SCIENTIFIC COMMUNIQUÉ II
Following the positive response to SCIENTIFIC COMMUNIQUÉ I, released in April 2018, CLINUVEL will now publish a longer SCIENTIFIC COMMUNIQUÉ series throughout the course of 2018. The goal of the SCIENTIFIC COMMUNIQUÉS is to share the current understanding and progress in proopiomelanocortin (POMC) science and photomedicine with various interested audiences. SCIENTIFIC COMMUNIQUÉ I provided an outline of the various physiological modifications taking place on proteins and the clinical relevance to our technology programs. In SCIENTIFIC COMMUNIQUÉ II we delve into how ligands bind to the various cellular receptors, signalling cascades and output to arrive at therapeutically meaningful applications. In SCIENTIFIC COMMUNIQUÉ III –scheduled for June – we will review the effects of afamelanotide, CLINUVEL’s lead drug, on the human genome, based on existing literature. These three pieces will then be used as a basis for further COMMUNIQUÉS later in the year. After the COMMUNIQUÉS we hope the reader will be able to grasp the, often opposing, opinions found in the abundance of literature on relevant topics. A library of scientific terms has been established on CLINUVEL’s website allowing the reader to follow in time. LIGANDS – MOORING AND BERTHING OF THE VESSEL CLINUVEL’s attention has long been on the family of melanocortin receptors, and specifically the melanocortin-1 receptor (MC1R). This receptor is expressed on a number of epidermal and dermal cells but also at various other locations in the human body. In our journey, we simultaneously started to investigate the endothelin-1, cKIT, CSF and MAP kinases. All of these deserve further discussion in a subsequent COMMUNIQUÉ, but it is sufficient here to state that our scientific teams are taking a broader direction when evaluating the applicability of the melanocortin family.
The study of the cellular membrane receptors, their expression, behaviour, and up and down regulation under various conditions, makes pharmacology and the area of pharmacogenomics a dynamic subject.
Figure 1: MC1 receptor
The MC1R consists of 317 amino acids and belongs to the family of G-protein coupled receptors (GPCRs), a receptor spanning the cell membrane over seven passes (seven winded pathways), also called transmembrane. Common to all GPCRs is that they pick up extracellular signals to pass them on to the cellular environment. Of high interest here are the signals provided by hormones, peptides which are transmitted through a cascade further into the cell. MC1R was first found in 1994 to encode for chromosome 16q24.3, and further genotyping followed years later. In reference to SCIENTIFIC COMMUNIQUÉ I, proteins undergo a wide variety of processes, and the protein receptor MC1R is subject to palmitoylation at one end of the molecule within the cell. The function of adding cysteine thiols on a transmembrane protein has been widely speculated, yet the consensus is that it regulates cell function and homeostasis and, critically, acts as a protein membrane associative signal. Conversely, the lack of the critical palmitoyltransferase (PAT) – an enzyme active during the process – is associated with various diseases of the nervous system and cancer progression. Humans are known to have 23 different palmitoyltransferases, and many proteins which are palmitoylated are associated with neuronal development.
Stepping back to the subject of the receptor itself, the further study of exploring a mooring and berthing site has proven essential to grasp the quality of the cellular signalling origin, in this case the melanocyte and other epi-/dermal cells, but also white blood cells. In the 1990’s, a number of research groups started to hone in on the importance of the MC1R quality and its possible consequences on the human response. In 2007, a Queensland research group started reporting its findings that loss-of-function alleles of the MC1R seen in Caucasian individuals made them more sensitive to ultraviolet (UV) radiation, a long-held view but now demonstrated in a series of human experiments. Large scale studies found that the polymorphisms of this receptor were mostly found in red haired individuals, this became later known in the field as the red hair colour (RHC)-blue eyes-freckled phenotype.
Figure 2: The quality of the MC1R resembles the optimum mooring site
MOORING AND SIGNALLING When reviewing the ability of drugs to bind to GPCRs, a number of analytical methods serve us to assess the strength of binding as well time to dislodge from the receptor. Classical assays were once the common method of evaluating the interaction between receptor and drug. Now we use methods such as free-energy perturbation (FEP) and thermodynamic integration (TI) which hold the promise of more accurately predicting the free energy of binding. FEP and TI are, however, still subject to debate as to their wider applicability in drug development and clinical interpretation of results.
In coming back to our main focus, the MC1R, CLINUVEL’s early knowledge gained on the strength of melanocortin binding and dissociation from the receptor has proven essential in choosing a clinical development program. Specific data and intelligence gained during investigative pharmacology ultimately decide the fate of a molecule. Thorough understanding of the differences of the pharmacology in vitro and in humans is essential to arrive at the desired application. In addition, the advances in modern medicine may confirm or negate one’s decisions on deploying new drugs; within CLINUVEL this is part of a scientific awareness which needs to be maintained at all times. An applicable example is how only recently the medical community has acknowledged the differences of MC1R function in various species of animals, and even in man.
Classically, scientists compared the binding strength of various drugs to each of the five melanocortin receptors, MC1R, MC2R, MC3R, MC4R and MC5R. However, individual assays are now able to compare ligands binding to the same receptor to make a faint prediction of how the molecule would behave in man. Outside the scope of this piece, the five melanocortin receptors are found in different parts and organs of the human body, and all regulate various functions such as obesity, thermoregulation, inflammation, immunomodulation, sexual behaviour, and pigmentation. As stated the MC1R is mainly found on the plasma membrane of the melanocyte – pigment producing cell in skin – but also on fibroblasts, cells lining blood vessels and white blood cells. How various molecules behave in interaction with the target receptors depends on a multitude of factors and is the subject of modelling studies attempting to replicate the desired or even undesired physiological effect(s). The distinction in modelling for use under physiological and pathological conditions is vast, and goes beyond this piece, but is relevant to CLINUVEL’s work. MC1R VARIANTS AND IMPACT Due to the thoughts of Dr T.B. Fitzpatrick, first published in 1975 and later expanded upon, dermatologists, photobiologists and physicists have followed his classification of skin types, consisting of skin types found in a sample of the general population. In this original classification, only skin types V and VI enjoy full melanoprotection. These thoughts are the focus of fierce debate in the relevant fields of dermatology, but are maintained due to lack of a better classification.
Figure 3: The Fitzpatrick skin types classification
Although our teams still work with the Fitzpatrick skin types classification it is deemed too crude to accurately describe a patient’s (in)tolerance to light and sun. In time, we strive to introduce a new classification in the field of photomedicine which will need to be more precise in reflecting the constitutive dermal and epidermal properties of patients of different melanin density, and tolerance to light and sun.
Essential work was performed for decades by various Australian, European and American universities on the role of receptor variants and risk of skin cancer. Simultaneously with our research, the understanding of the ability of alpha-melanocyte stimulating hormone to ‘override’ partial-loss-of-functions with new ligands has grown. One of the research objectives has been to
investigate the signalling functions of MC1R in models, but mostly relevant to man.
Figure 4: Variety in skin complexions, with lower melanin density increasing the propensity to ‘burn’ in response to UV radiation
When looking at the functionality of the MC1R and risk for incurring skin cancer(s), we tend to define the MC1R gene as one which is polymorphic and gives rise to allelic variants of the receptor. During the past two decades we have come to know the most frequent MC1R variants, Arg160Trp, Asp294His, Arg151Cys, Asp294His and Val92Met, but there are many more. These variants cause the receptor to function in a slightly deficient manner and, as highlighted at the start, partial loss of function results in greater human susceptibility to both various disease and damage from UV radiation. This is the subject of genetic and epigenetic studies, whereby major scientific leaps have been made the past decades. It is predicted that the individual susceptibility to various skin cancers will become relevant in finding targeted treatments, and this is highly relevant to our fields of research and interest. However, to add to another part of the puzzle, it was Dwyer and colleagues who had demonstrated a decade ago that Caucasian men living in Australia, those with 0–1 melanin density (MD) units, had six or more times risk of skin cancer than those with three or more MD units. Put simply, greater melanin equals greater protection and this in turn depends on good signalling of the melanocyte.
In summary, increasing evidence points to the MC1R, the quantum of melanin output, and constitutive propensity to respond to UV radiation, as main determinants of an individual’s skin cancer risk. OUTPUT Melanocytic activity – originating from MC1R and cAMP (intracellular messenger) – aims to provoke eumelanisation of the skin (see SCIENTIFIC COMMUNIQUÉ I). While eumelanin is seen as photoprotective, the counterpart pheomelanin is largely viewed as photoreactive, mostly in response to UVA (wavelength 320-400nm). In photophysics we investigate the absorptive, reflective, refractive and other optical properties of various pigments. In medical applications, we collectively have researched the ability to block emitted photons for some time, but also looked at using the absorptive capacity in techniques such as photodynamic therapy in superficial tissue lesions. Eumelanin comes in various forms, granules, particles and ‘dust’, depending on constitutive skin type. Therefore, the photoabsorptive properties differ between individuals and skin types.
Our work focused on thoroughly understanding UV-induced cellular damage due to radical and reactive oxygen species – mostly abbreviated as ROS – and oxidative injury. The current work and future technology will most certainly address these highly relevant issues. Cellular and proteonomic work is progressing, and understanding of the function of receptor, cellular environment, genomic mechanisms, and external variables are coming together in CLINUVEL’s next generation of pharmaceutical products.
In deepening our understanding, we typically find that MC1R protein expression is typically low, with approximately 700 units expressed per healthy melanocyte, and significantly higher numbers on mutated cells such as melanoma cells.
Figure 5: Melanin distinction to produce either photoprotective eumelanin or photoreactive pheomelanin RELEVANCE TO CLINUVEL’S TECHNOLOGY Historically our scientific professionals paid attention to the pigmentary response induced by UV exposure. The irradiation of the epi-/dermis brings about a number of instantaneous and delayed changes which are of great relevance.
In contrast, as we have discussed previously, the use of a hormonal analogue elicits various responses, of which one is the melanogenic response without UV radiation. Our scientific teams view this application of the analogue as a form of biomimicry, a way to emulate the biological response seen to light and UV irradiation under daily conditions.
We understood, early on, the potential and limitations of the melanogenic system and its pharmacological applications. Questions arose on the desired and ideal depth, length, location, and origin of pigmentation. By systematically working along decision trees, we arrived at working hypotheses and further applications of our hormonal therapy targeting MC1R. Decisions are continuously taken as CLINUVEL’s teams generate more data to further our knowledge. The colloquial “tip of the iceberg” is applicable here.
In the past, we discussed the strengths and weaknesses of pioneering in technology, but for this purpose it suffices to state that our collective attitude is to incrementally, and more so concentrically, expand our applications from the most recent knowledge gained. This approach reduces the risk of venturing into costly R&D projects with all too uncertain outcomes.
Figure 6: Concentric expansion of CLINUVEL’s attention
Originally, the scientific community debated the function of MC1R and its association with the pigmentary output; as discussed in SCIENTIFIC COMMUNIQUÉ I.
Whether the melanocytic output is eumelanin (brown) or pheomelanin (red-yellow) is predominantly determined by our constitution and genetic makeup, however – as we now know – activity of the cell through strong receptor binding is important to generate the required cellular output.
In the past decade our scientific understanding has progressed by finding epidemiological and statistical relations between the functioning of MC1R and individual sensitivity to UV radiation and skin cancer. This link between MC1R function and skin cancer has been of immense importance to forewarn individuals at high risk of contracting the disease at a later age.