SPC3649 ( LNA- locked nucleic acid) - a new hope for hepatitis C.....

When  I was working with Innovasynth Technologies, Khopoli, I had an opportunity to do literature survey about  Lock Nucleic Acids (LNAs) and Peptide Nucleic Acids (PNAs) (we were supposed to work on the preparation of  some of the LNAs & PNAs for US based companies). In my opinion though these class of compounds (including oligonucleotides) are  still emerging,  I think in the days to come there will be more and more drugs from oligonucleotides, Locked Nucleic Acids (LNAs) and Peptide Nucleic Acids, (PNAs) class of compounds.

LNAs : A locked nucleic acid (LNA) (see the right side general structure), is a modified RNA nucleotide. Ribose moiety of an LNA nucleotide is modified with an extra bridge

connecting the 2' oxygen and 4' carbon. The bridge "locks" the ribose  in the 3'-endo (North) conformation, which is often found in the A-form of DNA or RNA. LNA nucleotides can be mixed with DNA or RNA bases in the oligonucleotide whenever desired. This locking  significantly increases the thermal stability (mp) of oligonucleotide.

LNA nucleotides are used to increase the sensitivity and specificity of expression in DNA microarrays, FISH probes, real-time PCR probes and other molecular biology techniques based on oligonucleotides. For the in situ detection of miRNA the use of LNA is currently the only efficient method. A triplet of LNA nucleotides surrounding a single-base mismatch site maximizes LNA probe specificity unless the probe contains the  G-T mismatch. We have already seen some antisense drugs (oligonucleotides from Geron & Isis) and some are into clinical trials.

Now its interesting to see that Santaris Pharma   is currently advancing LNA based compounds within infectious diseases, metabolic disorders, oncology, inflammatory and rare genetic disorders.

Santaris Pharama, has developed a LNA,  SPC3649 - which captures a small RNA molecule in the liver, called microRNA122, that is required for HCV replication.

As per the claim by the company, SPC3649 works by altering the environment in the host liver cell to inhibit viral replication rather than inhibiting the virus itself. This subtle difference (in comparison  with other therapies) may have significant implications, as it may reduce the risk of the virus becoming resistant to therapy – a major concern with current therapies.

As per the claim by Dr. Robert Lanford,  (who has collaboration with Santaris Pharma), that in a preclinical study  SPC3649 successfully inhibited miR-122, a liver-expressed microRNA important for Hepatitis C viral replication. By inhibiting miR-122, SPC3649 dramatically reduced Hepatitis C virus in the liver and in the bloodstream in chimpanzees chronically infected with the Hepatitis C virus. Four HCV chronically infected chimpanzees were treated weekly with 5 or 1 mg/kg of SPC3649 for 12 weeks followed by a treatment free period of 17 weeks. The two animals that received the 5 mg/kg dose had a significant decline in viral levels in the blood and liver of approximately 2.5 orders of magnitude or approximately 350 fold. Hope the new therapy could potentially replace interferon (interferon and ribavirin is  approved by FDA for hepatitis C and this treatment is very toxic, requires 48 weeks with 50% success) in future cocktails, since it provides a high barrier to resistance. This antiviral could be used alone to treat disease progression and there are indications that it can convert interferon non-responders to responders, so that non-responders to the current therapy could be treated with the combination of this drug with interferon.   More....

Those interested  can see the video demo with the link

Biguanide mechanism discovered

For fifty years, one of the few classes of therapeutics effective in reducing glucose production has been the biguanides, which include phenformin and metformin, the latter the most frequently prescribed drug for type-2 diabetes. Nonetheless, the mechanism of action of biguanides remains imperfectly understood. The suggestion a decade ago that metformin reduces glucose synthesis through activation of the enzyme AMP-activated protein kinase (AMPK) has recently been challenged by genetic loss-of-function experiments. Here we provide a novel mechanism by which metformin antagonizes the action of glucagon, thus reducing fasting glucose levels. In mouse hepatocytes, metformin leads to the accumulation of AMP and related nucleotides, which inhibit adenylate cyclase, reduce levels of cyclic AMP and protein kinase A (PKA) activity, abrogate phosphorylation of critical protein targets of PKA, and block glucagon-dependent glucose output from hepatocytes. These data support a mechanism of action for metformin involving antagonism of glucagon, and suggest an approach for the development of antidiabetic drugs....

Biguanide mechanism discovered

A new experimental drug "antagomir" (antisense oligonucleotide) as an anti- miR-21 agent..

MicroRNAs are small scraps of RNA comprising around 20 nucleotides and it is only recently that scientists have discovered their power which is they can regulate the expression (switching on and off) of a large number of human genes (they are like "master controllers"). And also these are the culprits (when microRNAs don't appear in the right place at the right time within cells) for diseases such as cancer, viral infections, inflammatory diseases and metabolic disorders. The potential to use them as targets for drugs is obvious and possibly explains why this is one of the fastest growing areas of development for new drugs and treatments.

Scientists already knew that microRNA was involved in switching genes on and off in the heart, but the underlying mechanisms and how they relate to the development of particular types of heart disease and their potential as drug targets were still relatively unknown.

Thum and colleagues discovered that miR-21 was expressed in the heart's fibroblast cells (cells that make the scaffolding of collagen or connective tissue that hold the shape of the organ) and were in greater numbers in lab mice bred to have heart failure and also in human tissue from patients who had heart failure.

In this study they showed that increasing expression of miR-21 changed the way that signals behaved in a previously unknown stress response pathway that involved the gene sprouty-1 and the MAP-kinase signaling pathway. In turn, increasing the activity of the MAP-kinase pathway led to a number of signs of heart failure, such as enhanced fibroblast survival, increased secretion of factors like fibroblast growth factor, tissue scarring (fibrosis), and cardiac dysfunction including cellular hypertrophy.

The researchers proved they could administer anti-miR-21 effectively to the heart by monitoring it with fluorescence staining. Then, in a mouse transaortic constriction model of human heart failure, they showed that anti-miR-21 silenced increased expression of miR-21 and corrected downstream changes in sprouty-1 and MAP-kinase signaling.

The interesting thing is their conclusion : Anti-miR-21, showed the most statistically significant improvement in the heart failure mouse model when given before induction of heart failure and for as long as three weeks afterward and it might be possible to target entire disease pathways with one drug. Contrats Dr. Thomas Thum.

Cancer drug can neutralize toxic RNA that causes myotonic dystrophy

A group of researchers has shown for the first time in cells and in a mouse model that a drug used to treat cancer can neutralize the toxic RNA that causes the prolonged muscle contractions and other symptoms of myotonic dystrophy type 1, the most common form of adult-onset muscular dystrophy. The researchers report their findings today Dec. 10, 2015 in the journal Cell Reports. (actinomycin-D)

"This finding opens a new avenue for a therapeutic strategy for this disease," said Andrew Berglund, Ph.D., a professor of biochemistry and molecular biology in the University of Florida College of Medicine. "This is the first evidence that specifically inhibiting transcription can be effective in knocking down the toxic material that causes the disease."

In myotonic dystrophy and other related neurological disorders, the symptoms stem from repeated individual nucleotides, or "building blocks," in the RNA in muscle tissue cells that can build up over time. These repeats, called CTG expansions in myotonic dystrophy type 1, become 'toxic' when transcribed from DNA. The expansions disrupt the RNA binding proteins responsible for splicing, the 'editing' needed so that the RNA can create appropriate proteins that allow muscles to function properly.

Cancer drug can neutralize toxic RNA that causes myotonic dystrophy

RNA interference approach for prevention and treatment of STDs ?

In my earlier blog “Diverse use of Nucleic acids”, did mention that there is much interest in the medical uses of nucleic acids. For example, antisense, ribozymes, aptamer and RNA interference (RNAi) technologies are all being developed for potential therapeutic applications. Lots of research is being done in each specified fields and in fact there are already few drugs in “antisense category” and this time something really interesting has been reported by a Post Doc., Dr. Kim Woodrow in the field of RNA interference category. The following lines briefly summerise, what actually RNAis..

RNA interference (RNAi) is a system within living cells that helps to control which genes are active and how active they are. Two types of small RNA molecules – microRNA (miRNA) and small interfering RNA (siRNA) – are central to RNA interference. RNAs are the direct products of genes, and these small RNAs can bind to specific other RNAs and either increase or decrease their activity, for example by preventing a messenger RNA from producing a protein. RNA interference has an important role in defending cells against parasitic genes, viruses and transposons – but also in directing development as well as gene expression in general

The RNAi pathway is found in many eukaryotes including animals and is initiated by the enzyme Dicer, which cleaves long double-stranded RNA (dsRNA) molecules into short fragments of ~20 nucleotides. One of the two strands of each fragment, known as the guide strand, is then incorporated into the RNA-induced silencing complex (RISC). The most well-studied outcome is post-transcriptional gene silencing, which occurs when the guide strand base pairs with a complementary sequence of a messenger RNA molecule and induces cleavage by Argonaute, the catalytic component of the RISC complex. This process is known to spread systemically throughout the organism despite initially limited molar concentrations of siRNA. The importance of the siRNA lies in the fact that “RNAi is selective on gene expression” and hence can be used in the similar fashion like the antisense drugs (already a few drugs by ISIS, Serono and others). I did work on a few oligonucleotides (phosparothiamidates), while working in Innovasynth Technologies Limited Khopoli and know how difficult is to get the precursors of the antisense drugs. In 2006, Andrew Fire and Craig C. Mello shared the Nobel Prize in Physiology or Medicine for their work on RNA interference in the nematode worm C. elegans.

Gene interference therapy is moving rapidly from basic research to application. The PLGA packaging these researchers chose is already approved as safe and non-toxic by the FDA, speeding the path to clinical trials for infectious agents such as HPV and HIV.

Congrats Dr.Kim and co workers for this achievement. The significance of this research is the fact that “a safe and effective administration of potential antiviral drugs - small interfering RNA (siRNA) molecules using densely-loaded nanoparticles made of a biodegradable polymer known as PLGA. The researchers created a stable "time release" vehicle for delivery of siRNAs to sensitive mucosal tissue like that of the female reproductive system.

Ref : http://www.nature.com/nmat/journal/vaop/ncurrent/abs/nmat2444.html

Drug compounds target multiple pathways associated with myotonic dystrophy type 1

Efforts to treat myotonic dystrophy type 1, the most common form of muscular dystrophy, are in their infancy. In a new study, researchers report they have added new capabilities to an experimental drug agent that previously defeated only one of DM1's many modes of action. Their retooled compounds interrupt the disease's pathology in three ways.

"We've rationally designed something to target multiple pathways, which is contrary to the traditional thinking in medicinal chemistry, where you have one target, one drug," said University of Illinois chemistry professor Steven Zimmerman, who led the research with graduate students Lien Nguyen and Long Luu. "People are slowly discovering that drugs that hit multiple targets are actually better."

The team reports its findings in the Journal of the American Chemical Society.

DM1 (but not Duchennes muscular dystrophy) results from a genetic error that causes expansion of a region of a particular gene, called DMPK. This gene includes a repeated, three-letter sequence of nucleotides, the gene's chemical building blocks. Normal cells contain as many as 35 of these repeats, but sometimes mutation pushes the number of repeats beyond 50, which can lead to symptoms of the disease. Mutant DMPK genes often continue to expand, amplifying the health problems that can result. In some people, the gene includes as many as 10,000 repeats.

Drug compounds target multiple pathways associated with myotonic dystrophy type 1

RNA molecule can be manipulated to generate more neurons from neural stem cells

A research team at UC San Francisco has discovered an RNA molecule called Pnky that can be manipulated to increase the production of neurons from neural stem cells.

The research, led by neurosurgeon Daniel A. Lim, MD, PhD, and published on March 19, 2015 in Cell Stem Cell, has possible applications in regenerative medicine, including treatments of such disorders as Alzheimer's disease, Parkinson's disease and traumatic brain injury, and in cancer treatment.

Pnky is one of a number of newly discovered long noncoding RNAs (lncRNAs), which are stretches of 200 or more nucleotides in the human genome that do not code for proteins, yet seem to have a biological function.

The name, pronounced "Pinky," was inspired by the popular American cartoon series Pinky and the Brain. "Pnky is encoded near a gene called 'Brain,' so it sort of suggested itself to the students in my laboratory," said Lim. Pnky also appears only to be found in the brain, he noted.

Co-first authors Alex Ramos, PhD, and Rebecca Andersen, who are students in Lim's laboratory, first studied Pnky in neural stem cells found in mouse brains, and also identified the molecule in neural stem cells of the developing human brain. They found that when Pnky was removed from stem cells in a process called knockdown, neuron production increased three to four times.

"It is remarkable that when you take Pnky away, the stem cells produce many more neurons," said Lim, an assistant professor of neurological surgery and director of restorative surgery at UCSF. "These findings suggest that Pnky, and perhaps lncRNAs in general, could eventually have important applications in regenerative medicine and cancer treatment."

RNA molecule can be manipulated to generate more neurons from neural stem cells

TSRI scientists develop first drug candidate that neutralizes disease-causing RNA repeats

Top, Venn diagram of substructures in compounds that were found to bind to RNA from the fluorescence screening assay showed in Fig. 1c. Data were compiled by using compounds that had a P value of <0.001 for binding to the RNA hairpins. Bottom, structures of compounds 1 and 2 that were the most avid for binding to AUAU and AAUU RNA hairpins.

In an important new study with implications for the treatment of dozens of incurable diseases, scientists from the Florida campus of The Scripps Research Institute (TSRI) have for the first time created a drug candidate that attacks and neutralizes the RNA structure that causes an incurable progressive, inherited disease involving a gradual loss of control over body movement.

The study, which was published June 1, 2016 in Nature Communications, showed the compound significantly improved several aspects of cells taken directly from patients with spinocerebellar ataxia type 10 (SCA10), a form of spinocerebellar ataxia.

“More than 30 diseases, all of them incurable, are caused by RNA repeats,” said TSRI Professor Matthew Disney, who led the study. “By a thorough basic science investigation, we identified small molecules that target RNA base pairs precisely. We then leveraged this information to design the first drug candidate that binds to disease-causing defects in SCA10. Application of the drug candidate returns certain aspects of those cells to healthy levels—it’s like the defect is not even there.”

SCA10 is caused by what is called a pentanucleotide repeat (a genetic sequence of five nucleotides repeated many more times than normal) affecting the mitochondria, the cell’s energy source. The new drug candidate, known as 2AU-2, targets these repeats by binding to RNA base pairs.

“The potent bioactivity of 2AU-2 to moderate the structurally induced toxicity in SCA10 strongly suggests that base-pair-targeting RNA modules could have broad applicability in our effort to develop other compounds that target different RNAs,” said TSRI Research Associate Wang-Yong Yang, the first author of the study. “More than 70 percent of RNA secondary structure is made up of base pairing.”

The Disney group has developed new tools to identify optimal interactions between RNA structures and drug candidates targeting them. A database of these interactions has already been used to design several small molecule drug candidates.

“We are in the process of developing tools that allow one to design small molecules to target any RNA structural motif in a complex cellular environment. That environment can contain millions of other RNAs. In this study, Wang-Yong has done an exceptional job tackling this previously-thought-to-be-impossible molecular recognition problem,” Disney said.

Pathogenic RNA repeats contribute to disorders including Huntington’s disease, fragile X-associated tremor ataxia syndrome and myotonic dystrophy type 1 and 2.

Ref : http://www.nature.com/ncomms/2016/160601/ncomms11647/full/ncomms11647.html

Scripps Florida Scientists Devise New Way to Dramatically Raise RNA Treatment Potency

"We're trying to make tools that can target any RNA motif," said Matthew Disney, a TSRI associate professor who authored the research with a research associate in his lab, Lirui Guan. "This study completely validates our design -- it validates that our compound targets the desired RNA sequence in a complex cellular environment that contains many hundreds of thousands of RNAs."

While targeting DNA has been used as a therapeutic strategy against cancer, few similar approaches have been attempted for disease-associated RNAs.

In the new study, the scientists created a small molecule that binds to the genetic defect in RNA that causes myotonic dystrophy type 1 and improves associated defects in cell culture.

Myotonic dystrophy type 1 involves a type of RNA defect known as a "triplet repeat," a series of three nucleotides repeated more times than normal in an individual's genetic code. In this case, the repetition of the cytosine-uracil-guanine (CUG) in the RNA sequence leads to disease by binding to a particular protein, MBNL1, rendering it inactive and resulting in a number of protein-splicing abnormalities.

To achieve the increase in the drug candidate's potency, Disney and his colleagues attached a reactive molecule (a derivative of chlorambucil, (see structure below) a chemotherapy drug that has been used to treatment a form of leukemia) to the small molecule they had identified. As a result, the new compound not only binds to the target, it becomes a permanent part of the target -- as if it were super glued to it, Disney said. Once attached, it switches off the CUG defect and prevents the cell from turning it back on.

Disney was surprised at the approximately 2,500-fold improvement in potency with the new approach.

"I was shocked by the increase," he said. "This takes the potency into the realm where one would like to see if the compound were to have real therapeutic potential."

As a result, the new compound, known as 2H-4-CA, is the most potent compound known to date that improves DM1-associated splicing defects. Importantly, 2H-4-CA does not affect the alternative splicing of a transcript not regulated by MBNL1, demonstrating selectivity for the CUG repeat and suggesting that it might have minimal side effects. "We can now use this approach to attach reactive molecules to other RNA targeted small molecules," Disney said.

The reactive molecule model also provides a potentially general method to identify cellular targets of RNA-directed small molecules. Such probes could also identify unintended targets, information that could be used to design and identify compounds with improved selectivity in an approach similar to activity-based profiling, Disney said.

Scripps Florida Scientists Devise New Way to Dramatically Raise RNA Treatment Potency

Idera's gene-silencing oligonucleotide studies published in Journal of Medicinal Chemistry

In continuation of my update on antisense drugs and nucleotides

Idera's gene-silencing oligonucleotide studies published in Journal of Medicinal Chemistry

Ref : JMC