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Ion Channels Open Doors to New Drugs Increased R&D Efforts Are Overcoming Obstacles and Showing Potential

  • Nina Flanagan
Ion channels make good drug targets—they reside on the cell surface and are fast switching mechanisms. They act like cell transistors, controlling many cell processes. There are close to 500 types of ion channels, yet many remain undiscovered. This was mainly attributed to technology restraints, however, with the recent introduction of HT patch clamping, as well as new assays that facilitate faster, more robust screening, there are more ion channel receptors being detected.

Researchers at the recent Society for Biomolecular Screening conference and CHI’s upcoming “Pharmacology Driven Assays for GPCRs and Ion Channels” shared information on a cornucopia of topics, including the latest enabling technologies, new screening paradigms, and novel approaches to generate GPCRs.

The IonFlux system from Fluxion Biosciences was recently beta tested by scientists at Novartis Institutes for Biomedical Research (NIBR). “Compounds, buffers, and waste are contained on a single 96-well plate, eliminating robotic handling. Air pressure drives experiments in microfluidic channels in a layer below the wells. This is a novel approach in automated electrophysiology,” explained Andrew Golden, Ph.D., post-doc fellow.

Robustness is enhanced via recordings taken from 20-cell ensembles (IonFlux HT), and pharmacology improved by recording a full range of concentrations from the same group of cells, according to the company. There are two available systems—the IonFlux 16, which uses 96-well plates, and the IonFlux HT, which uses 384-well plates.

Analysis of the prototype (alpha and beta testing) was initially focused on whether IonFlux could reproduce results demonstrated on other platforms. “The microfluidic approach could be helpful for ligand-gated ion channels—especially for subsets of those for fast desensitizing ligand-gated ion channels where you only add a short pulse of the ligand or neurotransmitter,” explained Mats Holmqvist, Ph.D., research investigator in the center for proteomic chemistry at NIBR.

In addition, Dr. Holmqvist said the hope for the new platform is that it should provide selectivity not only by target but also by function. “You can utilize ‘use dependency’—the accumulation of inhibition with repetitive depolarizations. If an ion channel is active, the drug may be much more potent.” With this new technology, one should be able to refine and understand how a compound affects an ion channel. However, it’s still too early to show whether this will be the case.

Since HT platforms for ion channels are fairly new, standardization across different instruments hasn’t been addressed. “There are different quality control parameters, including the way of recording a single cell per well or ensemble recording in parallel. Some machines use Oracle database versus file formats. We’ve been trying to address that in safety profiling. A quick answer is that we make a summary PDF file of every compound in each experiment that can be accessed any time,” noted Dr. Holmqvist.

Parallel Screening

The traditional screening paradigm involves one target for primary HTS. However, this process “wastes a considerable amount of time to get results, and also wastes efforts on compound management in order to get those compounds ready for testing,” said Peter Hodder, Ph.D., senior director of lead identification for the translation research institute at the Scripps Institute, Florida.

His group uses a parallel screening process that screens compounds against the target and antitarget simultaneously. “Antitarget is an all-encompassing name for any assay you would run that’s different from the target—usually to remove compounds from further consideration,” Dr. Hodder explained. “We found most of those compounds are junk compounds anyway.” The antitarget becomes important for the hit compounds, because it provides information on whether it is something specific to the target or whether it is something nonspecific to the assay format.

Time saved via parallel screening can be four to five weeks per target. In addition, and what is more important and what is harder to gauge, he noted, is saved efforts following false trails, which result in smaller, cleaner datasets. Relevant structure  activity relationships emerge early in a campaign. For example, Dr. Hodder performed an SF1 (transcription factor) assay and ran the antitarget ROR against it and found potent compounds. “If we had relied on primary screening alone, those compounds would not have been selected.”

The parallel-screening format is not specific to any target class. “What’s more important is how to apply it to different target classes or different assay formats.” His group was successful in screening ion channels, including TRPML3 with TRPN1 as the antitarget (TRP is transient-receptor potential). HTS probes confirmed that the target is not located on plasma membranes in native cells.

Dr. Hodder added that this approach can be used to help focus on the most important compounds for drug or probe discovery, but it’s key is in choosing the right antitarget. “If it’s too close in relationship to the target, you’re going to start throwing out compounds you don’t want to during the campaign.”

His group is now performing more sophisticated screening using two or three antitargets and trying to find the overlap of hits that are specific in all three versus two or one of those targets and antitargets. “This challenges us to think about how we present and analyze our data.”

Novel Assays

Some of the challenges of working with ion channels include controlling activity, whether with a small molecule ligand or voltage. Many ion channels inactivate within milliseconds, making HTS difficult.

David Weaver, Ph.D., director at Vanderbilt Institute of Chemical Biology HTS, has been focusing his research efforts on ion channels—especially 7TM (7-Transmembrane) receptors.

“We are interested in looking at some of the effector systems that are more physiologically relevant and one of these is the GIRK (G-protein regulated inwardly rectifying potassium (K+) channel).” His group developed this assay to measure the activity of GI-coupled 7TM receptors. “The idea was whether we could see any differences in the pharmacology and the fact that we may be using a more physiologically relevant end effector rather than using mutant G proteins to couple the change in intracellular calcium.”

The success of the GIRK assay encouraged Dr. Weaver to examine ion channels as end effectors that could be used to generate new assays with physiological relevance. Preliminary data demonstrates the ability to detect changes in M-current (muscarinic-modulated potassium current, usually studied in the brain and peripheral nervous system) activity.

He developed an HTS-compatible assay that can measure and quantify the modulation of M-current downstream from the 7TM receptor using thallium-flux. This optical assay platform can use a commercially available kinetic imaging plate reader.

According to Dr. Weaver, the only nonstandard part of the assay is that he extracts a slope from the initial measurement, instead of fitting a peak amplitude. His hope is to use this assay to further understand the pharmacology of 7TM receptors. “It’s my intent that we can demonstrate that these are good, robust assays for use in HT screens to discover novel modulators of 7TM receptors or the ion channels we’re using as effectors.”

Novel Targets

“Ion channels are terrific molecular targets, and many drugs have been targeted to them,” stated David Clapham, M.D., Ph.D., Aldo R. Castenada professor of cardiovascular research at Children’s Hospital Boston. Yet, one of the biggest challenges is the gold standard assay—the patch clamp.

This is a time-consuming technique—single cell membranes must be broken open and the current must be recorded while controlling voltage in the cell. Although HT assays exist, not all ion channels are suited to them. “The most promising are the very fast, voltage-dependent channels with large, rapid changes and ones less amenable are ones that are similar to each other in their properties, like TRP channels—these are more difficult.”

Dr. Clapham also presented information on what he thought were good, fairly recent, ion-channel targets and included some recent data on some of his work with these targets.

Many TRP channels are involved in sensory functions, like smell, taste, and hearing. TRPV3 is an ion channel that is well expressed in skin. Dr. Clapham demonstrated that both skin barrier formation and some aspects of hair formation are altered by this ion-channel’s activation or block.

It is activated by subtle temperature changes—temperatures about 32ºC—indicating TRPV3 is sensing heat at the skin surface and relating that to the nerves. This indicates it may help regulate body temperature. Growth factors such as EPGR potentiates TRPV3 to bring calcium into karatinocytes, and, in turn, TRPV3 potentiates EPGR, so there’s a positive feedback loop.

“This is important for the proper formation of skin barriers, so that there is normally a cycle of karatinocytes maturing from deeper in the skin to the surface of the skin.” Dr. Clapham added that TRP channels are difficult to work with because they are fairly slow and their properties are often difficult to distinguish. In addition, they are often small in size, and there is a lack of known ways to activate them.

Additional ion channels that Dr. Clapham thought were worth pursuing were the NAV1.7 to NAV1.9 pain targets, which are voltage-gated sodium channels. A new chloride channel, TMAM16-A, and the ORAI channel, which is important in the immune system, were also on the list. An interesting new target for contraception, called CATSPER, is an ion channel only present in mature sperm and required for male fertility. “This may be a good method of contraception without hormones,” said Dr. Clapham.

“Our job is to find new targets and new molecules, and then other people can work with those molecules to target diseases.”

New Approach

There are many challenges for the generation of new GPCRs, said Michel Bouvier, Ph.D., professor and chairman in the department of biochemistry at the University of Montreal. These include selectivity and ligand-biased signaling, where one receptor can couple to different signaling pathways in a cell.

“The problem with this is that you are trying to monitor the efficacy of a compound toward one signaling pathway, but since there are multiple ones, we don’t necessarily know which one to follow that will correlate with a disease or particular activity.” His approach is to develop one assay that could encapsulate in one reading all the signaling pathways and by dissecting the signatures, provide information about the pathways being engaged by a receptor.

Utilizing Roche’s label-free xCELLigence platform, his group is able to measure cell impedence. Each well of the plate has electrodes. As the cells grow, the impedance increases, and when the cells are treated with compounds that bind to receptors, many different pathways are triggered.

The readout reflects changes in impedance from the compound over time—providing a global assessment of the various pathways. Different compounds generate different curve shapes. “We can use this technology to differentiate classes of compounds that have different relative selectivity toward different pathways. It’s generating a simpler way to classify compounds in different efficacy profiles toward different signaling pathways.”

Dr. Bouvier added that they can now, using selective inhibitors of different pathways  such as the generation of cyclic AMP, show how the inhibition influences the shape of the impedance curve. “Not only can we start classifying the ligands in different categories or compounds, but we can start making predictions on which pathways these compounds will be actively inhibiting. His group is planning to develop algorithms to apply to the curve and thus, provide a response as to which pathway is being affected. “We first need to confirm which portion of the curve informs us about each pathway.”

This approach can be used for almost any receptors, reported Dr. Bouvier. It provides a big time savings—one assay instead of four or five. However, he added, “we don’t know yet if all signaling pathways will respond to changes in impedance—from our data so far, we haven’t encountered such a pathway.”

source: genengnews.com

Rising to the Challenge in R&D

There has long been a consensus within the pharmaceutical industry that innovation and productivity is a critical focus. Nevertheless, the number of new molecular entities (NMEs) approved each year by FDA shows that industry has not been improving its output, despite progress in development and implementation of enabling technologies.

Over the past 20 years there has actually been a decline in NMEs approved by FDA. Furthermore, many of the NMEs approved are “me-too” molecules for disease states where first-in-class drugs are already on the market. Granted, there are other reasons for the dearth of product innovation—including regulatory issues, an increasing focus on short-term returns by some shareholders, and corporate restructuring—but the fact remains that pharmaceutical companies need NMEs with novel mechanisms and better safety and efficacy than offered in currently available drugs. Clearly, new chemistry allowing access to well known targets that have been intractable to older chemistries could provide a kick-start to the malaise in drug discovery.

A New Kind of Chemistry: Allosteric Modulation
Even as biologics, RNAi, and gene and cell therapies may provide value to patients in the short-to-medium term, small molecule drugs may one day offer patients many of the same benefits in a format that is more patient friendly (i.e. oral administration) and, potentially, with easier manufacturing and/or lower costs compared to non-pharmaceutical drugs. Allosteric modulators are an emerging class of orally available small molecule drugs that may have multiple advantages compared to traditional orthosteric drugs, including biologics.

Allosteric modulators have been shown to achieve greater selectivity, successfully modulating previously intractable therapeutic targets. In addition, orally available small molecule allosteric modulators have been discovered for targets for which only injectable biologic drugs are available. It is easier to achieve selectivity when targeting more heterogeneous allosteric binding sites on targets with therapeutic potential—such as G-Protein Coupled Receptors (GPCRs) and cytokine receptors—than an “active site,” which is often highly conserved across multiple related receptors.

Simply put, the active site on receptors acts as a switch that controls turning receptor signaling. Unlike orthosteric drugs, which turn receptors completely on or off, allosteric modulators act like a dimmer switch to mediate the intensity and frequency of receptor signaling. However, the trigger for signaling remains under the control of the endogenous ligand, which binds the target according to the physiological rhythm determined by the body. In many cases, allowing the body to retain control over initiating signaling while simply increasing or decreasing the amplitude of that signaling may offer a competitive advantage over other approaches. Although it has often been attempted with orthosteric drugs, comparable functional control over receptor signaling cannot be achieved simply by modifying the dose or delivery of orthosteric drugs.

Key Advantages of Allosteric Modulation

  • Because they do not compete for the endogenous ligand binding site and exert their effects even in the presence of endogenous ligands, lower doses of allosteric modulators may have greater potency than orthosteric molecules with similar affinity for the same target. Lower dosing often leads to fewer side effects.
  • Allosteric modulators can be devoid of activity in the absence of endogenous ligands, offering a less disruptive way to influence the functioning of biological systems and therefore could lead to greater safety and fewer tolerability issues.
  • Because they bind on a distinct site, it is possible to create new chemical entities with unfettered intellectual property that re-address well validated GPCR targets for which there are marketed products. In such cases, the goal would be that the allosteric mechanism offer clear differentiation in terms of efficacy and/or side effects.
  • It follows that highly selective allosteric modulators can be made for targets where it has been difficult to make selective orthosteric modulators. For example, orally available small molecule allosteric modulators against GLP-1 and FSH receptors—for which only peptide, protein or hormonal therapies are available—have been discovered.
  • Because they bind at a separate site, it is possible to combine allosteric modulators with orthosteric drugs. For example, a positive allosteric modulator, or PAM, could be used to potentiate an orthosteric agonist. This could alleviate side effects associated with off-target effects seen at high doses of some orthosteric drugs or simply reduce cost of goods for other orthosteric drugs, especially with biologics.

History of Allosteric Modulators
The concept of allosteric modulation is not new; scientists have been discussing it since the first half of the 20th century, and some suspected such a mechanism even earlier. In the 1960s, Roche introduced the tranquilizer Valium, which later was discovered to act by allosteric modulation of gamma-aminobutyric acid (GABA) receptors. More recent allosteric modulators include Sensipar (cinacalcet, from Amgen), a calcium-sensing receptor PAM, and Selzentry (maraviroc, from Pfizer), a CCR5 NAM.

But these first-to-market drugs were found more through serendipity than through focused searches for allosteric modulators. Indeed, the industrialization of allosteric drug discovery is something that many pharma companies and venture capitalists have shied away from due to the risks and the magnitude of investment.

The search for new drugs has long focused on GPCRs, but of roughly 850 known GPCRs less than 200 have been drugged. Compounds identified through screening have typically worked at the orthosteric site, but after finding the so-called “low hanging fruit,” this approach delivers fewer and fewer hits. In the late 1990s, researchers made some breakthroughs, identifying mGluR selective ligands that didn’t bind to the active sites on glutamate receptors, including allosteric modulators, targeting the metabotropic glutamate receptor 5 (mGluR5), which was discovered by researchers at SIBIA Neurosciences in collaboration with Novartis.

The goal soon became finding similar allosteric drugs; and for this, a new type of screening assay was needed. In the mid-1990s, screening assays evolved to include biological function. When the resulting compounds started to show different types of effects on the receptor, researchers concluded allosteric modulation may be playing a role.

In 2001, Vincent Mutel, CEO of Addex Pharmaceuticals, was a pharmacologist at Roche. Almost by chance, he and his colleagues discovered an allosteric molecule that enhanced the activity of the metabotropic glutamate receptor 1 (mGluR1). This glutamate receptor subtype was not tied to any particular disease, but the finding convinced Mutel that allosteric molecules could enhance an effect as well as block.

Addex was founded in 2002 and initial discovery work focused on targeting mGluR5 for addiction. As mGluRs had been intractable to orthosteric chemistry, Dr. Mutel and his team developed biological screening tools that would detect allosteric modulators of mGluR5 and other mGluR subtypes. It turned out that the tools developed could be adapted to almost any GPCR, and eventually to other types of receptors, like cytokine receptors. GPCRs are the targets of more than 30 percent of all medicines currently on the market . The company has disclosed discovering receptors in all three GPCR families and, more recently advances in the discovery of small molecules targeting receptors such as TNF-R1, IL-1R1, GIPR and GLP-1R, targets that have previously only been addressed by injectable protein or peptide therapeutics .

Future of Allosteric Modulators
The role of specific receptor sub-types has been elucidated in many diseases; however, in many cases, it has been challenging to develop sub-type specific drugs. These cases are the low hanging fruit for allosteric modulators. For example, metabotropic glutamate receptor 5 (mGluR5) has been implicated and clinically or preclinically validated in multiple diseases for more than two decades. But it took Big Pharma more than 20 years after the cloning of the mGluR5 receptor to identify and begin testing selective molecules against this high value target. In the end most if not all the molecules targeting mGluR5 are allosteric modulators. These molecules have progressed into the clinic and are now showing efficacy in humans in a variety of indications.

Addex’s lead compound ADX10059, a negative allosteric modulator of mGluR5, has shown efficacy in separate early Phase II studies for gastroesophageal reflux disease (GERD) and migraines. Clinical and preclinical data from Addex and other groups suggest that the product also has potential in Parkinson’s disease, and certain chronic forms of anxiety and depression. Other companies already are working on mGluR5 inhibitors to treat Parkinson’s disease, Fragile X, and neuropathic pain.

The allosteric drugs also could be combined with conventional orthosteric drugs against the same target to maximize the efficacy of the orthosteric and/or allow use of lower doses. This could be a desirable strategy to minimize dose-related, off-target side effects associated with the orthosteric product while potentially also reducing the cost of goods (especially if it is a biologic).

Allosteric modulators may become a life-cycle management strategy for biologics drugs. In the future, orally available small molecule allosteric modulator may be able to replace or complement many biologic drugs. The cost of a prescription allosteric modulator could, in some cases, obviate the opportunity for bio-generic competition while preserving the profit margin of the prescription biologic.

Allosteric drug discovery and development has only just begun. Many skeptics are being won over and it is beginning to become a mainstream approach. With more than 70,000 potential allosteric modulators in its unique biased library and a growing number of proprietary biological screening tools, Addex is leading the field. Its growing pipeline and partnerships serve as increasingly irrefutable validations. The approach, however, is much bigger than one company, with many in the industry predicting that allosteric modulation will become a new therapeutic class in the medical armamentarium.
Source: findpharma.com

AIDS Drugs – HIV

In the early 1980s, the human immunodeficiency virus (HIV) was identi­fied as the etiologic agent of acquired immune deficiency syndrome (AIDS). More than 3 million people worldwide died from HIV/AIDS in 2003, according to a July 2004 United Nations report. During the same period, about 5 million people contracted the human immunodeficiency virus, bringing the total number of people living with HIV worldwide to 38 million. Although AIDS was called the «gay men’s disease» at the be­ginning of the outbreak, it was soon discovered that sexual intercourse was not the only way of transmission. Blood transfusions and mother-to-baby transmission also spread the virus.

In comparison to the scourges caused by other viruses in history, we were more prepared and have achieved astonishing milestones against AIDS, thanks to our accumulated knowledge and efforts around the globe. HIV was identified and shown to be the cause of AIDS in less than 2% years. It took only another 2 years for blood tests to become commercially available. In 1987, the first anti-HIV drug, AZT, was introduced. With the arrival of the HIV protease inhibitors and triple drug therapy (the cocktail therapy) in 1995, many patients who would otherwise have died are still alive. In 1996, Time magazine named AIDS researcher David Ho «Man of the Year» for his revolutionary idea of the cocktail therapy.

Who discovered HIV was such a contentious is­sue that it took the President of the United States and the Premier of France to settle the dispute.

In 1983 Francoise Barre-Sinoussi and Luc Montagnier, in the laboratory led by Montagnier at the Institut Pasteur de Paris, first detected and later iso­lated a retrovirus, lymphadenopathy-associated virus (LAV), which they believed was the cause of AIDS. During their research on the virus, Montagnier’s lab­oratory collaborated with Robert C. Gallo, a renowned virologist at the National Cancer Institute (NCI), who was one of the most widely referenced scientists in the world in the 1980s and 1990s. Montagnier and Gallo frequently exchanged virus sampies and information. In April 1984, Gallo held a press conference an­nouncing that his laboratory had isolated a retrovirus, human T-lym-photrophic virus (HTLV-III), that he believed to be the cause of AIDS. Gallo was basking in scientific glory and was widely considered a leading contender for the Nobel Prize. Soon it was confirmed that Gallo’s HTLV-III and Montagnier’s LAV were identical. In 1986, a nomenclature com­mittee was set up, chaired by Harold Varmus, an expert in avian retrovirus and then director of the NIH. The NIH committee settled on the name of human immunodeficiency virus (HIV).

In April 1984, Gallo’s laboratory filed a patent on an HIV blood test kit using his HTLV-IIIB-ELISA (enzyme-linked immunosorbent assay), which was issued in a record 13 months via a special category involving na­tional security. Although Institut Pasteur had filed a patent in the United States much earlier, in December 1983, it was not granted until a later date. Gallo’s HIV test kit was approved by the FDA in 1985. An acrimonious le­gal battle ensued for the priority of the discovery of the HIV between the French and American teams. The contentious scientific and legal contro­versies came to an end in March 1987 when a historic agreement was signed by the directors of the NIH and the Institut Pasteur and ratified by Ronald Reagan and Jacques Chirac. The patents would become the joint properties of the two institutions, which would share the royalties. The three inventors from the NIH, including Gallo, would receive $100,000 annually from the royalties earned.

Even the intervention by two heads of state did not put the matter to rest. In November 1989, a Pulitzer Prize-winning investigative reporter, John Crewdson, published a 50,000-word article in the Chicago Tribune on the Montagnier-Gallo priority dispute. He concluded that Gallo had either stolen or allowed his samples to be contaminated with Montagnier’s virus. The controversy generated resulted in congressional investigations. In the end, it was found that Mikulas (Mika) Popovic from Czechoslovak­ia, a cell biologist in Gallo’s laboratory, had isolated HTLV-III from a pool by mixing several blood samples from different sources, including Montagnier’s sample, which contained LAV. Pooling blood samples was an unusual practice in virology. In 1991, Gallo admitted in Nature that he had not discovered the new virus. In 1996, he left the NCI, where he had worked for 30 years, to become the director of the Institute of Hu­man Virology at the University of Maryland Biotechnology Institute in Baltimore.

In 1987, the first anti-AIDS drug, AZT, was introduced by Burroughs Wellcome. AZT, which blocks HIV reverse transcriptase activity, stands for azidothymidine, with the generic name of zidovudine and the trade name of Retrovir. Popular media often give the credit to Gertrude Elion of Burroughs Wellcome for having discovered AZT. In fact, al­though Elion and George Hitchings (see chapter 1, page 19) developed the concept of using nucleotides as antimetabolites in treating cancers, AZT itself was synthesized by a group led by Jerome Horowitz of the Detroit Institute of Cancer Research in 1964 as a possible anticancer drug. Horowitz, now a professor at Wayne State University, published his syn­thesis as a note in the. Journal of Organic Chemistry in 1964.

Since its birth, AZT had a checkered life as a drug looking for a disease to treat. AZT did not show efficacy in treating cancers; the drug also failed to prolong the lives of leukemic animals. In 1974, a German laboratory found it effective against viral infection in mice—Wolfram Ostertag of the Max Planck Institute for Experimental Medicine showed that leukemia helper virus (LLV-F) replication by AZT occurred via phosphorylation of AZT to the corresponding triphosphate, which cannot be incorporated into the growing strand of DNA. Ostertag correctly concluded that AZT-triphosphate worked by binding to the growing strand of DNA. Bur­roughs Wellcome acquired AZT and explored the possibility of using it to treat the herpes virus under the guidance of Gertrude Elion, although it did not make it to the market.