Source: https://sussexdrugdiscovery.wordpress.com/2017/03/
Timestamp: 2019-04-23 00:14:54+00:00

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Telomeres are present at the ends of chromosomes and consist of tandem 5’-TTAGGG-3’ repeats. These form structures that maintain genome stability by protecting DNA ends from degradation or fusing with other chromosomes. Replication of chromosomes does not continue to their ends, so the telomere is slightly shortened during each round of replication. In somatic cells, which have no means of lengthening their telomeres, this means the number of times a cell can divide is limited.
For cancer cells to have unlimited replicative potential, the replicative limit placed on cells by telomere attrition, or Hayflick limit, must be overcome. There are two main ways in which this is achieved. Often cells reactivate telomerase. Telomerase is an enzyme active in stem cells and uses an RNA template to synthesise telomeric DNA so reactivation allows telomeres to be regenerated and replication to continue. The second route to overcoming the Hayflick limit is known as Alternative Lengthening of Telomeres, or ALT. This is defined as lengthening mechanisms that do not rely on telomerase (Pickett and Reddel 2015).
Alternative Lengthening of Telomeres is proposed to take place via a mechanism whereby another telomere is used as a DNA template for replication of new telomeric DNA. Telomeric DNA ends are thought to invade a homologous template ( Figure 1, Step 1) and undergo synthesis (Step 2) before the recombination intermediate formed is resolved (Step 3).
Figure 1 from Pickett and Reddel 2015.
This process involves proteins central to DNA replication and recombination and seems to be prevented in normal cells by telomere binding proteins such as POT1 and also by chromatin structure.
Since the ALT process has been estimated to occur in 10-15% of cancers, including some with high mortality rates, it is obviously of therapeutic interest (Cesare and Reddel 2010, Scarpa, Chang et al. 2017). However, the involvement of components of recombination and replication machinery in this process is not completely defined.
A recent paper (Dilley, Verma et al. 2016) has defined the molecular requirements for ALT using an assay that induces damage specifically at telomeres and then observes DNA incorporation into telomeres.
Figure 2 from Dilley, Verma et al. 2016.
Probing for telomeres allows newly synthesised telomeres to be quantified.
This system monitors break induced replication at telomeres. By combining this system with siRNA the group were able to define the requirements for break induced replication at telomeres and determine several replication factors involved in the process. They demonstrated that for this process, ATR and Rad51 were not required, and that replication factor C, proliferating cell nuclear antigen and DNA polymerase δ. Identification of how this process differs from S-phase replication may provide novel targets for therapeutics.
Cesare, A. J. and R. R. Reddel (2010). “Alternative lengthening of telomeres: models, mechanisms and implications.” Nat Rev Genet 11(5): 319-330.
Dilley, R. L., P. Verma, N. W. Cho, H. D. Winters, A. R. Wondisford and R. A. Greenberg (2016). “Break-induced telomere synthesis underlies alternative telomere maintenance.” Nature 539(7627): 54-58.
Pickett, H. A. and R. R. Reddel (2015). “Molecular mechanisms of activity and derepression of alternative lengthening of telomeres.” Nat Struct Mol Biol 22(11): 875-880.
Scarpa, A., D. K. Chang, K. Nones, V. Corbo, A.-M. Patch, P. Bailey, R. T. Lawlor, A. L. Johns, D. K. Miller, A. Mafficini, B. Rusev, M. Scardoni, D. Antonello, S. Barbi, K. O. Sikora, S. Cingarlini, C. Vicentini, S. McKay, M. C. J. Quinn, T. J. C. Bruxner, A. N. Christ, I. Harliwong, S. Idrisoglu, S. McLean, C. Nourse, E. Nourbakhsh, P. J. Wilson, M. J. Anderson, J. L. Fink, F. Newell, N. Waddell, O. Holmes, S. H. Kazakoff, C. Leonard, S. Wood, Q. Xu, S. H. Nagaraj, E. Amato, I. Dalai, S. Bersani, I. Cataldo, A. P. Dei Tos, P. Capelli, M. V. Davì, L. Landoni, A. Malpaga, M. Miotto, V. L. J. Whitehall, B. A. Leggett, J. L. Harris, J. Harris, M. D. Jones, J. Humphris, L. A. Chantrill, V. Chin, A. M. Nagrial, M. Pajic, C. J. Scarlett, A. Pinho, I. Rooman, C. Toon, J. Wu, M. Pinese, M. Cowley, A. Barbour, A. Mawson, E. S. Humphrey, E. K. Colvin, A. Chou, J. A. Lovell, N. B. Jamieson, F. Duthie, M.-C. Gingras, W. E. Fisher, R. A. Dagg, L. M. S. Lau, M. Lee, H. A. Pickett, R. R. Reddel, J. S. Samra, J. G. Kench, N. D. Merrett, K. Epari, N. Q. Nguyen, N. Zeps, M. Falconi, M. Simbolo, G. Butturini, G. Van Buren, S. Partelli, M. Fassan, I. Australian Pancreatic Cancer Genome, K. K. Khanna, A. J. Gill, D. A. Wheeler, R. A. Gibbs, E. A. Musgrove, C. Bassi, G. Tortora, P. Pederzoli, J. V. Pearson, N. Waddell, A. V. Biankin and S. M. Grimmond (2017). “Whole-genome landscape of pancreatic neuroendocrine tumours.” Nature 543(7643): 65-71.
I would like to begin this post on a personal note if I may. The truth is that as scientists involved in research it is sometimes easy to get fixated on the daily challenges of compound synthesis, assays and biological targets, inhibition results, efficacy and physiochemical properties without remembering why we and other research groups undertake the research that is so important. It’s not intentional but easy to sometimes forget the patients and families who need the progression of therapeutics to help them battle disease and improve quality of life. I find it’s nice to take a step back from the bench once in a while and look at the bigger picture.
As with many people, I have been witness to the devastating effects of neurodegenerative disease. The implications not only directly to the patients but on those around them who day to day have to see a loved ones’ health degrade in the knowledge that there is very little they can do.
In the last few years a close friend’s parent was diagnosed with early onset Alzheimer’s disease (AD) and after knowing them for many years it was sad to find that last time we met they didn’t know who I was. Even more heart breaking is that sooner or later the close family will also have the same experience. It is unfortunately inevitable.
Currently treatment focuses on temporary symptomatic relief with Acetylcolinesterase inhibitors and methyl-D-Aspartate receptor agonists rather than targeting the amyloid plaque formation believed root cause of AD.
Recently failures of both Janssen and Pfizers Bapineuzumab and Eli Lilly’s Solanezumab in phase III clinical trials has given a further knockback to AD therapeutics. Both of these drugs were developed to target β-amyloid (Aβ) of which high levels in the brain can promote the formation of amyloid plaques leading to neuronal death. The moderation of Aβ levels should reduce the formation of amyloid plaques and hence the neuronal death in neurodegenerative disease. Unfortunately, these drugs did not prove significantly efficacious in the clinical trials.
In response to the Aβ targeting therapeutics a recent publication from Van-Hai Hoang et al. describes their research targeting Aβ and the employment of rational design to develop potential anti-Alzheimer’s treatments. In the paper they explain the possible ineffective outcomes of Bapineuzumab and Solanezumab were perhaps related to the multi functionality of Aβ peptides and their diverse structural features. They suggest targeting specific Aβ peptides that are neurotoxic and prone to aggregation may prove therapeutically more effective.
The paper goes on to explain that certain N-terminal Aβ peptides are of significant levels in AD patients and these are prone to cyclisation by glutaminyl cyclase (QC) to form Pyroglutamate. These cyclic Aβ peptides are more neurotoxic, rapidly aggregate and are seed for both amyloid and tau plaques. It has been reported that QC is overexpressed in the brains of AD patients and inhibition of QC in animal models reduces the amount of cyclic Aβ peptides and Aβ plaques.
Van-Hai Hoang et al. use a pharmacophoric model based upon the N-terminal of Aβ3E-42 to develop a known QC inhibitor (compound 1) (Fig 1.).
A-region consists of a zinc binding group, in this case a 5-methyl imidazole, which binds to an active zinc ion.
B-region, H-bond donor site which consists of a thiourea motif.
D-region was postulated to require a mimic to the arginine motif.
The group had previously reported the SAR based upon Compound 1 (fig 1) giving an IC50 of 58 nM.  Keeping the A, B and C regions consistent with compound 1 the D-region was investigated via a number of synthesised analogues containing linked nitrogen containing groups. The length of linker and the nitrogen containing arginine mimic group were both varied to provide a large SAR landscape.
The group found that most analogues showed improved in vitro inhibitory activity against human QC compared to that of compound 1. For all compounds synthesised with the added D-region they saw 5-40 fold increases in potency and noted that nitrogen arrangement in this portion was key to the binding.
Compound, 212 (fig 2) a 3-carbon linked amino pyridine gave vast improvement in activity (4.5 nM) and when screened against an isozyme of QC also showed selectivity yielding a 100 fold decrease in IC50. In an acute model mice compound 212 confirmed penetration and efficacy reducing both Aβ and cyclic Aβ concentrations.
To test therapeutic effects long term in vivo efficacy studies of 212 were established in two transgenic AD model mice where not only concentrations of Aβ were reduced but cognitive functions of the mice were also restored.
Molecular modelling studies on the x-ray crystal structure of QC’s active site were also undertaken to try and rationalise the improvement in potency with the addition of the D-region. By docking a selection of their most potent compounds into the active site it was found that all had favourable binding to the first three regions and compound 212 formed strong interactions with its D-region amino pyridine to a key glutamic acid residue.
The publication from Van-Hai Hoang et al. is a great example of utilising rational thought processes and logical steps to re-asses the idea of targeting β-amyloid via inhibition of glutaminyl cyclase. The paper backs up its findings with both successful in vivo and in silico modelling.
Another piece in the puzzle for β-amyloid inhibitors perhaps, but most importantly a step in the right direction for the patients and their families who rely on this research.
Finally, I would like to thank the family of whom I spoke at the beginning of this blog who gave me their complete support to publish this post and increase awareness around Alzheimer’s disease. As researchers in drug discovery, it is this family and those like them who in the end we should remember.
 Hardy, J.; Selkoe, D. J. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 2002, 297, 353−356.
 Tran, P. T.; Hoang, V. H. et al, J.Structure-activity relationship of human glutaminyl cyclase inhibitorshaving an N-(5-methyl-1H-imidazol-1-yl)propyl thiourea template.Bioorg. Med. Chem. 2013, 21, 3821−3830.
Last month Cancer Research UK announced that it had awarded significant grants to several projects under its Grand Challenge Scheme. This scheme was set up by CRUK to fund ‘game changing research’ to try to address some of the leading problems in cancer research.
Challenge 1 – Develop vaccines to prevent non-viral cancers.
Challenge 2 – Eradicate EBV-induced cancers from the world.
Challenge 3 – Discover how unusual patterns of mutation are induced by different cancer-causing events.
Challenge 4 – Distinguish between lethal cancers that need treating, and non-lethal cancers that don’t.
Challenge 5 – Find a way of mapping tumours at the molecular and cellular level.
Challenge 6 – Develop innovative approaches to target the cancer super-controller MYC.
Challenge 7 – Deliver biologically active macromolecules to any and all cells in the body.
The intention was to fund one project, but CRUK were so inspired by the applications that from a total of 56 bids, a total of 4 international teams have received funding of up to £20 million over five years.
Professor Mike Stratton’s team are working on identifying preventable causes of cancer by studying DNA mutational fingerprints from patients. Changes can occur in the DNA due to damage caused by environmental factors such as UV exposure or lifestyle behaviours like smoking and drinking alcohol. These leave a ‘scar’ in the DNA. The causes of some mutational fingerprints have already been identified (as shown in the figure) but there are many more where the causes are currently unknown.
This ambitious project plans to study patient samples from over five continents to attempt to identify causes of more of these fingerprints in the hope that many common cancers may be prevented.
This research could also be extremely important in the longer term for oncology drug discovery. By gaining more information about the causes of DNA damage and how it is repaired, as well as identifying the early mutations that drive tumour growth, new targets for cancer treatments could be identified.
Live Cell Imaging – a cell Biologist’s Dream?
Biological science is renowned for is intrinsic variability that as Biologists we try to account for with controls galore. The cell biologist has the task of getting to know a huge variety of cell lines and primary cultures with their own preferences for nutrients, density and transfection conditions and individual tolerances to stress.
We’re often left guessing at what the cells are up to between splitting or after treatment; what the best time-point is for end-point measurements; when a protein begins to be expressed.
What if we could watch our cells Big Brother style 24-7?
We are trialling the IncuCyte® ZOOM from Essen bioscience through April.
This live-cell analysis system allows continuous monitoring of cells within the incubator. Monitoring can be label-free phase-contrast imaging to assess growth or coupled with fluorescent labels to assess cell death, protein expression or transfection efficiency.
The following paper gives an example of the advantages of live cell imaging for accurately and sensitively quantifying drug-induced cell death by multiplexing staining for lives cells with staining for cells undergoing cell death by apoptosis.
They effectively measure drug-induced apoptosis controlling for differences in cell number between samples due to proliferation effects and dead cell detachment with a rapid, zero-handling method producing data consistent with gold standard methods in the field.
I’m looking forward to spying on my cells during the InCuCyte trial and hopefully rapidly generating some high quality data of my own.
Numerous potentially harmful particles constantly enter our lungs. To guard against this, they are lined with a physical barrier called airway epithelium. In the conducting airways, this pseudostratified layer consists predominantly of mucus secreting goblet cells and ciliated cells. Their joint function is to trap and physically propel these particles out of the lung-a tightly regulated innate defence mechanism known as mucociliary clearance (MCC) 1.
Ciliated cells play a dual role in this process via ciliary beating and ion transport. The former is based on a coordinated wave-like motion of cilia. These are located on the apical membrane of the cell, layered by the periciliary liquid (PCL), which hydrates the airways and enables their smooth movement. Formation of PCL is a result of the water movement from serosa onto the apical surface through the tight junctions and is mediated by ion flow across the epithelium (Fig.1). A defect in this hydration mechanism results in conditions such as asthma, cystic fibrosis and chronic obstructive pulmonary disease1.
Currently, one of the main therapeutic targets is the ion channel TMEM16A, a member of Calcium Activated Chloride Channel family (CaCC). In the lung, upregulation of its Cl– and HCO3– secretion would promote PCL formation and re-establish airway homeostasis2. However, it has been demonstrated that a family of goblet-cell-derived proteins, known as Calcium-activated chloride channel regulators (CLCA), can regulate CaCC-mediated chloride currents. Chloride channel accessory 1 (CLCA1), one of the family members, and TMEM16A were found to be upregulated in response to inflammatory mediators, especially in conditions such as asthma and COPD, where they contribute to excessive mucus production3.
However, a recently published study by Sala-Rabanal et al. (2015) was the first to functionally link the two proteins, and specifically identify CLCA1 as a secreted modifier of TMEM16A. The hypothesis is that this effector protein acts in a paracrine fashion and exerts its effect via stabilising the TMEM16A channel dimer on the cell surface. As a result, it increases its surface expression and potentially elevates calcium dependent chloride currents, which could therefore increase MCC (Fig.2)4.
This makes both the CLCA1 and its site of interaction with TMEM16A, promising, and perhaps optimal, therapeutic targets for chronic obstructive airway diseases. Especially, since very few of the molecular players involved in mucus overproduction, driven by mucous cell metaplasia (MCM), have been identified so far5. Nevertheless, much more information will be required regarding the CLCA1-TMEM16A structure and interaction, within the MCC and MCM pathway.
Hollenhorst, M. I., Richter, K., Fronius, M. (2011). Ion Transport by Pulmonary Epithelia. Journal of Biomedicine and Biotechnology, 2011.
Caputo, A., Caci, E., Ferrera, L., Pedemonte, N., Barsanti, C., Sondo, E., Pfeffer, U., Ravazzolo, R., Zeagara-Moran, O., Galietta, L.J.. (2008). TMEM16A, a membrane protein associated with calcium-dependent chloride channel activity. Science, 590-594.
Alevy, Y.G., Patel, A.C., Romero, A.G., Patel,D.A., Tucker, J., Roswit, W.T., Miller, C.A., Heier, R.F., Byers, D.E., Brett, T.J., Holtzman, M.J. (2012), IL-13–induced airway mucus production is attenuated by MAPK13 inhibition. The Journal of Clinical Investigation, 122 (2); 4555-4568.
Sala-Rabanal, M., Yurtsever, Z., Nichols, C.G., Brett, T.J. (2015). Secreted CLC1 modulates TMEM16A to activate Ca2+ -dependent chloride currents in human cells. eLife.
Brett, T.J. (2015). CLCA1 and TMEM16A: the link towards a potential cure for airway diseases. Expert Review of Respiratory Medicine, 503-506.
Molecular diversity is a crucial feature in bioactive compound libraries. This makes sense as it would expand the chemical space around the studied biological targets or processes and therefore increase the chance to find hit compounds. During the 1990S and early 2000S, combinatorial chemistry was very popular within big pharmas as a privileged method to quickly generate diversity by simultaneously preparing multiple compound libraries; especially using the solid-phase synthesis techniques (i.e., functionalized lanterns and Merrifield resin beads).1 Yet, the major drawback is the lack of structural diversity (i.e., poor scaffold diversity) within the chemical series. Without throwing the baby out with the bathwater, combinatorial chemistry greatly contributes to fuel up many high-throughput screening campaigns and could be useful to assess quickly structure-activity relationships of different compounds having similar backbones. However, how can we efficiently achieve scaffold diversity? How can we navigate simultaneously into different regions of biologically relevant chemical space?
In my opinion, diversity-oriented synthesis (DOS) could be a potential answer to those questions.
The DOS approach considers the efficient and simultaneous synthesis of structurally different compounds with the purpose to probe large portions of the bioactive small molecules space.2 Compared to the target-oriented synthesis where each step is performed sequentially to yield a final product, DOS starts from simple and similar building blocks towards complex and diverse compounds, usually in few steps (Fig. 1). To be fruitful, four parameters have to be considered to create high molecular diversity: i) the building blocks, ii) the stereochemistry, iii) the functional groups and iv) the molecular skeleton, which is the most important criterion.
Figure 1. Synthetic approach in combinatorial synthesis and DOS.
Obviously, DOS heavily depends on reliable, atom-economic and high-yielding reactions and must work on a wide range of susbtrates as well as functional groups. Reactions such as multicomponent reactions (Ugi, Passerini, Petasis), tandem/domino and pericyclic processes as well as ring-closing metathesis (RCM) amongst others are now widely used in DOS. Recently, Nielsen and Schreiber have noticed that several DOS methodologies followed three distinct phases: Build/Couple/Pair (B/C/P).3 The Build part correspond to the synthesis of the starting materials, the Couple part refer to coupling reactions to form linear precursors. Finally, the Pair phase refer to folding reactions that trigger intramolecular pairing between compatible functional groups.
As a good example, Marcaurelle et al. have reported an aldol-based B/C/P strategy for the generation of structurally diverse macrocyclic histone deacetylase (HDAC) inhibitors.4 Using different asymmetric syn– and anti-aldol reactions in the Build phase, four stereoisomers of a Boc-protected g-amino acid were generated. On the other hand, chiral amine partners consisted in both stereoisomers of O-PMB-protected alaninol. Thus, in the Couple phase, eight chiral amides were prepared by coupling the chiral acid and amine starting materials. The resulting amides were then reduced to generate the related secondary amines. The fun part starts in the Pair phase where three different reactions – a nucleophilic aromatic substitution (SNAr), a [3+2] azide-alkyne cycloaddition and a ring-closing metathesis (RCM) – were used to greatly diversify the whole matrix, thus providing a variety of macrocycles of different size (8- to 14-membered rings, Figure 2). Finally, the combinatorial diversification of the scaffolds resulting from the RCM reaction, further yielded a 14 400 macrolactams library. This has led to the discovery of a novel class of HDAC inhibitors.
Figure 2. Aldol-based DOS strategy towards novel macrolactams inhibiting the HDAC enzyme by Marcaurelle et al.
Hence, by pushing the synthetic boundaries always further, DOS could serve as the perfect tool to rapidly interrogate the medicinally relevant chemical space.
(1) Carroll, J. Will Combinatorial Chemistry Keep Its Promise? Biotechnol. Healthc. 2005, 2 (3), 26–32.
(2) Galloway, W. R. J. D.; Isidro-Llobet, A.; Spring, D. R. Diversity-Oriented Synthesis as a Tool for the Discovery of Novel Biologically Active Small Molecules. Nat. Chem. 2010, 1, 80.
(3) Nielsen, T. E.; Schreiber, S. L. Towards the Optimal Screening Collection: A Synthesis Strategy. Angew. Chem. Int. Ed. 2008, 47 (1), 48–56.
(4) Marcaurelle, L. A.; Comer, E.; Dandapani, S.; Duvall, J. R.; Gerard, B.; Kesavan, S.; Lee, M. D.; Liu, H.; Lowe, J. T.; Marie, J.-C.; Mulrooney, C. A.; Pandya, B. A.; Rowley, A.; Ryba, T. D.; Suh, B.-C.; Wei, J.; Young, D. W.; Akella, L. B.; Ross, N. T.; Zhang, Y.-L.; Fass, D. M.; Reis, S. A.; Zhao, W.-N.; Haggarty, S. J.; Palmer, M.; Foley, M. A. An Aldol-Based Build/Couple/Pair Strategy for the Synthesis of Medium- and Large-Sized Rings: Discovery of Macrocyclic Histone Deacetylase Inhibitors. J. Am. Chem. Soc. 2010, 132 (47), 16962–16976.
Translational research is all about finding novel therapeutic targets and seeing if they’re relevant to a disease. One of the most challenging (and possibly nerve-wracking) scenarios for a drug-discovery team can be investigating a ‘promising’ new target – the scientific and commercial benefits can be profound. In some circumstances, standard biological target validation methods such as knock-out mice, antibodies, etc. which could give confidence in the concept might not be possible. In which case the future of the project may well rely on getting chemical validation in an animal.
To a medicinal chemist this sort of programme will look very different to your standard me-too-we-want-a-slice-of-the-pie-type drug discovery project where the expected outcome is an exquisitely drug-like and squeaky clean molecule ready for the clinic (ideally!). The emphasis will be more on quickly finding a ‘tool compound’ that ‘finds and binds’ your new target so you can see if it has the desired effect in an in-vivo disease model. Finding a compound which binds is usually the easier part, but because increasingly more challenging targets [i.e. with fussy active sites] are being explored the search for a tool with great binding AND great pharmacokinetics can be a nightmare [I’m thinking getting Bcl-2 inhibitors into the brain, for instance]. You now have a couple of options – use skill and tenacity (and time and money) to optimise your compound’s PK, or use a pharmacokinetic ‘get out of jail free card’ to help by-pass the most frequently encountered barriers….
The Gut Wall: For a poorly absorbed compound (low solubility/permeability) the gut can be by-passed by simply injecting a compound (obvious!), which probably results in significantly better exposure. If i.v. injection doesn’t provide sustained drug levels however (perhaps due to high clearance), an osmotic pump1 can be implanted subcutaneously. Osmotic pumps consist of a capsule containing drug and osmogens, coated with a semipermeable membrane. As the core absorbs water, hydrostatic pressure pushes the drug solution out at a controllable rate through the delivery ports – hey presto, sustained delivery!
2. The Liver: The hepatic reductase null mouse (HRN) is a transgenic mouse developed by Cancer Research UK, which has the Por gene knocked-out. Since this gene encodes the reductase that regenerates the entire cytochrome p450 system, these mice have no hepatic p450 activity. If first-pass metabolism is the cause of poor PK, then using HRN mice can markedly increase circulating drug levels. [NB: comparison of circulating drug levels in HRN versus wild-type mice can also indicate whether hepatic clearance is your issue and whether efficacy or toxicity is caused by formation of a metabolite]. A comparable chemical cytochrome knock-out can be achieved by pre-administration with the pan-Cyp inhibitor 1-aminobenzotriazole3.
Figure 2. Mean concentration time profiles of (A) docetaxel, (B) midazolam, and (C) theophylline in HRN and WT mice (from Boggs JW et. al. Mol Pharm. 2014 Mar 3;11(3):1062-8).
3. The Blood-Brain Barrier: There are some really exotic methods under investigation to circumvent the blood-brain barrier [including hyperosmotic blood-brain barrier opening and ‘trojan horse’ methods using antibodies or surface-modified liposomes which hijack endothelial cell transcytotic mechanisms]4. However, the P-glycoprotein, an ATP-driven drug efflux transporter is most commonly responsible for restricting access of experimental compounds into the brain5. Again, transgenic and chemical tools are available which can by-pass this obstacle to improve delivery of intrinsically permeable compounds to CNS targets. For example, inhibition of P-glycoprotein by the inhibitor Valspodar increases taxol levels in the brain by ten-fold allowing efficacy against glioblastoma6. However, Elacridar is a more commonly used tool to inactivate P-gp at the intestinal or blood-brain barrier, since it has better PK.
Any one of these tools may just give your compound wings and help you better investigate your novel target.
Osmotic Drug Delivery System as a Part of Modified Release Dosage Form. ISRN Pharm. 2012, 528079. doi: 10.5402/2012/528079.
In vitro and in vivo characterization of CYP inhibition by 1-aminobenzotriazole in rats. Biopharmaceutics & Drug Disposition. 2016, 37, 1099. doi: 10.1002/bdd.2000.
A review of nanocarrier-based CNS delivery systems. Curr Drug Deliv. 2006;3:219–232.
Blood-brain barrier drug targeting: the future of brain drug development. Mol Interv. 2003; 3: 90–105. doi: 10.1124/mi.3.2.90.
Transport of paclitaxel (Taxol) across the blood-brain barrier in vitro and in vivo. J Clin Invest 2002;110:1309–1318. doi: 10.1172/JCI15451.
Normal viability and altered pharmacokinetics in mice lacking mdr1-type (drug-transporting) P-glycoproteins. Proc Natl Acad Sci U S A. 1997, 94(8), 4028-33.
Figure 1. – Schematic representation of the main elements of the ECS.
Some interesting results have been achieved with the synthesis of a novel class of potent and selective MGL inhibitors tested in mice suffering from experimental autoimmune encephalomyelitis (EAE), a rodent demyelinating disease model universally accepted as an animal model of MS.(4) As proposed, in vivo administration of MGL inhibitors reduces the clinical severity of the EAE, induces re-myelinisation of damaged neurons and diminishes neuroinflammation. These encouraging results support the hypothesis of a tight intersection between the ECS and MS, suggesting MGL inhibition as an innovative therapeutic approach for treating MS.
Other recent investigations have addressed the involvement of ECS and ECBs levels in autism.(5) Being this uniquely human, there are only a few validated animal models useful to clarify the effects of ECBs-metabolizing enzymes inhibitors. However, an inherited disorder called Fragile X syndrome (FXS), caused by mutations in the fmr1 protein, produces autistic features in a high percentage of patients affected by this pathology. Thus, fmr1 knockout mice provide a good animal model to identify novel targets for autism. It has been reported that the ablation of fmr1 gene also causes dysfunctions on 2-AG metabolism. Then, stimulation of 2-AG signalling could be a useful treatment for mitigating FXS symptoms, since it is able to restore synaptic activity through type I metabotropic glutamate activation. These evidences highlight once again how important is the role of ECS in neurological disorders, pointing out the usefulness of efficient ECS tone modulators.

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