

Enhanced detection of receptor constitutive activity in the presence of regulators of G protein signaling: applications to the detection and analysis of inverse agonists and low-efficacy partial agonists.

Fusion proteins between the human 5-hydroxytryptamine (5-HT)(1A) receptor and either wild type or certain pertussis toxin-resistant forms of G(o1)alpha and G(i1)alpha display constitutive GTPase activity that can be inhibited by the inverse agonist spiperone. Addition of recombinant regulator of G protein signaling (RGS) 1 or RGS16 to membranes expressing these fusion proteins resulted in elevation of this constitutive GTPase activity without significantly altering the binding affinity of antagonist/inverse agonist ligands. For a 5-HT(1A) receptor-(Cys(351)Ile)G(o1)alpha fusion protein the increase in basal GTPase activity was greater than 4-fold. Enzyme kinetic analysis demonstrated that the effect of RGS1 was as a GTPase-activating protein for the fusion construct. In the presence of the RGS proteins, both agonists and inverse agonists produced much more robust regulation of high-affinity GTPase activity than in their absence. This allowed detection of the partial agonist nature of WAY100635, which has been described previously as a neutral antagonist at the 5-HT(1A) receptor. Of a range of ligands studied, only haloperidol functioned as a neutral ligand in the presence of RGS1. These studies show that addition of a recombinant RGS protein provides a simple and novel means to elevate the fraction of basal membrane GTPase activity contributed by the constitutive activity of a receptor. By so doing, it also greatly enhances the ability to detect and analyze the effects of inverse agonists and to discriminate between neutral ligands and those with low levels of positive intrinsic efficacy.