Melatonin Research May Aid in Adjusting Biological Clocks

For the first time, scientific collaborators at the Jacobs School of Medicine and Biomedical Sciences, the University of North Carolina School of Medicine (UNC) and the University of California San Francisco (UCSF) created molecules to selectively bind to MT1 melatonin receptors on the surface of cells and modulate circadian rhythms.

“This discovery allows us to now focus on the development of unique new molecules to generate a response that will help bring sleep patterns and other biological rhythms in line with environmental light and dark cycles, providing the sense of well-being that is only experienced when such rhythms are in sync,” says Dubocovich, SUNY Distinguished Professor of pharmacology and toxicology and senior associate dean for diversity and inclusion.

The research, published Feb. 10 in Nature, will greatly facilitate the development of targeted therapies that can either mimic or counteract the actions of melatonin, which is implicated in numerous circadian disorders ranging from depression, blindness, seasonal affective disorder and sleep disorders to difficulties experienced as a result of jet lag and shift work.

Dubocovich is senior author and one of three corresponding authors. The others are Brian K. Shoichet, PhD, professor in the Department of Pharmaceutical Chemistry at UCSF, and Bryan L. Roth, MD, PhD, Michael Hooker Distinguished Professor in the Department of Pharmacology at UNC.

They note that the new research represents a remarkable confluence of major, complementary achievements and state-of-the-art expertise at three institutions. All three are members of the Clinical and Translational Science Awards Program of the National Center for Advancing Translational Sciences of the National Institutes of Health.

These discoveries are:

• molecules — never made before or present in nature — unrelated to any known melatonin receptor ligand through the computational docking (simulating three-dimensional binding) of more than 150 million diverse, “make-on-demand” library of molecules (UCSF)
• molecules (UCSF 7447 and UCSF 3384) that attach with high strength and selectivity to human or mouse MT1 melatonin receptors — they generate cellular responses opposite to that of melatonin, which provided the rationale for the mouse circadian behavior (UNC)
• translation of cellular responses seen in vitro directly to in vivo function, slowing down adjustment to a new environmental light/dark period in the mouse model of jet lag, with one unexpected finding demonstrating that they mimic melatonin to modulate rhythms in the absence of light cues (Jacobs School)

This work was further facilitated by the publication of the first crystal structure of the MT1 receptor, providing the team with the “template” to fit new melatonin molecules into the receptor pocket.

“For us, it was at first exciting to see the novelty of the new ligands that emerged from fitting members of an ultra-large chemical library into the receptor structure,” Shoichet says. “That’s what one always hopes for in a structure-based program: finding new chemistries not imaginable from knowing the endogenous ligand (here, melatonin). What made this doubly exciting was to see the new chemistries lead to new signaling, in the Roth lab experiments, and to unexpected mouse pharmacology in the Dubocovich lab.”

Roth adds: “My UNC lab spent more than a year characterizing the pharmacology and drug-like properties of the molecules before we could hand them off for animal testing in the Dubocovich lab. We were all excited to see that the new compounds Brian and I had discovered had interesting properties in mice.”

The new research caps what Dubocovich says has been her 15-year search to discover MT1 ligands.

“Ever since we demonstrated that melatonin’s effect of resetting biological clocks in vivo circadian models occurs through actions at the MT1 receptors, we have focused through various collaborations on searching for ligands that would better fit the human melatonin receptor,” Dubocovich says. “Our hope has always been to find selective MT1–type molecules, either one that works to modulate circadian rhythm responses as with melatonin, or its opposite as with the molecules discovered in this study.”

The ultimate goal, she says, was always to develop drugs that could address all the disorders that disrupted circadian rhythms can cause.

“When Brian Shoichet called to ask about our interest in testing in our circadian mouse models the novel molecules they had identified from his ultra-large library of over 150 million compounds, we were eager to collaborate!” she adds.

The availability of the UCSF vast virtual library was a critical aspect of the research. Dubocovich describes it as a “gold mine” of millions of molecules with distinct shapes, many of which have never been synthesized or seen in nature, and all of them available for mining and “docking” (fitting) into the pocket of the targeted receptor. The team advanced the research directly from discovery of these molecules to the assessment of their ability to mimic or oppose the effect of melatonin, to in vivo demonstrations of how these molecules impact the animals’ circadian function.

The team found it especially interesting that the two molecules discovered in this study generate two distinct and opposite mouse circadian responses that are dependent on clock time and the environmental light conditions that the animals experience.

In the experiments where the onset of dark is advanced — known as reentrainment or the jet lag model — the molecules slow this reentrainment, an effect opposite to that of melatonin. However, when mice were exposed to constant dark, the two molecules demonstrate an effect identical to that of melatonin.

“This could be potentially useful to entrain rhythms to the 24-hour day in populations removed from natural light/dark exposure, including the blind as well as some shift workers, submarine workers or those working in extreme environments, such as polar explorers,” Dubocovich says.

This can also help those who travel between time zones and suffer from jet lag.

“When the body is exposed to an abrupt change in the light/dark cycle — like what we experience when we travel across continents — there isn’t sufficient time for the biological clock to adjust upon reaching the destination,” Dubocovich explains.

“Giving these new molecules at the appropriate clock time under a light/dark cycle would allow us to decelerate our ability to adjust to the new environment, potentially providing a treatment for certain types of jet lag and, more importantly, addressing other conditions affected by circadian rhythm disruptions, such as shift work, sleep disorders and depression,” she adds.

This finding reinforces the increased interest in chronopharmacology, the premise of which is that drugs given at the time when a patient’s biological clock is ready to receive them — given conditions of a precise time of day and environmental lighting — will produce a more effective outcome.

Dubocovich says the next step will be to identify the molecular and signaling pathways that translate the response exerted by these molecules from the time they interact with the receptors in the biological clock to the ultimate circadian behavior expressed in a mouse or human.

Dubocovich had high praise for others on the research teams.

“Each team included talented and hardworking junior scientists contributing to the projects with precision and high standards,” she says.

Grant C. Glatfelter, PhD, a doctoral candidate in pharmacology in the Dubocovich lab at the time of the research and now a postdoctoral research fellow at the National Institute on Drug Abuse, is a co-first author.

“Grant worked for three full days and nights at the time to assure each single mouse received the drugs at the time the biological clock was ready to adjust clock time,” Dubocovich says.

Anthony J. Jones, PhD, a doctoral candidate in neuroscience in Dubocovich’s lab at the time of the research and now a postdoctoral associate of pharmacology and toxicology, is a co-author.

Reed Stein, a doctoral candidate in the UCSF Department of Pharmaceutical Sciences and Pharmacogenomics program, is lead first author.

“Reed discovered the virtual molecules and gathered and assembled the data from each team, checking every number and every table and every figure before emailing back the most in-depth and challenging questions — at times not easy to answer,” Dubocovich says.

John McCorvy, PhD, now at the University of Wisconsin, and Hye Jin Kang, PhD, both first co-authors and postdoctoral fellows at UNC at the time of the research, tested each molecule — defining the pharmacological properties and determining the direction of the responses — before they were delivered to Dubocovich’s team for further in vitro testing by Jones and in vivo testing by Glatfelter.

The research was supported by National Institutes of Health awards.