Epigenetics: The sins of the father


In the recent paper in Nature (2014, vol 507, p. 22-24), Virginia Hughes reports the experiments carried out by Dr Dias and Dr Ressler from the University of Atlanta in recent years. They have studied the involvement in inheritance imprints in mice as a result of a fear-based reaction associated to acetophenone. As a result, they found a larger than normal expression of M71 glomeruli receptors in their offspring’s noses. These receptors are encoded by a single gene, known as Olfr151.

This elegant, but still inconclusive cause-effect mechanism approach, brings a possible explanation to a controversial observation back to the 19th century when French biologist Jean-Baptiste Lamark pointed out the pass of acquired traits to future generations. Since then, scientists have observed this phenomenon in plants, animals and even humans.

Although some scientists are still sceptical about the transmitance method, nobody denies the phenomenon. Finding an explanation to this complex problem would involve a deeper study on reproductive biology and to study both mother and father lines over few generations.

The strong suggestion that this heriditary transmission of environmental factors is due to epigenetics, a concept introduced in the 2000’s, where there are some changes in the way that DNA is packed and expressed without altering its sequence, is one of the strong lines of thought, where chemical tags (methylation) on DNA can turn genes on and off.

But even if epigenetics is directly involved in the inheritance, through marks on the material contained in the sperm, the first question to be addressed would be to understand how the effects of environmental/ health legacy get embedded into the animal’s germ cells.

Epigenetics is still unable to explain how this observed phenomenon gets passed down through multiple generations, surviving several rounds of genetic re-programing. Other suggested agents might involve histones (proteins which has been observed that they can be passed down through generations) or short RNA molecules which role would be to latch on DNA and affect further into gene expression.

Scientists are optimistic about finding a cause-effect relationship in the years to come for a phenomenon which has proved elusive for researchers in the past hundred years.

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Predicting cancer targets modulated by Ayurvedic medicines


The recent availability of databases that provide both phenotypic descriptions and the chemical structures of the constituent compounds in traditional Chinese and Indian medicines, have enabled Bender et al  (J. Chem. Inf. Model. 2013 (53) 661 – 673, DOI: 10.1021/ci3005513 , http://www.andreasbender.de/) to develop a cool algorithm to predict the mode of action (MOA) of these compounds and to predict novel protein targets for cancer therapies.

Traditional medicine has been utilised by human for thousands of years and normally viewed as complementary or alternative to mainstream therapies.  However, both Chinese and traditional Indian medicine (Ayervedic) have provided us with important drugs for instance Artemisinin an antimalarial drug and reserpine an antihypertensive drug.

From 1981 to 2007, 67% of the pharmaceuticals or new molecular entities (NMEs) introduced into the market were natural product based or a derivative there of.  These natural products often have desirable properties which make them good drugs; they are soluble despite breaking Lipinski’s Rule of Five, they embody privileged structures that are more frequently found to bind a variety of proteins in different organisms, and they are safe and well-tolerated, often having been commonly used for centuries.

However, there are major challenges that need to be resolved that enable the development of a new drug from a traditional medicine.  These include the isolation of the active constituents, the synthesis of the active constituents, the elucidation of the mode of action and finally the development of the compound as a “drug”.

The recent availability of databases that provide chemical structures and their corresponding phenotypes have enabled Bender et al to predict the MOA of compounds found in TCM and Ayurveda addressing one of these major challenges.  First they developed a classifier using bioactive compounds from the ChEMBL database, ChEMBL biological targets, ECFP_4 fingerprints for each compound and a Naïve Bayes classifier.

FP4

Figure 1: The compounds were represented using the Extended Connectivity Fingerprints, with a diameter of 4 bonds ECFP_4.  The ECPF is derived from the Morgan algorithm and was implemented in Sitegic’s Pipeline Pilot (Accelrys Inc).  Each atom identifier contains topological information on the atom that includes the number of immediate heavy atoms, the atom’s mass, its charge, the number of hydrogens attached, the valance minus the number of hydrogens and whether it is part of a ring.

This was used to predict which compound (fingerprint) would inhibit each protein target.  Then by creating fingerprints for each traditional medicine compound they could predict which protein targets they would hit.  For example they predicted the protein targets for some of the active ingredients of Panaz ginseng

FP5Figure 2:  Predicted targets for some of the active ingredients in Panax Ginseng

Next they correlated different proteins targets with different phenotypes. Predicting which molecular targets were modulated by the compounds in each different phenotype.  This enabled them to identify the protein targets most frequently modulated by Ayurvedic medicines, with possible anti-tumour effects.  The 10 most enriched protein targets are shown in the table below. The progesterone receptor currently has over 10 inhibitors with FDA approval.  Other proteins identified by this methods include regulators of other well-known cancer targets.

FP6

Figure 3:  Top 10 cancer targets in predicted to be inhibited by Ayurvedic medicines.

Promising New Frontiers for RNAi Therapy


In 2001 Elbashir and Tuschl (1) published they had managed to silence gene expression in mammalian cells using small interfering RNA (siRNA). This was the catalyst for an explosion of research using siRNA to demonstrate the effect of knocking out targets without the need to identify compounds capable of doing this job. Potentially highly specific, the opportunity to use these as therapeutics was rapidly explored. Ten years later the first clinical trials are coming through; impressively fast.

Probably the largest obstacle to therapeutic siRNA has been delivery, which often is either toxic, or not very effective – as any of us who have tried to transfect siRNA into primary cells will be able to appreciate. Advances in materials science are seemingly solving this problem encapsulating siRNA in nanoparticles, resulting in safe and effective delivery. CALAA-01 (Calando Pharmaceuticals) lead the way  being the first to deliver siRNA therapeutically demonstrating phase I efficacy and safety, as well as localisation to melanoma metastases.

One particular advantage of siRNA is that once delivery has been optimised, it is possible for several different siRNAs for different targets to be contained within on package. This could enable simultaneous delivery of different targets simultaneously effecting different aspects of the same disease, i.e. metastasis and well as tumour growth. By also hitting a known resistance pathway this duel delivery could enable the chemotherapy to be more effective.

A recently published article (2) has done just this, delivering two siRNAs in lipid nanoparticles (known as ALN-VSP) for both VEGF-A (vascular endothelial growth factor-A) and kinesin spindle protein (KSP) for the treatment of advanced solid tumours with liver metastases.  KSP is involved in cancer proliferation and VEGF-A in the growth of new blood vessels.

The study was phase I, dose escalation, on patients who had already been heavily pre-treated with chemotherapy and/or anti VEGF/VEGFR agents with the ALN-VSP administered as 15 minute IV every 2 weeks for 1 month.

The safety profile was very encouraging with ALN-VSP being generally well-tolerated with mainly low-grade fatigue, nausea and fever noted in 15-24% of patients. The lipid nanoparticle of ALN-VSP distributes primarily to the liver and spleen and the delivery was also excellent. Liver biopsies were performed on 12 patients before the first dose and then at 2 and 7 days post dose. qPCR identified VEGF siRNA present in all 12 patients and KSP siRNA present in 11 of the 12.

Of the patients treated, 7 had no disease progression (measured by computerized tomography [CT] scan) after the treatment cycles and continued onto an extension study. One patient in particular with endometrial cancer achieved a complete response after 20 months of treatment

This is clearly fantastic progress for ALN-VSP, and specifically for the handful of patients who were positively affected from participation in the trial. The results from this study also demonstrate the ability for safe delivery of multiple siRNA to specific sites tumour and this extends the promising start for these methods of siRNA delivery which may open up previously un-druggable targets.

 

1.           Elbashir SM, Harborth J, Lendeckel W, Yalcin a, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411: 494–8, 2001.

2.           Tabernero J, Shapiro GI, Lorusso PM, Cervantes A, Schwartz GK, Weiss GJ, Paz-Ares L, Cho DC, Infante JR, Alsina M, Gounder MM, Falzone R, Harrop J, Seila White AC, Toudjarska I, Bumcrot D, Meyers RE, Hinkle G, Svrzikapa N, Hutabarat RM, Clausen V a, Cehelsky J, Nochur S V, Gamba-Vitalo C, Vaishnaw AK, Sah DWY, Gollob J a, Burris H a. First-in-Man Trial of an RNA Interference Therapeutic Targeting VEGF and KSP in Cancer Patients with Liver Involvement. Cancer discovery (January 28, 2013). doi: 10.1158/2159-8290.CD-12-0429.

 

 

 

Eph in ALS


A recent publication from Van Hoecke and colleagues (Van Hoecke et al., 2012) suggests a novel therapeutic approach to the treatment of amyotrophic lateral sclerosis (ALS; also called Lou Gehrig’s disease). ALS is a progressive degenerative disorder that affects 1-2/100,000 people per year and results in death, normally by respiratory failure, 3-5 years after onset. It is caused by a loss of the motor neurons that control muscle movement. There is a hereditary component in about 10% of all ALS cases and in these familial ALS subjects, a variety of genes have been implicated (Al Chalabi et al., 2012), including SOD1, TARDBP and FUS and which encode superoxide dismutase 1 (SOD-1; which was the initial gene reported to be associated with familial ALS in 1993) TAR DNA binding protein (TDP-43) and the Fused in Sarcoma protein, respectively. Recently, a hexanucleotide repeat expansions that occurs in the chromosome 9 open-reading frame 72 gene (C9ORF72) has been described to be associated with ALS with frontotemporal dementia (DeJesus-Hernandez et al, 2011; Renton et al, 2011).

The huge advances in our understanding of the genetics underlying the familial form of ALS have yet to result in breakthrough therapies for this disorder and Riluzole remains the only FDA-approved treatment for ALS. It was approved in 1995 on the basis of clinical studies that demonstrated that it increased survival times in patients, yet the effects are relatively modest and there is a clear need for new and improved treatments for ALS. Since Riluzole was approved, there have been over 30 clinical trials of new treatments but for a variety of reasons (including poor clinical trial design and drug delivery or dose selection issues) none have reached the market, although dexpramipexole, which enhances mitochondrial function, is currently undergoing Phase III trials sponsored by Knapp (Cudcowicz et al, 2011).

A key challenge to the development of new drugs based upon the genetic information derived from familial ALS, as well as genes associated with sporadic ALS, is to understand how mutations in the various genes produce a similar clinical and pathological phenotype. In other words, what is the final common pathway by which these genetic mutations produce ALS? Generic explanations such as mitochondrial dysfunction or alterations in protein degradation pathways have been suggested but how these processes are affected by genetic influences remain vague. However, it is not necessary to understand the mechanism if one can develop a screen that rescues the phenotype produced by different mutations, and this is what Van Hoecke and colleagues did. Hence, they screened for different morpholinos (antisense oligos in which ribose or deoxyribose is replaced by a morpholine ring) that rescued a SOD-1 induced axonopathy in zebra fish. The most protective morpolino targeted the zebra fish Rtk2 gene, which has 67% identity to the human EPHA4 gene that encodes for the Epha4 receptor tyrosine kinase that can bind both type A and type B ephrins. Knock down of the Rtk2 gene rescued the phenotype in zebra fish with various SOD1 mutants (A4V, G37R and G93A) and SOD-1-induced axonopathy could also be rescued pharmacologically by inhibition of Epha4 using 2,5-dimethylpyrrolyl benzoic acid.  Importantly, knockdown of Rtk1, which is a paralog with 83% identity to human Epha4, was able to rescue the axonopathy induced by either mutant SOD-1, TDP-43 or knockdown of Smn1 in zebra fish, indicating that inhibition of EphA4 is protective against motor neuron degeneration irrespective of the genetic determinant of vulnerability. Having identified Epha4 as a potential modifier of SOD1-mediated pathology, the authors also studied the effects of a deletion of the Epha4 gene in mice overexpressing the G93A mutant SOD1 and were able to show that in heterozygotes, a 50% reduction in Epha4 was able to prolong survival.

As regards ALS itself, EphA4 mRNA expression in total blood was inversely collected to the age of onset such that patients with lower levels of EphA4 expression had an age of onset older than those with higher levels of expression. Suggesting that reduced EphA4 expression is associated with a reduced disease severity. Collectively, these data shed light onto an intriguing pathway in which rescue of the axonopathy is achieved irrespective of the genetic cause. A further understanding of the mechanism by which Epha4 exerts these effects could provide the basis for novel therapeutic approaches to treating ALS.

References:

Al-Chalabi, A., Jones, A., Troakes, C., King, A., Al-Sarraj, S. and van den Berg, L.H. (2012) The genetics and neuropathology of amyotrophic lateral sclerosis. Acta Neuropathol., 124:339-352.

Cudkowicz, M., Bozik, M.E., Ingersoll, E.W., Miller, R., Mitsumoto, H., Shefner, J., Moore, D.H., Schoenfeld, D., Mather, J.L., Archibald, D., Sullivan, M., Amburgey, C., Moritz, J. and Gribkoff, V.K. (2011) The effects of dexpramipexole (KNS-760704) in individuals with amyotrophic lateral sclerosis. Nat. Med., 17:1652-1656.

DeJesus-Hernandez, M., Mackenzie, I.R., Boeve, B.F., et al. (2011) Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron, 72:245-256.

Renton, A.E., Majounie, E., Waite, A., et al., A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron, 72:257-268.

Van Hoecke, A., Schoonaert, L., Lemmens, R., Timmers, M., Staats, K.A., Laird, A.S., Peeters, E., Philips, T., Goris, A., Dubois, B., Andersen, P.M., Al-Chalabi, A., Thijs, V., Turnley, A.M., van Vught, P.W., Veldink, J.H., Hardiman, O., Van Den Bosch, L., Gonzalez-Perez, P., Van Damme, P., Brown, R.H. Jr., van den Berg, L.H. and Robberecht, W. (2012) EPHA4 is a disease modifier of amyotrophic lateral sclerosis in animal models and in humans. Nat. Med., Aug 26. doi: 10.1038/nm.2901. [Epub ahead of print]