12 research outputs found
AI is a viable alternative to high throughput screening: a 318-target study
: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery
Anti-inflammatory and anti-oxidant neuroprotection in the prevention of Alzheimer's disease
Current therapy for Alzheimer's disease addresses the symptoms of the disease and results in moderate improvements in cognitive functions. Acetylcholinesterase inhibitors, the main treatment of choice, increase the availability of acetylcholine in the brain, thereby enhancing synaptic transmission and improving cognition. This approach however, does not prevent or delay the onset of the disease. The new class of drug in the treatment of Alzheimer's disease, NMDA-receptor antagonists, do offer some protection of neurons against excitotoxic insult, but are only effective following diagnosis and do not possess anti-inflammatory properties.\ud
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The elucidation of the inflammatory processes responsible for Alzheimer's disease has demonstrated similarities to other inflammation-associated diseases. It is therefore not surprising that therapies used for the treatment of other medical conditions, namely Nonsteroidal anti-inflammatory drugs, statins and antioxidants, may be of benefit in Alzheimer's disease. Closer inspection however, reveals that only selected drugs within these groups appear to provide neuroprotection. The aim of this study was to determine whether this effect translates to the in vitro situation and if so, the causes of the disparity between drugs within the same class.\ud
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A co-culture model of Alzheimer's disease was designed containing human microglia and fluorescent neurons, to allow determination of neuronal viability separately from microglial viability. The establishment of stably-expressing fluorescent neurons for this purpose required significant optimization of transfection and stable selection, resulting in a methods paper [1]. The primary outcome of this article is that although linearization of DNA can increase the production of stable clones, it is dependent on the site of restriction enzyme digestion and requires plasmid-specific optimization.\ud
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Initially, the aim of this study was to assay anti-inflammatory compounds in three distinct culture models of neurodegeneration. The development of these models highlighted important aspects of neurodegeneration in vitro. Transferral of activated microglial media to neurons and activation of microglia when separated from neurons in co-culture, both failed to induce neuron death in the presence of pro-inflammatory mediators Lipopolysaccharide and Interferon-γ. These mediators however, induced significant neuron death when microglia and neurons were co-cultured with direct cell to cell contact. Thus, neuron death in vitro is dependent on microglial proximity and is likely to be due to short-lived toxic factors such as free radicals, as opposed to long-lived cytokines and other inflammatory mediators. Furthermore, inhibitors of Nitric oxide synthase were found to rescue neurons from microglial insult, indicating that the free radical nitric oxide is highly involved in the induction of neurodegeneration.\ud
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The direct co-culture system found to induce neuron death was utilized to assay numerous compounds, from the non-steroidal anti-inflammatory drug, statin and antioxidant classes, for their neuroprotective abilities. In agreement with in vivo studies, it was found that not all compounds within a drug class shared neuroprotective properties. Moreover, the neuroprotection conveyed by ibuprofen, indomethacin and sulindac sulphide in this in vitro system coincides with epidemiological observations that suggest these therapies provide greater protection against the onset of Alzheimer's disease compared to other non-steroidal anti-inflammatory drugs. Selected therapeutics from the statin and nutraceutical antioxidant classes also provided neuroprotection and although the pathways or targets responsible for neuroprotection were not determined, it is clear that the inhibition of Nitric oxide via direct or antioxidant mechanisms plays a role.\ud
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The findings in this study indicate that some currently available anti-inflammatory therapies protect neurons against inflammation-dependent degeneration in vitro. In the clinical setting, this neuroprotective action may translate to a delay in the onset and perhaps progression of Alzheimer's disease. The relatively safe toxicity profiles and ease of access to currently available anti-inflammatory and nutraceutical therapies renders them attractive as interim therapies until more specific therapies for Alzheimer's disease are developed. Furthermore, investigation into the shared targets of these anti-inflammatory therapies that are responsible for neuroprotection may assist in the identification of candidate targets for future drug development
Optimizing the generation of stable neuronal cell lines via pre-transfection restriction enzyme digestion of plasmid DNA
Transfection of mammalian cell lines is a widely used technique that requires significant optimization, including transfection method or product used, DNA vector, cell density, media composition and incubation time. Generation and isolation of stable transfectants from the large pool of untransfected or only transiently transfected cells can be laborious and time-consuming. Transfection of DNA is usually performed with a non-linearized plasmid, since it is assumed that cutting the plasmid beforehand leads to a lower efficiency of transfection or the degradation of linearized DNA by cytosolic nucleases. However, the transfected circular plasmid will be linearized by a random cut within the cell and it might be possible that sensitive parts of the plasmid such as the resistance gene or the gene of interest are destroyed upon linearization. On the other hand, linearizing a plasmid before transfection by a single, defined cut with a selected restriction enzyme in a non-coding area of the gene has the advantage of ensuring the integrity of all necessary gene elements of the plasmid. In this study, we have compared these different methods in order to increase both transient and stable transfection efficiency in mammalian cells. We report that linearization of plasmid DNA prior to transfection can increase both the efficiency of stable clone generation and target gene expression, but is dependant on the site of linearization within the vector
The molecular basis of the prevention of Alzheimer’s disease through healthy nutrition
The Alzheimer’s disease (AD) brain shows numerous pathological phenomena, including amyloid plaques, neurofibrillary tangles, elevated levels of advanced glycation endproducts and their receptor, oxidative damage and inflammation, all of which contribute to neurodegeneration. In this review, we consider these neuropathologies associated with AD and propose that inflammation and oxidative stress play major pathogenic roles throughout disease progression. It is believed that oxidative stress and inflammation not only play major roles early in the disease, but that they act in a reinforcing cycle, amplifying their damaging effects. Therefore, epidemiological studies indicate that anti-inflammatory, antioxidant and neuroprotective agents including those from medicinal plants and health promoting foods may protect against AD, possibly through scavenging of reactive oxygen species, cytokine downregulation and strengthening the neurons antioxidant defense. This concept is further supported by evidence that certain diets (such as a Mediterranean diet) have been associated with a lower incidence of AD. This review highlights specific foods and diets thought to lower the risk of developing AD and discusses the potential of healthy nutrition in disease prevention
Neuroprotective therapies for Alzheimer's disease
One of the major age-related damaging agents are reactive oxygen species (ROS). The brain is more vulnerable to oxidative stress than other organs as concomitant low activity and capacity of antioxidative protection systems allow for increased exposure of target molecules to ROS. Since neurons are postmitotic cells, they have to live with cellular damage accumulated over many decades. Increased levels of ROS (also termed "oxidative stress"), produced by normal mitochondrial activity, inflammation and excess glutamate levels, are proposed to accelerate neurodegenerative processes characteristic of Alzheimer's disease. This review presents evidence of the importance of oxidative stress in the pathogenesis of these diseases and explains the nature of different types of ROS mediating neuronal damage. Furthermore, the potential beneficial effects of neuroprotective treatments, including antioxidants and anti - glutamatergic drugs are discussed
Induction of novel cytokines and chemokines by advanced glycation endproducts determined with a cytometric bead array
Advanced glycation endproducts (AGEs) accumulate on long-lived protein deposits, e.g. those composed of β2-microglobulin (in dialysis-related amyloidosis) or β-amyloid peptide (in Alzheimer's disease). When AGEs bind to the "receptor for advanced glycation endproducts", they activate redox-sensitive transcription factors such as NF-κB, and subsequently induce the expression of pro-inflammatory cytokines such as IL-1, IL-6 and TNF-α. Using a cytokine bead array, we have further analyzed the Bovine Serum Albumin (BSA)-AGE induced expression of selected cytokines/chemokines in two murine cell lines, RAW 264.7 macrophages and N-11 microglia. Our study showed that monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor (TNF-α) were both released in a time-dependent manner from both RAW 264.7 macrophages and N-11 microglia upon stimulation with BSA-AGE or lipopolysaccharide (LPS), which was used as a positive control. Interestingly, MCP-1 was also constitutively expressed by unstimulated cells, although at a lower levels. Much higher levels of IL-6 were secreted by RAW 264.7 macrophages than by N-11 microglia in response to both stimuli. IL-12p70, interferon-γ and the anti-inflammatory cytokine IL-10 were not induced by either LPS or BSA-AGE. Our results indicate a very similar pattern of chemokine and cytokine expression induced by such different ligands as AGEs and LPS indicating similar or convergent downstream signaling pathways
The thiamine analogue and advanced glycation endproducts crosslink breaker ALT-711 does not interfere with transketolase activity
The enzyme transketolase (sedoheptulose-7-phosphate:D-glyceraldehyde-3-phosphate glycolaldehydetransferase, EC 2.2.1.1) is involved in the pentose phosphate pathway (PPP) and catalyses the transfer of a 2-carbon fragment from a 5-carbon keto sugar (xylulose-5-P) to a 5-carbon aldo sugar (ribose-5-P) to form a 7-carbon keto sugar (sedoheptulose- 7-P) and a 3-carbon aldo sugar (glyceraldehyde-3-P). Transketolase requires thiamine pyrophosphate as a co-factor. Advanced glycation endproducts (AGEs) are implicated in the complications of diabetes and aging, primarily via adventitious and crosslinking of tissue proteins. ALT-711 is an AGE crosslink breaker and has been tested as an intervention therapy in established complications of diabetes. It has been noticed that it has a similar structure to that of thiamine and it was hypothesized that it might inhibit transketolase by replacing the active co-factor rendering the enzyme inactive. In this study, we have established a novel microtiter plate format transketolase assay which determines the concentration of NADH by measuring its fluorescence. Using this assay, it was found that ALT-711 does not inhibit the activity of transketolase up to concentration of 5 mM. We conclude that ALT-711 does not interfere with transketolase activity at clinically relevant concentrations
The Thiamine Analogue and Advanced Glycation Endproducts Crosslink Breaker ALT-711 does not Interfere with Transketolase Activity
Methylglyoxal impairs glucose metabolism and leads to energy depletion in neuronal cells—protection by carbonyl scavengers
Advanced glycation end products (AGEs) are found in various intraneuronal protein deposits such as neurofibrillary tangles in Alzheimer's disease and Lewy bodies in Parkinson's disease. Among the many reactive carbonyl compounds and AGE precursors, methylglyoxal is most likely to contribute to intracellular AGE formation, since it is extremely reactive and constantly produced by degradation of triosephosphates. Furthermore, methylglyoxal levels increase under pathophysiological conditions, for example, when trisosephosphate levels are elevated, the expression or activity of glyoxalase I is decreased, as is the case when the concentration of reduced glutathione, the rate-determining co-factor of glyoxalase I, is low. However, the effects of methylglyoxal on mitochondrial function and energy levels have not been studied in detail. In this study, we show that methylglyoxal increases the formation of intracellular reactive oxygen species and lactate in SH-SY5Y neuroblastoma cells. Methylglyoxal also decreases mitochondrial membrane potential and intracellular ATP levels, suggesting that carbonyl stress-induced loss of mitochondrial integrity could contribute to the cytotoxicity of methylglyoxal. The methylglyoxal-induced effects such as ATP depletion and mitochondrial dysfunction can be prevented by pre-incubation of the cells with the carbonyl scavengers aminoguanidine and tenilsetam. In a clinical context, these compounds could not only offer a promising therapeutic strategy to reduce intracellular AGE-accumulation, but also to decrease the dicarbonyl-induced impairment of energy production in aging and neurodegeneration
Lipoic acid as a novel treatment for Alzheimer's disease and related dementias
Alzheimer's disease (AD) is a progressive neurodegenerative disorder that destroys patient memory and cognition, communication ability with the social environment and the ability to carry out daily activities. Despite extensive research into the pathogenesis of AD, a neuroprotective treatment – particularly for the early stages of disease – remains unavailable for clinical use. In this review, we advance the suggestion that lipoic acid (LA) may fulfil this therapeutic need. A naturally occurring precursor of an essential cofactor for mitochondrial enzymes, including pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (KGDH), LA has been shown to have a variety of properties which can interfere with pathogenic principles of AD. For example, LA increases acetylcholine (ACh) production by activation of choline acetyltransferase and increases glucose uptake, thus supplying more acetyl-CoA for the production of ACh. LA chelates redox-active transition metals, thus inhibiting the formation of hydroxyl radicals and also scavenges reactive oxygen species (ROS), thereby increasing the levels of reduced glutathione. Via the same mechanisms, downregulation redox-sensitive inflammatory processes is also achieved. Furthermore, LA can scavenge lipid peroxidation products such as hydroxynonenal and acrolein. The reduced form of LA, dihydrolipoic acid (DHLA), is the active compound responsible for most of these beneficial effects. R-α-LA can be applied instead of DHLA, as it is reduced by mitochondrial lipoamide dehydrogenase, a part of the PDH complex. In this review, the properties of LA are explored with particular emphasis on how this agent, particularly the R-α-enantiomer, may be effective to treat AD and related dementias