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Re-creating cardiac tissue

Re-creating cardiac tissue

A Heart in a Petri Dish

For medical research in the 21st century, regenerative medicine offers one of the most promising futures and prospects for further development. Revolutionary results have already been achieved by the efforts of genetic engineering, although ethical and regulatory aspects mean that such methods are unlikely to see widespread deployment.

A complementary approach is now being pursued by the application of small-molecule ­compounds: a rapidly growing number of organic molecules are now available, which can influence the maturation processes by which ­precursor cells become specific types of tissue. This opens up the door to treatment techniques that are based on the basic principles of “normal” pharmaceuticals.

Depending on the type of tissue involved, the human body varies greatly in its ability to regenerate damaged organs. While skin and the liver possess highly advanced healing mechanisms, for example, at the other end of the spectrum we have neurons and cardiac muscle cells, which have very limited capabilities for self-repair. Such distinctions are increasingly playing a role due to a trend towards rising life expectancy, especially in developed countries: a fulfilling life in old age can only be ensured if as many bodily functions as possible are in good working order. As a consequence, particular attention is being made to research work that addresses the regeneration of organ function after suffering damage. For some disease conditions found in civilised societies (such as heart failure, neurodegenerative diseases and diabetes), there are currently no treatment options available that lead to a complete recovery of original organ function.


New insights into cell differentiation

Regenerative medicine concerns itself precisely with these research topics and objectives. During recent years, ground-breaking results have been achieved in this field. On the one hand, we have considerably improved our understanding of the processes by which precursor cells differentiate into mature tissue and organs (see sidebar: “Precursor cell types”). On the other hand, these insights have led to new pathways to treatment options that restore the original tissue function. Most of the approaches in this area to date have followed strategies based on “re-programming” cells by the targeted introduction of transcription factors. Thanks to these studies, our knowledge about cell differentiation has dramatically increased over the last few years. Genetic engineering of this kind is generally suitable only as a research tool, however: the likelihood of broad therapeutic application seems questionable, given the ethical aspects and regulatory conditions governing its use.

Yet, due to our growing knowledge of differentiation processes in cells at a molecular level and the related screening techniques that are now becoming available, recent years have witnessed the discovery of a steadily increasing number of small-molecule compounds capable of influencing cell maturation towards specific types of tissue. As these kinds of compounds resemble standard active pharmaceutical ingredients (APIs), they offer considerable advantages in terms of their broad application in medicine: (i) When developing an active ingredient of this type, one can draw on an established body of knowledge from pharmaceutical chemistry. (ii) Well-established approval processes exist for small-molecule drug substances, for the market launch of these APIs as products following an appropriate review of their benefit-risk profile. (iii) Treatment can be administered over a specific period without making genetic modifications to the organism being treated.


Tissue from the test tube. The treatment of the patient's own cellular material with small-molecule APIs could lead to the functional repair of damaged organs in the foreseeable future. Photo: VUT

Lead substances for cardiac muscle cells

The regeneration of damaged cardiac muscle tissue is of particular significance for industrialised nations, since heart failure and its consequential effects make up one of the three leading causes of death in such countries. In this context, the discovery of Cardiogenol C [1] as a hit compound by a team of researchers in the USA truly catalysed work in this area (see sidebar: “Small molecules for tissue regeneration”). This compound is capable of inducing embryonic stem cells to differentiate into cardiac muscle cells. While this effect had already been discovered for naturally-occurring substances such as retinoic acids, the latter compounds often trigger non-selective differentiation into a range of tissue types.

Basing our work on these results with Cardiogenol C, we have spent the last few years conducting a systematic investigation of this class of compounds. Medicinal chemistry techniques such as “bioisostery” featured strongly in such work. This method replaces structural aspects of the lead substance with functional groups possessing certain similarities but also slight variations in terms of their physical chemical properties: the goal is thus to fine-tune the compound’s pharmacological effect. Synthesis procedures driven by automation were also used extensively (microwave chemistry, continuous-flow chemical synthesis): these enable rapid access to highly-focused substance libraries.


fig. Interventional goals for tissue regeneration using small-molecule APIs of primary interest for their relevance in the treatment of lifestile diseases.

In the course of such work to date, we have succeeded in significantly increasing hit compound activity in a mouse model. In addition, the substance VUT-MK142 establishes a new lead compound that is capable of inducing differentiation into cardiac muscle cells not only from embryonic stem cells (P19) but, beyond this, to achieve the same by “re-programming” pre-existing, pre-differentiated precursor cells for skeletal muscle (C2C12). This constitutes a key step towards future use as a treatment method, since it obviates the direct dependency on embryonic stem cells, which are not only difficult to obtain but are also potentially subject to ethical restrictions. In a second generation of modifications, more radical changes were then made to the lead substance (including alterations to its underlying heterocyclic ring system), which culminated in triazines [2] as a new structural class featuring an altered activity profile. With the VUT-MK142 compound, it ultimately proved possible to produce autonomously beating cell clusters of functional cardiomyocytes [3].

Another interesting “side effect” of this research work was the discovery of a class of compounds that had the effect of speeding up the differentiation of precursor cells into skeletal muscle tissue [4]. In the long term, this might offer the possibility of accelerating the treatment of muscle fibre injuries. This discovery is all the more important for showcasing the extraordinary diversity that can be seen in the influence of small molecule compounds on cellular development processes. In this context, the availability of sensitive, high-throughput procedures for substance testing is of central importance, as they facilitate the identification of potent new lead compounds.


Notwithstanding these achievements, differentiation into target cells via induction by small-molecule APIs is only half the story in terms of a potential future therapy, since this technique is always dependent on the availability of a sufficient quantity of precursor cells. In this context, it is interesting that discovery of the compound reversine also identified a lead substance that permits the inversion of cell differentiation for mature tissue cell lines into more “pliable” developmental stages (progenitor cells). This inversion has been successfully demonstrated by “reverse differentiation” of skeletal muscle cells, followed by their subsequent differentiation into osteoblasts or, alternatively, adipocytes [5].

Taken together, these API candidates offer the prospect of actually being able to set our sights on regenerative tissue treatments that are based on easily-extractable cell types that are available in sufficient quantities. It is of course true that the majority of results achieved to date must be duly transferred from the animal cell model to human cell lines, which will require particular efforts in basic research. The regeneration of organ function also presents potential obstacles in the form of constructing complex three-dimensional structures from a range of tissue types (perhaps aided by bio-compatible polymers), followed by successful re-implantation into the patient. While solutions to such obstacles will be needed, the potential diversity shown in the restoration of damaged tissue is nonetheless reflected in the fact that, alongside the regeneration of cardiomyocetes, other synthetic compounds have been identified. These can stimulate differentiation into neurons, for example [6], or can positively influence the functionality of pancreatic ß cells in terms of their insulin production.

In summary, the rapid progress made over the last few years in research into the induction of regenerative processes by small-molecule substances has thrown open the door to potential applications in medicine. On the basis of the scientific evidence it provides, we can contemplate novel forms of therapy that, mere decades ago, would have been dismissed as science fiction.

Portions of the research work presented here were completed in close collaboration with colleagues from the Medical University of Vienna.

Bibliography
[1] Wu, X. et al. (2004), J. Am. Chem. Soc. 126, 1590
[2] Mihovilovic, M.D. et. al., Austrian Pat. Appl. AT 511441; PCT Pat. Appl. PCT/AT2012/050140
[3] Koley, M. et al. (2013), Med. Chem. Commun. 4, 1189
[4] Schnürch, M. et al. (2011), Bioorg. Med. Chem. Lett. 21, 2149
[5] Chen, S. et al. (2007), Proc. Natl. Acad. Sci. U S A. 104, 10482
[6] Williams, D. R. et al. (2007), Am. Chem. Soc. 129, 9258; Warashina M. et al. (2006), Angew. Chem. Int. Ed. 45, 591
[7] Fomina-Yadlin, D. et al. (2010), Proc. Natl. Acad. Sci. U S A. 107, 15099

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L&M orient 1 / 2014

The articles are publishes in issue L&M orient 1 / 2014.
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