Our article is written for a bioscience audience and offers guidance on best practice in chemical probe selection, evaluation and use.

I have previously postedabout how small molecule chemical probes of high quality are crucial for investigating the function of proteins in cells and organisms and also for validating them (or not) as drug targets. This applies to all areas of biomedical research see articles by Stephen Fryeand Mark Bunnage and colleagues.

My colleague Julian Blagg and I have today published a Perspective on choosing and using chemical probes in the journal Cancer Cellthat is written specifically with the audience of biologists in mind.

There are numerous examples of how the use of fit-for-purpose chemical probeshas led to important discoveries in biomedical research. The value of chemical probes is particularly well demonstrated in the field of cancer research.

A good case in point is the relatively recent rapid growth in our understanding of the biology and pharmacology of bromodomains that was triggered by the discovery of potent chemical probes such as JQ1and I-BETand their closely matched inactive partner compounds used as controls.

Yet in my earlier blogI also drew attention to how loose standards in the selection and use of chemical probes are leading to serious errors in biomedical research studies.

To be effective as chemical probes, small molecule agents need to be cell permeable and to bind potently (i.e. strongly at low concentrations) to the desired protein target and modulate its function in the cell as measured by direct target interaction and appropriate downstream biomarker changes. And they also need to bind selectively, meaning that they dont interact with and modulate other cellular targets or more realistically that they only affect an acceptable number of additional relevant proteins.

However, use of poorly selective, or otherwise unsuitably flawed even frankly dreadful chemical compounds is widespread. This is sloppy science and contributes to what is often referred as a crisis in the reproducibility and robustness of biological findings.

Moreover, recent calculations have suggested that spending $150 on a poor quality, out-of-date chemical compound from a vendor catalogue, instead of buying a high quality chemical probe, can cost the scientific community billions of dollars. There are at least200 historic compoundsthat are often used and should be replaced with better probes.

So misuse of chemical probes is wasting scientists time and money and in many instances is undoubtedly leading to delays in the discovery of much needed medicines.

I explained in my previous posthow, in a Commentary article in the journal Nature Chemical Biology by Arrowsmith et alpublished in August 2015, an international panel of chemical biology scientists (of which I was a member) had issued a call-to-arms aimed at eliminating the use of substandard research tools in biomedical research and promoting best practice. That call was linked with the launch of a new community-based, 'TripAdvisor-style' online resource available at the Chemical Probes Portal.

The non-profit Portal works by offering online expert annotation and recommendations for use of chemical probes for particular molecular targets. These are provided by a Scientific Advisory Board (for full disclosure I am a member of this and a Board Director) with about 400 probes assessed to date.

Theres no doubt that great progress has been made in discovering high quality tools for cancer biology and target validation. Unfortunately two years on now from the publication of the Arrowsmith et al paper and the initial launch of the Chemical Probes Portal it is abundantly obvious that bad practice in the selection and use of chemical probes is still very widespread in biomedical research, including numerous, continuing high profile examples in cancer.

Its clear that biologists commonly choose chemical probes based on querying search engines such as Google which will lead them to vendor catalogues that provide variable levels of information, do not prioritize probes based on quality, and sometimesrecommend the same compound as a probe for different protein targets.

Alternatively, use of search engines like Google Scholar will return as top hits publications that are the most highly cited, but that also describe the oldest chemical probes. Such searches are less likely to find the best, usually more recent tools. For example, when Chemical Probes Portal staff looked at 10 compounds, randomly selected from the 200 no longer recommended historical probes listed on the Portal website, they found that since 2016 these past-their-sell-by date reagents have been used in 2,090 publications.

A specific illustration is the still very frequent use of one of the above historical compounds, LY294002 an initially valuable early inhibitor of the phosphoinositide 3-kinase lipid kinases (PI3 kinase) that was originally described in 1994. Although a usefulpathfinder probe,LY294002 exhibits only weak, micromolar potency for PI3 kinases and through chemoproteomic studies it was subsequently found to be active against numerous members of the PI3 kinase family, and also other unrelated proteins including bromodomains.

LY249002 has been cited in over 30,000 publications; moreover despite its poor potency and selectivity and its supersedence by severalsuperior compounds as chemical probes for PI3K, a recent search for LY294002 on Google Scholar returned 1,190 publications for the year 2016 alone and this now outdated and flawed probecontinues to be sold by many commercial vendors.

Its clear then that we need to find a way make things change and especially influence behaviour of biological research community which is the main user group for chemical probes. This is why Julian Blagg and I have written our Perspectivein way that we hope will get the message out to biologists.

As we say in the Abstract of the Perspective:

Small-molecule chemical probes or tools have become progressively more important in recent years as valuable reagents to investigate fundamental biological mechanisms and processes causing disease, including cancer. Chemical probes have also achieved greater prominence alongside complementary biological reagents for target validation in drug discovery. However, there is evidence of widespread continuing misuse and promulgation of poor-quality and insufficiently selective chemical probes, perpetuating a worrisome and misleading pollution of the scientific literature. We discuss current challenges with the selection and use of chemical probes, and suggest how biologists can and should be more discriminating in the probes they employ.

Despite the efforts so far within the chemical biology community, we point out that we have been guilty of: largely preaching to the choir [meaning chemical biology specialists] and failing to connect to a really critical audience: namely, the wider cancer biology community who rely upon small-molecule tool compounds, often in harness with biological reagents, to interrogate cancer cell biology and who frequently draw important and highly impactful biological interpretations, whether correct or misleading, from such studies.

A topical example that we highlight in our Perspectiveis the initially erroneous discovery and validation of the proposed target MTH1 in cancer. MTH1 has a role in breaking down damaged metabolites called nucleotides in cells and thus preventing them from being incorporated into DNA and was first published as a cancer target in very high profile publications in the journal Nature.

Small molecule agents that were originally used to validate MTH1 include compounds TH287 and TH588 as well as S-crizotinib. We discuss in our Perspectivethe elegant publicationfrom AstraZeneca scientists that identifies three different chemical series of potent and highly selective chemical probes that clearly inhibit MTH1 in cancer cells but despite this have no therapeutic effect on cancer cells.

Furthermore, the same article shows that neither small interfering RNA (siRNA) reagents that deplete MTH1 nor CRISPR-mediated removal of MTH1 had any beneficial effect of cancer cells, pointing to off-target activity with the original siRNA reagent as well the chemical compounds used.

Furthermore, the blogger Derek Lowehas just a couple of days ago updated this story by discussing a new publication from researchers at Bayerwho discovered BAY-707, yet another highly potent and selective inhibitor of MTH1, and found it to have no therapeutic effect on cancers cells. Hence at this stage the balance of opinion strongly indicates that MTH1 is not a valid target for cancer treatment.

One piece of evidence in the AstraZeneca study that was particularly critical in invalidating the initial chemical probes was the demonstration that both TH287 and S-crizotinib killed cancer cell lines lacking MTH1 and subsequent protein screening workshowed that the binding of TH287 and TH588 to tubulin is responsible for their cytotoxic effects.

We provide in the Perspectiveseveral other examples of how the close integration of orthogonal chemical and biological tools can be very powerful, as in the case of studies on SWI/SNF chromatin remodelling complex components, the transcription factor HIF2 and the Jumonji family of histone lysine demethylases. In addition, we describe cautionary tales of the problems arising with uncritical use of claimed chemical probes for proteins including poly ADP ribose polymerases (PARPs; for which a flawed PARP compound progressed to the clinic and failed); the molecular chaperone HSP70; KRAS-regulated autophagy; and pan-steroid receptor co-activators.

We discusshow a gold-standard test to validate the functional on-target response to a chemical probe is to demonstrate reversal of the cellular effects of a proposed small molecule probe compound by mutation in the protein target that abrogates compound binding.

Another useful technique is to engineer the target to interact with chemical probes not recognized by the natural (so-called) wild-type protein. An additional approach now becoming common is to determine the effects of the chemical probe in cells where the proposed protein target has been removed by CRISPR technology.

We point outthat although Small molecules are from Mars, biological tools are from Venus, they are nevertheless part of the same overall universe, providing orthogonal and complimentary approaches to understanding biology and target validation a very powerful, multidisciplinary and essential toolkit for modern biomedical research.

Also in our Perspectivewe highlight and explain an important aspect of target binding selectivity that is rarely articulated in discussion of chemical probes that it is absolutely to be expected that most small molecules will generally interact with multiple biological targets in cells and organisms. By contrast, biological reagents, for example siRNA oligonucleotides and antibodies, are intrinsically more likely than small molecules to bind selectively to the desired biological target as a result of the greater breadth, complexity and thus specificity of their combined intermolecular interactions.

Of course there are also major problems with the use of insufficiently selective biological reagents and greater rigour in their use is important too as elegantly discussed recently by Bill Kaelin but biologists need to be even more critical in their use of small molecule probes because their smaller size and lower complexity means that at least some degree promiscuity is likely to be the rule rather than the exception. This tendency can be mitigated by careful design and optimization of the probe but even then rigorous and broad experimental testing for selectivity is essential.

Indeed, we strongly advisethe maxim of caveat emptor let the buyer beware! when choosing and using chemical probes for biological exploration and target validation.

In discussing the challenge of selectivity, we illustrate how the off-target effects can range from an interaction with one or two proteins perhaps but necessarily related to the target of interest through binding to tens of other targets, all the way to the extreme end of unacceptability where compounds are frequent hitters or chemical impostersthat have totally unacceptable features like indiscriminate chemical reactivity, aqueous insolubility and self-aggregation that make them worthless for biological research.

Hard to believe, but there are even isolated examples of vendors supplying the incorrect chemical compound and routine checking for evidence of authenticity is advisable. Related to this, in our Perspectivewe call for further efforts in the community to eliminate the especially egregious behaviour of publishing biological results without disclosing compound structures which of course means that the suitability of a probe cannot be assessed, nor can the claims be independently checked. Reviewers of submitted papers and grant applications as well as journal editors should be especially vigilant about this.

We recognize that for many if not most biologists these considerations of the selectivity of chemical probes are not part of their training or expertise. They may not have ready access to advice from chemical biology or medicinal chemistry colleagues. And they may find articles in the specialist chemical biology literature off-putting and full of jargon as most scientific disciplines are.

So in our Perspective we provide what we hope will be useful tools for biologists using chemical probes. Firstly, we include as Box 1a Glossary so that that any specialist terminology that cannot really be avoided is not too much of a turn-off.

We provide in Box 2a comparison of the desired selectivity profiles of chemical probes with those of approved drugs making the point that in comparison to drugs, chemical probes generally need to be even more selective than drugs so that probes can be used with confidence to modulate the intended target of interest.

In Box 3we summarize the factors that determine the fitness and quality of chemical probes and in Figure 2we present an overview of Dos and Donts for their selection and use. In particular, we strongly recommend taking a routinely sceptical approach, including the use of orthogonal chemical and biological reagents; the use of at least two different chemical series (chemotypes) of probe along with inactive control compounds; demonstration of potency and selectivity; and obtaining evidence for selective target engagement and modulation in cells (e.g. using thePharmacological Audit Trail).

We advise (on page 13 of the Perspective) strongly against a common and dangerous practice, which is to expose cells with ever increasing concentrations of a chemical probe until a desired cell effect (phenotype), usually cell death, is seen and then attributing this phenotype to the specific effect of the probe on the protein target under investigation.

Higher probe concentrations increase the likelihood of off-target effects and the general range that should not be exceeded is 10-20 micromolar to minimize non-specific effects. Accompanying biomarker evidence of target modulation is also important.

Alongside the general guidance provided in our Cancer Cell Perspective, we strongly recommend the use of the Chemical Probes Portalfor expert advice and ratings for specific probes and targets.

We likenthe provision of advice on the selection and use of chemical probes to ensuring the biological researcher avoids being equipped with the equivalent of a defective global positioning/satellite navigation system, as illustrated in the cartoon below:

Download a larger version of Professor Julian Blagg's cartoon (PDF, 51KB)

Caption: The right way and wrong way with chemical probes

We finish the Perspectivewith the following strong new call-to-action:

We need to maximize the promise and minimize the peril of chemical probes and this requires the broad research community to use high-quality chemical probes that have been critiqued with equivalent rigor to biological reagents. It is time to put our house in order and biologists as well as chemists have an important responsibility to do so.

Im grateful to my colleague and joint senior co-author of our Cancer Cell PerspectiveProfessor Julian Blaggfor his excellent collaboration and insights. We developed the content of the Perspective very much in partnership. I also thank Julian for drafting the cartoon illustration.

In addition, I thank many colleagues and collaborators for helpful discussions and input, including the anonymous reviewers of the Perspective, and those in the field whose outstanding work we have built upon.

Excerpt from:
Call to bioscientists: choose and use your chemicaI probes very carefully - The Institute of Cancer Research (blog)

July 10, 2017 by Jade Boyd Researchers from Rice University and Baylor College of Medicine have shown they initiate a process called tubulogenesis that is crucial to the formation of blood-transporting capillaries. In microscopic images taken a different times during a weeklong experiment, researchers tracked the changes in cells (green) and cell nuclei (orange) using fluorescent markers. Credit: Jeff Fitlow/Rice University

In their work toward 3-D printing transplantable tissues and organs, bioengineers and scientists from Rice University and Baylor College of Medicine have demonstrated a key step on the path to generate implantable tissues with functioning capillaries.

In a paper published online in the journal Biomaterials Science, a team from the laboratories of Rice bioengineer Jordan Miller and Baylor College of Medicine biophysicist Mary Dickinson showed how to use a combination of human endothelial cells and mesenchymal stem cells to initiate a process called tubulogenesis that is crucial to the formation of blood-transporting capillaries.

The work is an important step with fragile endothelial cells (ECs) made from "induced pluripotent stem cells," or iPSCs, a type of cell that can potentially be made from the cells of any human patient. Because iPSCs can be patient-specific, researchers hope to find ways of using them to generate tissues and replacement organs that can be transplanted without risk of rejection by a patient's immune system. But the fragility of endothelial cells during laboratory growth has limited the utilization of this critical cell type, which is found in all vasculature.

"Our work has important therapeutic implications because we demonstrate utilization of human cells and the ability to live-monitor their tubulogenesis potential as they form primitive vessel networks," said study lead author Gisele Calderon, a graduate student in Miller's Physiologic Systems Engineering and Advanced Materials Laboratory.

"We've confirmed that these cells have the capacity to form capillary-like structures, both in a natural material called fibrin and in a semisynthetic material called gelatin methacrylate, or GelMA," Calderon said. "The GelMA finding is particularly interesting because it is something we can readily 3-D print for future tissue-engineering applications."

Tissue engineering, also known as regenerative medicine, is a field aimed at integrating advances in stem cell biology and materials science to grow transplantable replacement tissues and organs. While tissue engineers have found dozens of ways to coax stems cells into forming specific kinds of cells and tissues, they still cannot grow tissues with vasculaturecapillaries and the larger blood vessels that can supply the tissues with life-giving blood. Without vascularization, tissues more than a few millimeters in thickness will die due to lack of nutrients, so finding a way to grow tissues with blood vessels is one of the most sought-after advances in the field.

Miller, who earned his Ph.D. at Rice in 2008, has studied vascularization in tissue engineering for more than 14 years. During his postdoctoral studies at the University of Pennsylvania, he also became heavily involved in the open-source 3-D printing movement, and his work at Rice combines both.

"Ultimately, we'd like to 3-D print with living cells, a process known as 3-D bioprinting, to create fully vascularized tissues for therapeutic applications," said Miller, assistant professor of bioengineering. "To get there, we have to better understand the mechanical and physiological aspects of new blood-vessel formation and the ways that bioprinting impacts those processes. We are using 3-D bioprinting to build tissues with large vessels that we can connect to pumps, and are integrating that strategy with these iPS-ECs to help us form the smallest capillaries to better nourish the new tissue."

Each of the trillions of living cells in the human body are constantly supplied with oxygen and nutrients by tiny blood vessels known as capillaries. Measuring just a few thousandths of a millimeter in diameter, some capillaries are so narrow that individual blood cells must squeeze through them in single-file. Capillaries are made entirely from networks of endothelial cells, the type of cell that lines the inner surface of every blood vessel in the human body.

In the process of tubulogenesisthe first step to making capillariesendothelial cells undergo a series of changes. First, they form small, empty chambers called vacuoles, and then they connect with neighboring cells, linking the vacuoles together to form endothelial-lined tubes that can eventually become capillaries.

"We expect our findings will benefit biological studies of vasculogenesis and will have applications in tissue engineering to prevascularize tissue constructs that are fabricated with advanced photo-patterning and three-dimensional printing," said Dickinson, the Kyle and Josephine Morrow Chair in Molecular Physiology and Biophysics at Baylor College of Medicine and adjunct professor of bioengineering at Rice.

In the study, Calderon, Rice undergraduate Patricia Thai and colleagues investigated whether commercially available endothelial cells grown from iPSCs had tubulogenic potential. The test examined this potential in two types of semisolid gelsfibrin and GelMA. Finally, the researchers also investigated whether a second type of stem cell, human mesenchymal stem cells, could improve the likelihood of tubulogenesis.

Calderon said fibrin was chosen for the experiment because it's a natural material that's known to induce tubulogenesis for wound healing. As such, the researchers expected endothelial cells would be induced to form tubules in fibrin.

Calderon said the first step in the experiments was to develop a third-generation lentivirus reporter to genetically modify the cells to produce two types of fluorescent protein, one located only in the nucleus and another throughout the cell. This permanent genetic modification allowed the team to noninvasively observe the cell morphology and also identify the action of each individual cell for later quantitative measurements. Next, the cells were mixed with fibrin and incubated for a week. Several times per day, Calderon and Thai used microscopes to photograph the growing samples. Thanks to the two fluorescent markers, time-lapse images revealed how the cells were progressing on their tubulogenic odyssey.

Calderon conducted advanced confocal microscopy at the Optical Imaging and Vital Microscopy Core facility at Baylor College of Medicine. Calderon and Thai then used an open-source software called FARSIGHT to quantitatively analyze the 3-D growth patterns and development character of the tubulogenenic networks in each sample. In fibrin, the team found robust tubule formation, as expected. They also found that endothelial cells had a more difficult time forming viable tubules in GelMA, a mix of denatured collagen that was chemically modified with methacrylates to allow rapid photopolymerization.

Over several months and dozens of experiments the team developed a workflow to produce robust tubulogenesis in GelMA, Calderon said. This involved adding mesenchymal stem cells, another type of adult human stem cell that had previously been shown to stabilize the formation of tubules.

Miller said that while clinical applications of 3-D bioprinting are expected to advance rapidly over the next few decades, even small tissue samples with working capillary networks could find use much more quickly for laboratory applications like drug testing.

"You could foresee using these three-dimensional, printed tissues to provide a more accurate representation of how our bodies will respond to a drug," Miller said. "Preclinical human testing of new drugs today is done with flat two-dimensional human tissue cultures. But it is well-known that cells often behave differently in three-dimensional tissues than they do in two-dimensional cultures. There's hope that testing drugs in more realistic three-dimensional cultures will lower overall drug development costs. And the potential to build tissue constructs made from a particular patient represents the ultimate test bed for personalized medicine. We could screen dozens of potential drug cocktails on this type of generated tissue sample to identify candidates that will work best for that patient."

Explore further: Giving blood to artificial organs

More information: G. A. Calderon et al. Tubulogenesis of co-cultured human iPS-derived endothelial cells and human mesenchymal stem cells in fibrin and gelatin methacrylate gels, Biomater. Sci. (2017). DOI: 10.1039/C7BM00223H

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Houston team one step closer to growing capillaries - Phys.Org

Dongeun Huh, PhD, from the University of Pennsylvanias Department of Bioengineering, is the recipient of theCancer Research Institutes (CRI) inauguralTechnology Impact Awardto advance the field ofimmuno-oncology.

Huh will receive a $1 million grant paid over three years to develop a microchip-based research model that mimics human cancer and immune cell interactions, a technological innovation that has the potential to accelerate the development of effective immunotherapies across different types of cancer.

There is an urgent need within the research community for new ways to model, observe, and interrogate complex interactions between the human immune system and tumorsa dynamic interplay that current two-dimensional cell cultures and animal models cannot characterize optimally, Jill ODonnell-Tormey, PhD, CRIs chief executive officer and director of scientific affairs, said in apress release.

Dr. Huhs microchip-based human cancer models represent a highly innovative intersection of cell biology and microengineering, which, when applied to cancer immunotherapy research, may spur advances in our understanding of how malignant human tumors interact with the immune system and surrounding tissues, providing researchers with new insights that will lead to improved cancer treatments, she added.

Huh has teamed up with two University of Pennsylvania immunologists to carry out the project: E. John Wherry, PhD, and G. Scott Worthen, MD. Together, they will oversee the investigation of cancer-immune cell interactions using Huhs technology.

Huh and his colleagues will use new bioengineering technology to apply microfabrication techniques originally developed for manufacturing computer chips to create a cancer-on-a-chip micro-device, which would enable cultures to be made of patient cancer cells.

The model will be engineered to form a network of living blood vessels that simulate the same vessels that immune cells use to circulate inside our bodies. Using this platform, Huh expects to study the interactions between cancer cells with key components of the immune system involved in cancer elimination, such as macrophages and T-cells.

Huh believes the research will lead to discoveries that could pave the way for new strategies to treat cancer with immunotherapies. In addition, the model could be developed into a screening platform to test and predict the effectiveness and safety of new drug candidates without having to test them on patients first.

Dr. Huhs proposal is truly exciting in terms of its potential to make an impact on how cancer immunotherapy research is conducted as well as how we predict patient response to immunotherapy, said Mark M. Davis, PhD, professor of immunology at Stanford Universitys School of Medicine and chair of the CRI Technology Impact Awards scientific committee.

This is sorely needed, and we also think this project could help us develop strategies that will extend the benefits of immunotherapy to more and more patients, he added.

The CRI Technology Impact Award is a new program designed to offer scientists and researchers a platform for multidisciplinary collaboration to transform the field of immuno-oncology. Organizers hope to fund technologies that improve our understanding of the antigenic profile, cellular interplay, and mechanistic pathways within the tumor microenvironment that are critical for an effective anti-tumor response. By supporting the development of highly innovative technologies, the program aims to enable researchers to develop the next generation of cancer immunotherapies.

See the rest here:
Cancer Research Institute Announces $1 Million Technology Impact Award Winner - Immuno-Oncology News