A newly designed molecule J2326 for Alzheimer's disease disaggregates amyloid fibrils and induces neurite outgrowth.

Alzheimer's disease is a neurodegenerative disorder characterized by deposition of ß-amyloid (Aß) fibrils accompanied with progressive neurite loss. None of the clinically approved anti-Alzheimer's agents target both pathological processes. We hypothesized that conjugation of a metal chelator to destabilize Aß fibrils (fAßs) and a long-chain fatty alcohol to induce neurite outgrowth may generate a novel molecular scaffold that targets both pathologies. The hydroxyalkylquinoline J2326 was designed and synthesized by joining an 11-carbon alcohol to 5-chloro-8-methoxyquinoline at the 2-position and its anti-neurodegenerative potentials in vitro and in vivo were characterized. It attenuated fAß formation and disaggregated the existing fAß zinc-dependently as well as zinc-independently. It also triggered extracellular signal-regulated kinase-dependent neurite outgrowth and increased synaptic activity in neuronal cells. In fAß-driven neurodegeneration in vitro, J2326 reversed neurite collapse and neurotoxicity. These roles of J2326 were also demonstrated in vivo and were pivotal to the observed improvement in memory of mice with hippocampal fAß lesions. These results show that the effectiveness of J2326 on fAß-driven neurodegeneration is ascribed to its novel scaffold. This might give clues to evolving attractive therapy for future clinical trials.

Novel acylureidoindolin-2-one derivatives as dual Aurora B/FLT3 inhibitors for the treatment of acute myeloid leukemia.

A series of 6-acylureido derivatives containing a 3-(pyrrol-2-ylmethylidene)indolin-2-one scaffold were synthesized as potential dual Aurora B/FLT3 inhibitors by replacing the 6-arylureido moiety in 6-arylureidoindolin-2-one-based multi-kinase inhibitors. (Z)-N-(2-(pyrrolidin-1-yl)ethyl)-5-((6-(3-(2-fluoro-4-methoxybenzoyl)ureido)-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide (54) was identified as a dual Aurora B/FLT3 inhibitor (IC50 = 0.4 nM and 0.5 nM, respectively). Compound 54 also exhibited potent cytotoxicity with single-digit nanomolar IC50 values against the FLT3 mutant-associated human acute myeloid leukemia (AML) cell lines MV4-11 (FLT3-ITD) and MOLM-13 (FLT3-ITD). Compound 54 also specifically induced extrinsic apoptosis by inhibiting the phosphorylation of the Aurora B and FLT3 pathways in MOLM-13 cells. Compound 54 had a moderate pharmacokinetic profile. The mesylate salt of 54 efficiently inhibited tumor growth and reduced the mortality of BALB/c nude mice (subcutaneous xenograft model) that had been implanted with AML MOLM-13 cells. Compound 54 is more potent than sunitinib not only against FLT3-WT AML cells but also active against sunitinib-resistant FLT3-ITD AML cells. This study demonstrates the significance of dual Aurora B/FLT3 inhibitors for the development of potential agents to treat AML.

Bioisosteric replacement of an acylureido moiety attached to an indolin-2-one scaffold with a malonamido or a 2/4-pyridinoylamido moiety produces a selectively potent Aurora-B inhibitor.

Bioisosteric replacement of acylureido moiety in 6-acylureido-3-pyrrolylmethylidene-2-oxoindoline derivatives resulted in a series of malonamido derivatives with indolin-2-one scaffold (11-14). Further conformational restrictions of the malonamido moiety led to 2-oxo-1,2-dihydropyridine (21-25) or a 4-oxo-1,4-dihydropyridine derivatives (31-36). 4-Oxo-1,4-dihydropyridine derivatives were more potent Aurora B inhibitors than their 2-oxo-1,2-dihydropyridine counterparts and demonstrated cytotoxicities against A549 and HepG2 cells in the submicromolar range. In A549 cells, 31h decreased phosphorylation of histone H3, triggered polyploidy, induced expression of pro-apoptotic Fas and FasL with subsequent activation of caspase 8, resulting into apoptosis. In a Huh7-xenograft mouse model, 31h demonstrated potent in vivo efficacy with a daily dose of 5 mg/kg.

Quinazolin-4-one derivatives as selective histone deacetylase-6 inhibitors for the treatment of Alzheimer's disease.

Novel quinazolin-4-one derivatives containing a hydroxamic acid moiety were designed and synthesized. All compounds were subjected to histone deacetylase (HDAC) enzymatic assays to identify selective HDAC6 inhibitors with nanomolar IC50 values. (E)-3-(2-Ethyl-7-fluoro-4-oxo-3-phenethyl-3,4-dihydroquinazolin-6-yl)-N-hydroxyacrylamide, 4b, is the most potent HDAC6 inhibitor (IC50, 8 nM). In vitro, these compounds induced neurite outgrowth accompanied by growth-associated protein 43 expression, and they enhanced the synaptic activities of PC12 and SH-SY5Y neuronal cells without producing toxic or mitogenic effects. Several of the compounds dramatically increased nonhistone protein acetylation, specifically of α-tubulin. Some of the more potent HDAC6 inhibitors decreased zinc-mediated ß-amyloid aggregation in vitro. N-Hydroxy-3-(2-methyl-4-oxo-3-phenethyl-3,4-dihydro-quinazolin-7-yl)-acrylamide, 3f, the most promising drug candidate, selectively inhibits HDAC6 (IC50, 29 nM), practically does not affect human ether-a-go-go-related membrane channel activity (IC50 >10 µM) or cytochrome P450 activity (IC50 >6.5 µM) in vitro, and significantly improves learning-based performances of mice with ß-amyloid-induced hippocampal lesions.

Discovery of N-arylalkyl-3-hydroxy-4-oxo-3,4-dihydroquinazolin-2-carboxamide derivatives as HCV NS5B polymerase inhibitors.

The metal ion chelating ß-N-hydroxy-γ-ketocarboxamide pharmacophore was integrated into a quinazolinone scaffold, leading to N-arylalkyl-3-hydroxy-4-oxo-3,4-dihydroquinazolin-2-carboxamide derivatives as hepatitis C virus (HCV) NS5B polymerase inhibitors. Lead optimization led to the identification of N-phenylpropyl carboxamide 9 k (IC(50) =8.8 µM). Compound 9 k possesses selectivity toward HCV1b replicon Ava.5 cells (EC(50) =17.5 µM) over parent Huh-7 cells (CC(50) =187.5 µM). Compound 9 k effects a mixed mode of NS5B inhibition, with NTP-competitive displacement properties. The interaction between 9 k and NS5B is stabilized by the presence of magnesium ions. Docking studies showed that the binding orientation of 9 k occupies the central portions of both magnesium-mediated and NTP-ribose-response binding sites within the active site region of NS5B. As a result, 3-hydroxy-4-oxo-3,4-dihydroquinazolin-2-carboxamide derivatives are disclosed herein as novel, mainly active site inhibitors of HCV NS5B polymerase.

Design, synthesis and biological evaluation of benzo[1.3.2]dithiazolium ylide 1,1-dioxide derivatives as potential dual cyclooxygenase-2/5-lipoxygenase inhibitors.

3-(4-Bromophenyl)-6-nitrobenzo[1.3.2]dithiazolium ylide 1,1-dioxide (5) was discovered as a new prototype for dual inhibitors of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX). Thus, the structure-activity relationships of benzo[1.3.2]dithiazolium ylide 1,1-dioxide skeleton were carried out. The 6-NO(2) group played an essential role in the inhibitory activity. In addition, moderate-sized lipophilic substituents at the para-position of the 3-aryl moiety were required for dual COX-2/5-LOX inhibitory activity. Among the identified potent dual inhibitors, 3-(4-tbutylphenyl) derivative 30c (IC(50) values of 0.27 µM and 0.30 µM against COX-2 and 5-LOX, respectively) and 3-(4-biphenyl) derivative 30f (IC(50) values of 0.50 µM and 0.15µM against COX-2 and 5-LOX, respectively) were the most potent dual COX-2/5-LOX inhibitors. Intraperitoneal administration of 30c at 100mg/kg demonstrated potent acute anti-inflammatory activity. As a result, benzo[1.3.2]dithiazolium ylide 1,1-dioxide represented a novel scaffold for the exploitation in developing dual COX-2/5-LOX inhibitors.

Adeno-associated virus-mediated human acidic fibroblast growth factor expression promotes functional recovery of spinal cord-contused rats.

BACKGROUND: Following spinal cord injury, the delivery of neurotrophic factors to the injured spinal cord has been shown to promote axonal regeneration and functional recovery. In previous studies, we showed that acidic fibroblast growth factor (aFGF) is a potent neurotrophic factor that promotes the regeneration of axotomized spinal cord or dorsal root ganglion neurones. METHODS: We constructed a recombinant adeno-associated virus (AAV) vector to express human aFGF and evaluated aFGF expression and function in AAV-aFGF-infected PC12 cells. We analyzed AAV-green fluorescent protein (GFP) tropism and AAV-mediated aFGF expression in contused spinal cords. Animals received behavioural testing to evaluate the functional recovery. RESULTS: Overexpression of aFGF was shown in AAV-aFGF-infected PC12 cells in a dose-dependent manner. Concurrently, neurite extension and cell number were significantly increased in AAV-aFGF infected cells. AAV-mediated GFP expression persisted for at least 5 weeks in contused spinal cords, and the most prominently transduced cells were neurones. Contusive injury reduced endogenous aFGF expression in spinal cords. Overexpression of aFGF was demonstrated in AAV-aFGF transduced spinal cords compared to AAV-GFP transduced spinal cords at 3 and 14 days post-injury. Evaluation of motor function revealed that the improvement of AAV-aFGF-treated rats was prominent. Both AAV-aFGF- and recombinant human aFGF-treated rats revealed significantly better recovery at 5 weeks post-injury, compared to vehicle- and AAV-GFP-treated rats. CONCLUSIONS: These data suggest that supplement of aFGF improve the functional recovery of spinal cord-contused rats and that AAV-aFGF-mediated gene transfer could be a clinically feasible therapeutic approach for patients after nervous system injuries.

An improved screening model to identify inhibitors targeting zinc-enhanced amyloid aggregation.

Zinc, which is abundant in senile plaques consisting mainly of fibrillar beta-amyloid (Abeta), plays a critical role in the pathogenesis of Alzheimer's disease. Treatment with zinc chelators such as clioquinol has been used to prevent Abeta aggregation in Alzheimer's patients; however, clioquinol produces severe side effects. A simple, easy, inexpensive, and versatile screen to identify zinc chelators for inhibition of Abeta aggregation is currently unavailable. We thus developed a high-throughput screen that identifies zinc chelators with anti-Abeta aggregation activity. The recombinant Abeta peptides, aggregated on solid-phase microplates, formed Abeta-immunopositive beta-sheet-containing structures in the presence of zinc. Formation of these Abeta fibrils was specifically blocked by metal ion chelators. This screening model improves identification of zinc-enhanced Abeta fibrils and anti-Abeta aggregation mediated by zinc chelating. The convenient system could qualitatively and quantitatively assay a large sample pool for Abeta aggregation inhibition and dissolution of Abeta aggregates. This screen is practical, reliable, and versatile for comprehensive detection of amyloid fibrillation and identification of inhibitors of Abeta aggregation.

Sensory and motor recovery after repairing transected cervical roots.

BACKGROUND: Adult mammal sensory axons avulsed through spinal dorsal root traction injuries, especially of the brachial plexus or cauda equina, cannot normally regenerate through axonal outgrowth from the DRG into the spinal cord, thus causing clinical conditions that require neuronal regeneration for sensory recovery and for which no successful treatment has yet been reported. METHODS: To evaluate the sensory recovery of the forelimb after transection of their left cervical dorsal and ventral roots (C6-C8) at their spinal cord junctions, 22 SD rats were randomly assigned to 3 groups: transection only (control 1); transection followed by repair using intercostal nerve grafts and fibrin glue (control 2); transection, repair, and application of aFGF and fibrin glue (experimental group). The following tests were reperformed after retransecting the repaired nerve roots to discount collateral innervation from adjacent nerve roots: motor function (grasping power), mechanical sensitivity to pain and touch (foot-withdrawal response to mechanical stimuli), temperature sensitivity (foot-withdrawal response to cold stimulus), and electrophysiologic sensory responses (measurement of cortical SEP). RESULTS: After transection and repair, the experimental group rats showed recovery in both motor (grasping power) and sensory (touch, pain, and temperature sensation) nerve functions. Neuronal regeneration was confirmed by the reappearance of cortical SEP and by its disappearance after retransection of the repaired cervical nerve roots. CONCLUSION: Using our strategy for repairing transected cervical nerve roots, motor and sensory recovery was achieved in adult rats. The success of our study highlights possible treatment options for humans with avulsion injuries of the dorsal roots from the spinal cord.

Laminin-incorporated nerve conduits made by plasma treatment for repairing spinal cord injury.

To better direct the repair of damaged axons following spinal cord injury (SCI), we designed a nerve conduit (NC) modeled after the intact spinal cord, which would enable the axons to cross the lesioned area to rejoin on the other side. The NC consisted of a porous chitosan scaffold and was incorporated with laminin (LN) on the inner surface through oxygen plasma treatment. According to the BBB, CBS, and treadmill analyses, we found that following the implantation of the laminin-coated NC (LN-NC) the rats showed a tendency towards behavior improvement and functional recovery. Histology and immunocytochemical analyses indicated that the NC groups were capable of leading the damaged axons through the lesioned area without triggering inflammation or apoptosis. Together with the significantly enhanced expression of local GAP-43 in the LN-NC groups, as evidenced by western blot analysis, axon re-growth mediated by LN-NC was found to compare better than that by NC group. These results suggest a new possible approach to repairing SCI and, in general, a model which will be useful for other multidisciplinary procedures for complex neurological situations.

Effect of enhanced prostacyclin synthesis by adenovirus-mediated transfer on lipopolysaccharide stimulation in neuron-glia cultures.

Prostacyclin (PGI2) is known as a short-lived, potent vasodilator and platelet anti-aggregatory eicosanoid. This work attempts to selectively augment PGI2 synthesis in neuron-glia cultures by adenoviral (Ad) gene transfer of PGI synthase (PGIS) or bicistronic cyclooxygenase 1 (COX-1)/PGIS and examines whether PGI2 confers protection against lipopolysaccharide (LPS) stimulation. Cultures released low levels of eicosanoids. Upon Ad-PGIS or Ad-COX-1/PGIS infection, cultures selectively increased prostacyclin release. Both PGIS- and COX-1/PGIS-overexpressed cultures contained fewer microglial numbers. Further, they significantly attenuated LPS-induced iNOS expression and lactate, nitric oxide, and TNF-alpha production. Taken together, enhanced prostacyclin synthesis in neuron-glial cultures reduced microglia number and suppressed LPS stimulation.