Structural basis of colchicine-site targeting acylhydrazones active against multidrug-resistant acute lymphoblastic leukemia

Tubulin is one of the best validated anti-cancer targets, but most anti-tubulin agents have unfavorable therapeutic indexes. Here, we characterized the tubulin-binding activity, the mechanism of action, and the in vivo anti-leukemia efficacy of three 3,4,5-trimethoxy-N-acylhydrazones. We show that all compounds target the colchicine-binding site of tubulin and that none is a substrate of ABC transporters. The crystal structure of the tubulin-bound N-(1′-naphthyl)-3,4,5-trimethoxybenzohydrazide (12) revealed steric hindrance on the T7 loop movement of β-tubulin, thereby rendering tubulin assembly incompetent. Using dose escalation and short-term repeated dose studies, we further report that this compound class is well tolerated to >100 mg/kg in mice. We finally observed that intraperitoneally administered compound 12 significantly prolonged the overall survival of mice transplanted with both sensitive and multidrug-resistant acute lymphoblastic leukemia (ALL) cells. Taken together, this work describes promising colchicine-site-targeting tubulin inhibitors featuring favorable therapeutic effects against ALL and multidrug-resistant cell2195109CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP305896/2013-0; 301596/2017-414/08247-8; 17/14737-6We thank Ganadería Fernando Díaz for calf brains for tubulin purification. The authors acknowledge networking contribution by the COST Action CM1407 “Challenging organic syntheses inspired by nature - from natural products chemistry to drug discovery.” J.F.D. is a member of the CIB Intramural Program “Molecular Machines for Better Life” (MACBET). N.M.C. was supported by a fellowship from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 14/08247-8, and 17/14737-6). J.A.Y. received a Productivity fellowship from the Brazilian National Counsel of Technological and Scientific Development (CNPq 305896/2013-0 and 301596/2017-4). This work was supported in part by grants BFU2016-75319-R (AEI/FEDER, UE) (J.F.D.) from Ministerio de Economía y Competitividad. The crystal structure work was supported by grants from the Swiss National Science Foundation (31003A_166608, to M.O.S.) and by the COST action CM1407 (to M.O.S.). Part of the in vivo work was supported by R01CA209829 and R01CA213912, Hyundai Hope On Wheels Scholar Grant, Bear Necessities Pediatric Cancer Foundation, Alex’s Lemonade Stand Foundation, the Four Diamonds Fund of the Pennsylvania State University College of Medicine, and the John Wawrynovic Leukemia Research Scholar Endowment (to S.D.

Sleep-Deprivation Regulates α-2 Adrenergic Responses of Rat Hypocretin/Orexin Neurons

We recently demonstrated, in rat brain slices, that the usual excitation by noradrenaline (NA) of hypocretin/orexin (hcrt/orx) neurons was changed to an inhibition following sleep deprivation (SD). Here we describe that in control condition (CC), i.e. following 2 hours of natural sleep in the morning, the α2-adrenergic receptor (α2-AR) agonist, clonidine, had no effect on hcrt/orx neurons, whereas following 2 hours of SD (SDC), it hyperpolarized the neurons by activating G-protein-gated inwardly rectifying potassium (GIRK) channels. Since concentrations of clonidine up to a thousand times (100 µM) higher than those effective in SDC (100 nM), were completely ineffective in CC, a change in the availability of G-proteins is unlikely to explain the difference between the two conditions. To test whether the absence of effect of clonidine in CC could be due to a down-regulation of GIRK channels, we applied baclofen, a GABAB agonist known to also activate GIRK channels, and found that it hyperpolarized hcrt/orx neurons in that condition. Moreover, baclofen occluded the response to clonidine in SDC, indicating that absence of effect of clonidine in CC could not be attributed to down-regulation of GIRK channels. We finally tested whether α2-ARs were still available at the membrane in CC and found that clonidine could reduce calcium currents, indicating that α2-ARs associated with calcium channels remain available in that condition. Taken together, these results suggest that a pool of α2-ARs associated with GIRK channels is normally down-regulated (or desensitized) in hcrt/orx neurons to only become available for their inhibition following sleep deprivation

Crystal structures of taxane-tubulin complexes: Implications for the mechanism of microtubule stabilization by Taxol

30 p.-9 fig.-1 tab.Paclitaxel (Taxol®) is a first-line chemotherapeutic drug that promotes the curved-to-straight conformational transition of tubulin, an activation step that is necessary for microtubule formation. Crystallization of Taxol bound to tubulin has been long elusive. We found that baccatin III, the core structure of paclitaxel which lacks the C13 side chain, readily co-crystallizes with curved tubulin. Tailor-made taxanes with alternative side chains also co-crystallized, allowing us to investigate their binding modes. Interestingly, these Taxol derived compounds lost their microtubule stabilizing activity and cytotoxicity but kept their full microtubule binding affinity, and all induced lattice expansion upon binding. Additional nuclear magnetic resonance studies propose that Taxol binds to a small fraction of straight tubulin present in solution. Our results suggest a mode of action of Taxol, where the core structure is responsible for the interacting energy while the bulky hydrophobic C13 side chain enables binding selectively to straight tubulin and promotes stabilization.N

Pharmacological characterization and differentiation of non-cholinergic nucleus basalis neurons in vitro

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Effects of Hypocretin/Orexin and Major Transmitters of Arousal on Fast Spiking Neurons in Mouse Cortical Layer 6B

Fast spiking (FS) GABAergic neurons are thought to be involved in the generation of high-frequency cortical rhythms during the waking state. We previously showed that cortical layer 6b (L6b) was a specific target for the wake-promoting transmitter, hypocretin/orexin (hcrt/orx). Here, we have investigated whether L6b FS cells were sensitive to hcrt/orx and other transmitters associated with cortical activation. Recordings were thus made from L6b FS cells in either wild-type mice or in transgenic mice in which GFP-positive GABAergic cells are parvalbumin positive. Whereas in a control condition hcrt/orx induced a strong increase in the frequency, but not amplitude, of spontaneous synaptic currents, in the presence of TTX, it had no effect at all on miniature synaptic currents. Hcrt/orx effect was thus presynaptic although not by an action on glutamatergic terminals but rather on neighboring cells. In contrast, noradrenaline and acetylcholine depolarized and excited these cells through a direct postsynaptic action. Neurotensin, which is colocalized in hcrt/orx neurons, also depolarized and excited these cells but the effect was indirect. Morphologically, these cells exhibited basket-like features. These results suggest that hcrt/orx, noradrenaline, acetylcholine, and neurotensin could contribute to high-frequency cortical activity through an action on L6b GABAergic FS cells

Cholinergic nucleus basalis neurons are excited by histamine in vitro

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Suprachiasmatic modulation of noradrenaline release in the ventrolateral preoptic nucleus.

As the major brain circadian pacemaker, the suprachiasmatic nucleus (SCN) is known to influence the timing of sleep and waking. We thus investigated here the effect of SCN stimulation on neurons of the ventrolateral preoptic nucleus (VLPO) thought to be involved in promoting sleep. Using an acute in vitro preparation of the rat anterior hypothalamus/preoptic area, we found that whereas single-pulse stimulations of the SCN evoked standard fast ionotropic IPSPs and EPSPs, train stimulations unexpectedly evoked a long-lasting inhibition (LLI). Such LLIs could also be evoked in VLPO neurons by pressure application of NMDA within the SCN, indicating the specific activation of SCN neurons. This LLI was shown to result from the presynaptic facilitation of noradrenaline release, because it was suppressed in presence of yohimbine, a selective antagonist of alpha2-adrenoreceptors. The LLI depended on the opening of a potassium conductance, because it was annulled at E(K) and could be reversed below E(K). These results show that the SCN can provide an LLI of the sleep-promoting VLPO neurons that could play a role in the circadian organization of the sleep-waking cycle

Rat Hypocretin/Orexin Neurons Are Maintained in a Depolarized State by TRPC Channels

In a previous study we proposed that the depolarized state of the wake-promoting hypocretin/orexin (hcrt/orx) neurons was independent of synaptic inputs as it persisted in tetrodotoxin and low calcium/high magnesium solutions. Here we show first that these cells are hyperpolarized when external sodium is lowered, suggesting that non-selective cation channels (NSCCs) could be involved. As canonical transient receptor channels (TRPCs) are known to form NSCCs, we looked for TRPCs subunits using single-cell RT-PCR and found that TRPC6 mRNA was detectable in a small minority, TRPC1, TRPC3 and TRPC7 in a majority and TRPC4 and 5 in the vast majority (,90%) of hcrt/orx neurons. Using intracellular applications of TRPC antibodies against subunits known to form NSCCs, we then found that only TRPC5 antibodies elicited an outward current, together with hyperpolarization and inhibition of the cells. These effects were blocked by co-application of a TRPC5 antigen peptide. Voltage-clamp ramps in the presence or absence of TRPC5 antibodies indicated the presence of a current with a reversal potential close to 215 mV. Application of the non-selective TRPC channel blocker, flufenamic acid, had a similar effect, which could be occluded in cells pre-loaded with TRPC5 antibodies. Finally, using the same TRPC5 antibodies we found that most hcrt/orx cells show immunostaining for the TRPC5 subunit. These results suggest that hcrt/orx neurons are endowed with a constitutively active non-selective cation current which depends on TRPC channels containing th