Therapeutic targets and potential of the novel brain- permeable multifunctional iron chelator-monoamine oxidase inhibitor drug, M-30, for the treatment of Alzheimer's disease

Novel therapeutic approaches for the treatment of neurodegenerative disorders comprise drug candidates designed specifically to act on multiple CNS targets. We have synthesized a multifunctional non-toxic, brain permeable iron chelator drug, M-30, possessing propargyl monoamine oxidase (MAO) inhibitory neuroprotective and iron-chelating moieties, from our prototype iron chelator VK-28. In the present study M-30 was shown to possess a wide range of pharmacological activities, including pro-survival neurorescue effects, induction of neuronal differentiation and regulation of amyloid precursor protein (APP) and beta-amyloid (Abeta) levels. M-30 was found to decrease apoptosis of SH-SY5Y neuroblastoma cells in a neurorescue, serum deprivation model, via reduction of the pro-apoptotic proteins Bad and Bax, and inhibition of the apoptosis-associated phosphorylated H2A.X protein (Ser 139) and caspase 3 activation. In addition, M-30 induced the outgrowth of neurites, triggered cell cycle arrest in G(0)/G(1) phase and enhanced the expression of growth associated protein-43. Furthermore, M-30 markedly reduced the levels of cellular APP and beta-C-terminal fragment (beta-CTF) and the levels of the amyloidogenic Abeta peptide in the medium of SH-SY5Y cells and Chinese hamster ovary cells stably transfected with the APP 'Swedish' mutation. Levels of the non-amyloidogenic soluble APPalpha and alpha-CTF in the medium and cell lysate respectively were coordinately increased. These properties, together with its brain selective MAO inhibitory and propargylamine- dependent neuroprotective effects, suggest that M-30 might serve as an ideal drug for neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases, in which oxidative stress and iron dysregulation have been implicated.     

Novel multifunctional neuroprotective iron chelator-monoamine oxidase inhibitor drugs for neurodegenerative diseases: in vitro studies on antioxidant activity, prevention of lipid peroxide formation and monoamine oxidase inhibition

Iron-dependent oxidative stress, elevated levels of iron and of monoamine oxidase (MAO)-B activity, and depletion of antioxidants in the brain may be major pathogenic factors in Parkinson's disease, Alzheimer's disease and related neurodegenerative diseases. Accordingly, iron chelators, antioxidants and MAO-B inhibitors have shown efficacy in a variety of cellular and animal models of CNS injury. In searching for novel antioxidant iron chelators with potential MAO-B inhibitory activity, a series of new iron chelators has been designed, synthesized and investigated. In this study, the novel chelators were further examined for their activity as antioxidants, MAO-B inhibitors and neuroprotective agents in vitro. Three of the selected chelators (M30, HLA20 and M32) were the most effective in inhibiting iron-dependent lipid peroxidation in rat brain homogenates with IC50 values (12-16 microM), which is comparable with that of desferal, a prototype iron chelator that is not has orally active. Their antioxidant activities were further confirmed using electron paramagnetic resonance spectroscopy. In PC12 cell culture, the three novel chelators at 0.1 microM were able to attenuate cell death induced by serum deprivation and by 6-hydroxydopamine. M30 possessing propargyl, the MAO inhibitory moiety of the anti-Parkinson drug rasagiline, displayed greater neuroprotective potency than that of rasagiline. In addition, in vitro, M30 was a highly potent non-selective MAO-A and MAO-B inhibitor (IC50 < 0.1 microM). However, HLA20 was more selective for MAO-B but had poor MAO inhibition, with an IC50 value of 64.2 microM. The data suggest that M30 and HLA20 might serve as leads in developing drugs with multifunctional activities for the treatment of various neurodegenerative disorders.     

Antioxidative and thrombolytic TMP nitrone for treatment of ischemic stroke

Ischemic stroke results from brain blood vessel blockage by thrombus, and produces neuronal cell damage and death. While thrombolytic therapy with tPA has achieved some success in clinic, the strategy of using neuroprotective agents to treat ischemic stroke has been disappointing thus far. In the present work, we synthesized TBN, a derivative of the clinically useful stroke drug TMP armed with a powerful free radical-scavenging nitrone moiety. TBN retains the thrombolytic activity of the parent TMP and possesses strong antioxidative properties. TBN demonstrates significant activity in the rat MCAo stroke model. The results suggest that design of molecules possessing both thrombolytic and neuroprotective properties may be a novel strategy for effective stroke therapeutics.     

Novel multifunctional neuroprotective iron chelator-monoamine oxidase inhibitor drugs for neurodegenerative diseases. In vivo selective brain monoamine oxidase inhibition and prevention of MPTP-induced striatal dopamine depletion

Several multifunctional iron chelators have been synthesized from hydroxyquinoline pharmacophore of the iron chelator, VK-28, possessing the monoamine oxidase (MAO) and neuroprotective N-propargylamine moiety. They have iron chelating potency similar to desferal. M30 is a potent irreversible rat brain mitochondrial MAO-A and -B inhibitor in vitro (IC50, MAO-A, 0.037 +/- 0.02; MAO-B, 0.057 +/- 0.01). Acute (1-5 mg/kg) and chronic [5-10 mg/kg intraperitoneally (i.p.) or orally (p.o.) once daily for 14 days]in vivo studies have shown M30 to be a potent brain selective (striatum, hippocampus and cerebellum) MAO-A and -B inhibitor. It has little effects on the enzyme activities of the liver and small intestine. Its N-desmethylated derivative, M30A is significantly less active. Acute and chronic treatment with M30 results in increased levels of dopamine (DA), serotonin(5-HT), noradrenaline (NA) and decreases in DOPAC (dihydroxyphenylacetic acid), HVA (homovanillic acid) and 5-HIAA (5-hydroxyindole acetic acid) as determined in striatum and hypothalamus. In the mouse MPTP (N-methy-4-phenyl-1,2,3,6-tetrahydropyridine) model of Parkinson's disease (PD) it attenuates the DA depleting action of the neurotoxin and increases striatal levels of DA, 5-HT and NA, while decreasing their metabolites. As DA is equally well metabolized by MAO-A and -B, it is expected that M30 would have a greater DA neurotransmission potentiation in PD than selective MAO-B inhibitors, for which it is being developed, as MAO-B inhibitors do not alter brain dopamine.     

Site-Activated Chelators Derived from Anti-Parkinson Drug Rasagiline as a Potential Safer and More Effective Approach to the Treatment of Alzheimer’s Disease

chelators can modulate β-amyloid accumulation, protect against tau hyperphosphorylation, and block metal-related oxidative stress, and thereby hold considerable promise as effective anti-AD drugs. At present, a growing interest is focusing on increasing the efficacy and targeting of chelators through drug design. To this end, we have developed a new class of multifunctional prochelators from three FDA- approved drugs rasagiline, rivastigmine, and donepezil or tacrine. HLA20 A was designed by merging the important pharmacophores of rasagiline, rivastigmine, and donepezil into our newly developed multifunctional chelator HLA20. M30D was constructed using the key pharmacophoric moieties from rasagiline, rivastigmine, and tacrine. Experiments showed that both compounds possess potent anti-acetylcholinesterase (AChE) activity in vitro with weak inhibition of butyrylcholinesterase (BuChE), and without significant metal-binding activity. M30D was found also to be a highly potent MAO A inhibitor with moderate inhibition of MAO B in vitro. Both HLA20 and M30D can be activated by inhibition of AChE to release active chelators HLA20 and M30, respectively. HLA20 and M30 have been shown to be able to modulate amyloid precursor protein regulation and beta-amyloid reduction, suppress oxidative stress, and passivate excess metal ions (Fe, Cu, and Zn). Compared with the activated chelator HLA20 or M30, both HLA20A and M30D exhibited lower cytotoxicity in SH-SY5Y neuroblastoma cells, substantiating the prochelator strategy for minimizing toxicity associated with poor targeted chelators.     

Effects of Vinpocetine on mitochondrial function and neuroprotection in primary cortical neurons

Vinpocetine (ethyl apovincaminate), a synthetic derivative of the Vinca minor alkaloid vincamine, is widely used for the treatment of cerebrovascular-related diseases. One of the proposed mechanisms underlying its action is to protect against the cytotoxic effects of glutamate overexposure. Glutamate excitotoxicity leads to the disregulation of mitochondrial function and neuronal metabolism. As Vinpocetine has a binding affinity to the peripheral-type benzodiazepine receptor (PBR) involved in the mitochondrial transition pore complex, we investigated whether neuroprotection can be at least partially due to Vinpocetine’s effects on PBRs.Neuroprotective effects of PK11195 and Ro5-4864, two drugs with selective and high affinity to PBR, were compared to Vinpocetine in glutamate excitotoxicity assays on primary cortical neuronal cultures. Vinpocetine exerted a neuroprotective action in a 1–50 μM concentration range while PK11195 and Ro5-4864 were only slightly neuroprotective, especially in high (>25 μM) concentrations. Combined pretreatment of neuronal cultures with Vinpocetine and PK11195 or Ro5-4864 showed increased neuroprotection in a dose-dependent manner, indicating that the different drugs may have different targets. To test this hypothesis, mitochondrial membrane potential (MMP) of cultured neurons was measured by flow cytometry. 25 μM Vinpocetine reduced the decrease of mitochondrial inner membrane potential induced by glutamate exposure, but Ro5-4864 in itself was found to be more potent to block glutamate-evoked changes in MMP. Combination of Ro5-4864 and Vinpocetine treatment was found to be even more effective.In summary, the present results indicate that the neuroprotective action of vinpocetine in culture can not be explained by its effect on neuronal PBRs alone and that additional drug targets are involved.     

Effects of Vinpocetine on mitochondrial function and neuroprotection in primary cortical neurons

Vinpocetine (ethyl apovincaminate), a synthetic derivative of the Vinca minor alkaloid vincamine, is widely used for the treatment of cerebrovascular-related diseases. One of the proposed mechanisms underlying its action is to protect against the cytotoxic effects of glutamate overexposure. Glutamate excitotoxicity leads to the disregulation of mitochondrial function and neuronal metabolism. As Vinpocetine has a binding affinity to the peripheral-type benzodiazepine receptor (PBR) involved in the mitochondrial transition pore complex, we investigated whether neuroprotection can be at least partially due to Vinpocetine’s effects on PBRs.Neuroprotective effects of PK11195 and Ro5-4864, two drugs with selective and high affinity to PBR, were compared to Vinpocetine in glutamate excitotoxicity assays on primary cortical neuronal cultures. Vinpocetine exerted a neuroprotective action in a 1–50 μM concentration range while PK11195 and Ro5-4864 were only slightly neuroprotective, especially in high (>25 μM) concentrations. Combined pretreatment of neuronal cultures with Vinpocetine and PK11195 or Ro5-4864 showed increased neuroprotection in a dose-dependent manner, indicating that the different drugs may have different targets. To test this hypothesis, mitochondrial membrane potential (MMP) of cultured neurons was measured by flow cytometry. 25 μM Vinpocetine reduced the decrease of mitochondrial inner membrane potential induced by glutamate exposure, but Ro5-4864 in itself was found to be more potent to block glutamate-evoked changes in MMP. Combination of Ro5-4864 and Vinpocetine treatment was found to be even more effective.In summary, the present results indicate that the neuroprotective action of vinpocetine in culture can not be explained by its effect on neuronal PBRs alone and that additional drug targets are involved.     

Levetiracetam protects hippocampal neurons in culture against hypoxia-induced injury

Many experimental studies indicate that some antiepileptic drugs possess neuroprotective properties in varied models of neuronal injury. Levetiracetam is a second-generation antiepileptic drug with a novel mechanism of action. In the present study, we evaluated the putative neuroprotective effect of levetiracetam on primary hippocampal cultures at seven day in vitro. Cell death was induced by incubation of neural cultures in hypoxic conditions over 24 hours. Neuronal injury was assessed by morphometric investigation of death/total ratio of neurons in light microscopy using Trypan blue staining and by evaluation of lactate dehydrogenase (LDH) release in the culture medium. Our results indicate that pre-conditioning of hippocampal cultures with high concentrations of levetiracetam (100 μM and 300 μM) protects neurons against hypoxia-induced death. Two-fold higher number of neurons remained viable as compared to control cultures without drug. Lack of neuroprotective action of the drug on hippocampal neural cultures was observed, when a low concentration (10 μM) of levetiracetam was used.     

New SIRT2 inhibitors: Histidine-based bleomycin spin-off

Bleomycin is considered to exert its antitumor activity via DNA cleavage mediated by activated oxygen generated from the iron complex in its chelator moiety. Spin-offs from this moiety, HPH-1Trt and HPH-2Trt, with anti-cancer activities were recently synthesized. In this paper, we developed inhibitors of nicotinamide adenine dinucleotide-dependent deacetylase isoform 2 of Sirtuin protein (SIRT2), based on HPH-1Trt/HPH-2Trt, and aimed to generate new anti-cancer drugs. HPH-1Trt and HPH-2Trt had in vitro anti-SIRT2 inhibitory activity with 50% inhibitory concentration (IC₅₀) values of 5.5 and 8.8?μM, respectively. A structural portion of HPH-1Trt/HPH-2Trt, a tritylhistidine derivative TH-1, had stronger activity (IC₅₀?=?1.7?μM), and thus, fourteen derivatives of TH-1 were synthesized. Among them, TH-3 had the strongest activity (IC₅₀?=?1.3?μM). Selective binding of TH-3 in the pocket of SIRT2 protein was confirmed with a molecular docking study. Furthermore, TH-3 strongly lowered viability of the breast cancer cell line MCF7 with an IC₅₀ of 0.71?μM. A structure-activity relationship study using cell lines suggested that the mechanism of TH-3 to suppress MCF7 cells involves not only SIRT2 inhibition, but also another function. This compound may be a new candidate anti-cancer drug.     

Modulation by Topiramate of AMPA and Kainate Mediated Calcium Influx in Cultured Cerebral Cortical,Hippocampal and Cerebellar Neurons

The effect of the antiepileptic drug topiramate on Ca2+ uptake through (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionate (AMPA) and kainate (KA) receptors was investigated in different cell culture systems consisting of neurons from the cerebral cortex, hippocampus, and cerebellum. Ca2+ influx was assayed using a fluorescent Ca2+ chelator to monitor changes in the intracellular Ca2+ concentration or cobalt staining to assess the effect of topiramate on Ca2+-permeable AMPA/KA receptors. In all types of neuronal cultures studied, AMPA and KA were found to elicit an influx of Ca2+ in a subset of the neuronal population. Topiramate, at concentrations of 30 and 100 microM, inhibited Ca2+ influx by up to 60%. Modulation of AMPA and KA-evoked Ca2+ influx may contribute to both the antiepileptic and neuroprotective properties of topiramate.     

Cerebral ischemia, cell cycle elements and Cdk5

Stroke is a devastating disorder that significantly contributes to death, disability, and healthcare costs. New therapeutic strategies have been recently focusing on the development of neuroprotective agents that could halt the underlying mechanisms of neuronal death leading to brain damage. Accumulating evidence implicates proteins that are normally involved in the regulation of the cell cycle to neuronal death following ischemic insult, suggesting that these proteins could be suitable targets for stroke therapy. In this brief review, we present in vitro and in vivo arguments linking cell cycle molecules, i.e., cyclins, mitotic cyclin-dependent kinases (Cdks), as well as non-mitotic Cdk5, to ischemic neuronal death. We also report the evaluation of the potential of Cdk inhibitors as neuroprotective strategy for ischemic injury.     

Valproate Administered after Traumatic Brain Injury Provides Neuroprotection and Improves Cognitive Function in Rats

Background

Traumatic brain injury (TBI) initiates a complex series of neurochemical and signaling changes that lead to pathological events including neuronal hyperactivity, excessive glutamate release, inflammation, increased blood-brain barrier (BBB) permeability and cerebral edema, altered gene expression, and neuronal dysfunction. It is believed that a drug combination, or a single drug acting on multiple targets, may be an effective strategy to treat TBI. Valproate, a widely used antiepileptic drug, has a number of targets including GABA transaminase, voltage-gated sodium channels, glycogen synthase kinase (GSK)-3, and histone deacetylases (HDACs), and therefore may attenuate a number of TBI-associated pathologies.

Methodology/Principal Findings

Using a rodent model of TBI, we tested if post-injury administration of valproate can decrease BBB permeability, reduce neural damage and improve cognitive outcome. Dose-response studies revealed that systemic administration of 400 mg/kg (i.p.), but not 15, 30, 60 or 100 mg/kg, increases histone H3 and H4 acetylation, and reduces GSK-3 activity, in the hippocampus. Thirty min post-injury administration of 400 mg/kg valproate improved BBB integrity as indicated by a reduction in Evans Blue dye extravasation. Consistent with its dose response to inhibit GSK-3 and HDACs, valproate at 400 mg/kg, but not 100 mg/kg, reduced TBI-associated hippocampal dendritic damage, lessened cortical contusion volume, and improved motor function and spatial memory. These behavioral improvements were not observed when SAHA (suberoylanilide hydroxamic acid), a selective HDAC inhibitor, was administered.

Conclusion/Significance

Our findings indicate that valproate given soon after TBI can be neuroprotective. As clinically proven interventions that can be used to minimize the damage following TBI are not currently available, the findings from this report support the further testing of valproate as an acute therapeutic strategy.     

A Multidimensional Strategy to Detect Polypharmacological Targets in the Absence of Structural and Sequence Homology

Conventional drug design embraces the “one gene, one drug, one disease” philosophy. Polypharmacology, which focuses on multi-target drugs, has emerged as a new paradigm in drug discovery. The rational design of drugs that act via polypharmacological mechanisms can produce compounds that exhibit increased therapeutic potency and against which resistance is less likely to develop. Additionally, identifying multiple protein targets is also critical for side-effect prediction. One third of potential therapeutic compounds fail in clinical trials or are later removed from the market due to unacceptable side effects often caused by off-target binding. In the current work, we introduce a multidimensional strategy for the identification of secondary targets of known small-molecule inhibitors in the absence of global structural and sequence homology with the primary target protein. To demonstrate the utility of the strategy, we identify several targets of 4,5-dihydroxy-3-(1-naphthyldiazenyl)-2,7-naphthalenedisulfonic acid, a known micromolar inhibitor of Trypanosoma brucei RNA editing ligase 1. As it is capable of identifying potential secondary targets, the strategy described here may play a useful role in future efforts to reduce drug side effects and/or to increase polypharmacology.     

Flux balance analysis of mycolic acid pathway: targets for anti-tubercular drugs

Mycobacterium tuberculosis is the focus of several investigations for design of newer drugs, as tuberculosis remains a major epidemic despite the availability of several drugs and a vaccine. Mycobacteria owe many of their unique qualities to mycolic acids, which are known to be important for their growth, survival, and pathogenicity. Mycolic acid biosynthesis has therefore been the focus of a number of biochemical and genetic studies. It also turns out to be the pathway inhibited by front-line anti-tubercular drugs such as isoniazid and ethionamide. Recent years have seen the emergence of systems-based methodologies that can be used to study microbial metabolism. Here, we seek to apply insights from flux balance analyses of the mycolic acid pathway (MAP) for the identification of anti-tubercular drug targets. We present a comprehensive model of mycolic acid synthesis in the pathogen M. tuberculosis involving 197 metabolites participating in 219 reactions catalysed by 28 proteins. Flux balance analysis (FBA) has been performed on the MAP model, which has provided insights into the metabolic capabilities of the pathway. In silico systematic gene deletions and inhibition of InhA by isoniazid, studied here, provide clues about proteins essential for the pathway and hence lead to a rational identification of possible drug targets. Feasibility studies using sequence analysis of the M. tuberculosis H37Rv and human proteomes indicate that, apart from the known InhA, potential targets for anti-tubercular drug design are AccD3, Fas, FabH, Pks13, DesA1/2, and DesA3. Proteins identified as essential by FBA correlate well with those previously identified experimentally through transposon site hybridisation mutagenesis. This study demonstrates the application of FBA for rational identification of potential anti-tubercular drug targets, which can indeed be a general strategy in drug design. The targets, chosen based on the critical points in the pathway, form a ready shortlist for experimental testing.     

Bisubstrate analogue structure-activity relationships for p300 histone acetyltransferase inhibitors

p300 and CBP are important histone acetyltransferases (HATs) that regulate gene expression and may be anti-cancer drug targets. Based on a previous lead compound, Lys-CoA, we have used solid phase synthesis to generate a series of 11 new analogues and evaluated these compounds as HAT inhibitors. Increased spacing between the CoA moiety and the lysyl moiety generally decreases inhibitory potency. We have found two substituted derivatives that show about 4-fold increased potency compared to the parent compound Lys-CoA. These structure-activity studies allow for a greater understanding of the optimal requirements for potent inhibition of HAT enzymes and pave the way for a novel class of anti-cancer therapeutics.     

Comparative folate metabolism in humans and malaria parasites (part I): pointers for malaria treatment from cancer chemotherapy

New inhibitors are urgently needed to overcome the burgeoning problem of drug resistance in the treatment of Plasmodium falciparum infection. Targeting the folate pathway has proved to be a powerful strategy for drug development against rapidly multiplying systems such as cancer cells and microorganisms. Antifolates have long been used for malaria treatment but, despite their success, much less is known about parasite folate metabolism than about that of the human host. In this article, we focus on folate enzymes used clinically as anticancer drug targets, in addition to those that have potential to be used as drug targets, for which there are inhibitors at various stages of development. We discuss how this information could lead to the identification of new targets in malaria parasites.     

Computational identification of putative common genomic drug and vaccine targets in Mycoplasma genitalium

Mycoplasma genitalium is an obligate intracellular bacterium that is responsible for several sexually transmitted infections, including non-gonococcal urethritis in men and several inflammatory reproductive tract syndromes in women. Here, we applied subtractive genomics and reverse vaccinology approaches for in silico prediction of potential vaccine and drug targets against five strains of M. genitalium. We identified 403 genes shared by all five strains, from which 104 non-host homologous proteins were selected, comprising of 44 exposed/secreted/membrane proteins and 60 cytoplasmic proteins. Based on the essentiality, functionality, and structure-based binding affinity, we finally predicted 19 (14 novel) putative vaccine and 7 (2 novel) candidate drug targets. The docking analysis showed six molecules from the ZINC database as promising drug candidates against the identified targets. Altogether, both vaccine candidates and drug targets identified here may contribute to the future development of therapeutic strategies to control the spread of M. genitalium worldwide.     

Transcriptome network analyses in human coronavirus infections suggest a rational use of immunomodulatory drugs for COVID-19 therapy

The recent outbreak of coronavirus disease 2019 (COVID-19) by SARS-CoV-2 has led to uptodate 24.3 M cases and 0.8 M deaths. It is thus in urgent need to rationalize potential therapeutic targets against the progression of diseases. An effective, feasible way is to use the pre-existing ΔORF6 mutant of SARS-CoV as a surrogate for SARS-CoV-2, since both lack the moiety responsible for interferon antagonistic effects. By analyzing temporal profiles of upregulated genes in ΔORF6-infected Calu-3 cells, we prioritized 55 genes and 238 ligands to reposition currently available medications for COVID-19 therapy. Eight of them are already in clinical trials, including dexamethasone, ritonavir, baricitinib, tofacitinib, naproxen, budesonide, ciclesonide and formoterol. We also pinpointed 16 drug groups from the Anatomical Therapeutic Chemical classification system, with the potential to mitigate symptoms of SARS-CoV-2 infection and thus to be repositioned for COVID-19 therapy.     

天然活性成分作用靶点识别及确证方法的研究进展

药物靶标的发现和验证是新药研发的关键环节,对新药创制具有源头创新意义。天然产物是新药创制的重要来源,识别其作用靶点不仅为临床预防治疗提供可能新策略,也为进一步阐释中草药及其复方的作用特点及分子机制提供参考依据。随着生命科学和信息学的发展,药物靶点的识别及确证方法不断涌现,生物信息学、网络药理学、蛋白质组学、亲和色谱、药物亲和稳定性、芯片技术、基因敲除技术、RNA干扰等技术的广泛应用,越来越多的天然活性成分的靶点得以识别和验证。因此,本文对近五年来天然活性成分作用靶点识别及确证方法做一简要综述,以供参考。