ReviewPharmacosynthetics: Reimagining the pharmacogenetic approach
Introduction
The gap between receptor mediated signaling and the ultimate functional output of the brain is shrinking. The past decade has witnessed the advent of multiple technologies that allow exquisite manipulation of neurons in an intact animal, providing the opportunity to definitively determine the neuronal correlates of complex brain function. The primary technologies are optogenetic – the modulation of transgenic receptors and channels via photons- and pharmacogenetic – the modulation of transgenic receptors via pharmacologic agents. This review will focus on pharmacogenetics.
First and foremost, the authors present an alternative name for this technology to disambiguate the topic from other uses of the word pharmacogenetic. The term “pharmacogenetic” is already an established MeSH (Medical Subject Headings) term, defined as “a branch of genetics which deals with the genetic variability in individual responses to drugs and drug metabolism.” This word has been well adopted, retrieving 3318 results from PubMed as of publishing date, has been in use for an extended period of time (Gonzalez-Vacarezza et al., 2012, LaDu, 1972, Weinshilboum et al., 1999) and is the foundation of personalized medicine (Cohen, 1997, Kohane, 2012). For similar reasons, the term “chemical genetics” (or its portmanteau chemicogenetic), while not being assigned its own MeSH term, has been defined as “the study of gene-product function in a cellular or organismal context using exogenous ligands” (Stockwell, 2000). Here we present alternative terminology and reimagination of pharmacogenetics (the modulation of transgenic receptors via pharmacologic agents) as pharmacosynthetics. This term integrates the true meaning and functional mechanisms of the technology: pharmaco- meaning drug and -synthetic meaning the combination of two or more parts in an artificial manner. Pharmacosynthetics provides a clear distinction from both pharmacogenetics and chemicogenetics, and as of publishing date, retrieves no results in PubMed or Wikipedia.
We present the formal definition of pharmacosynthetics as “a branch of biology which deals with the creation of pharmacological modulation using artificial components”. While it is possible to equate conventional drugs with pharmacosynthetics (or having been developed through pharmacosynthesis), there are distinctions within the semantics that should be explored to provide clarification. A chemical is synthesized to have a particular pharmacology, and this pharmacology is based on the system that the chemical interacts with. On the other hand, a pharmacosynthetic approach creates a pharmacological response within a system using artificial components. While a pharmacological agent may be synthesized, at no point in this effort is the pharmacology of the agent created—instead, it is measured. In one way of thinking about it, a pharmacology (as defined as the study of drug action) is synthesized for an otherwise inert chemical by engineering a receptor and inserting the receptor into a living system. On the other hand, when a novel chemical is synthesized, its pharmacology in a living system is studied to determine whether or not it is a drug.
The pharmacosynthetic tools currently utilized include the Designer Receptors Exclusively Activated by Designer Drug (DREADDs), the latest iteration of a long-standing concept of creating orthologous ligand–receptor pairs to remotely control cellular GPCR signaling (Conklin et al., 2008). The original DREADDs were human muscarinic acetylcholine receptors engineered to be activated by clozapine N-oxide (CNO), an otherwise inert pharmacological agent. Additionally, DREADDs are insensitive to the endogenous ligand, acetylcholine. There are currently three DREADDs in common use—the hM3Dq that activates Gαq signaling, the hM4Di that activates Gαi signaling, and the rM3Ds that activates Gαs signaling. These three DREADDs share the same point mutations (Fig. 1) that simultaneously engender CNO efficacy and acetylcholine inefficacy (Armbruster et al., 2007). The rM3Ds was engineered to couple Gαs by replacing intracellular loops 2 and 3 of the hM3Dq with those from the turkey β1-adrenergic receptor (Guettier et al., 2009). With these three DREADDs, it is possible to control the most common types of G protein signaling found in the mammalian brain.
Connecting receptor mediated signaling to overt brain function is the defining challenge of neuropsychopharmacology research. The hypothesis that aberrant neuronal activity underlies neuropsychiatric disease and the knowledge that drugs modulate neuronal activity via receptors has fueled the persistence of this challenge. To date, small molecule therapeutics are the first line treatments for debilitating mental illness including schizophrenia, Parkinson's disease, and depression, to name a few. DREADDs offer a unique opportunity to study the neurophysiological correlates of therapeutic efficacy due to the nature of the DREADD technology and therapeutic mechanisms of efficacy. First and foremost, DREADDs are G protein coupled receptors—a drug target class of which 36% of all currently approved drugs either directly or indirectly modulate (Klabunde and Hessler, 2002). Furthermore, DREADDs are modulated in a drug-like fashion since the small-molecule ligand exhibits drug-like pharmacokinetics. Finally, it has been observed that therapeutic efficacy is most often obtained through modulation of diffusely expressed albeit specific drug targets (Roth et al., 2004). These three characteristics can only be mimicked via systemic injection of drug and genomic transgene-driven dispersed expression of the DREADD. This similarity to conventional therapeutics will allow an immediate crossover of insights gleaned from research utilizing DREADDs to the physiological phenomena responsible and necessary for therapeutic efficacy.
DREADDs are capable of providing non-invasive temporal control of neuronal signaling for three important reasons that clearly distinguish DREADDs from previous pharmacosynthetic technology. The first is the two-way selectivity of the receptor–ligand pair, in that CNO does not modulate other known effectors in a biological system and that the engineered receptor is not activated by effectors present in the biological system. Second, DREADDs do not exhibit constitutive activity—i.e., in the absence of CNO, the DREADDs do not modulate neuronal signaling. The variant rM3Ds has been shown to exhibit constitutive activity in pancreatic beta cells (Guettier et al., 2009), though constitutive activity was not observed in striatal neurons (Farrell et al., In revision). Finally, the drug used to activate the DREADD is bio-available and drug-like, meaning that a simple administration method (injection, drinking water, food, etc.) can be used to modulate DREADD activity. These advancements are perhaps the most important in terms of the ultimate goal of neuropsychopharmacology, as it permits the investigation of specific signaling states on changes in overt animal behavior with minimal invasiveness.
For a review of the primary research and development of pharmacosynthetics, the reader is directed to Rogan and Roth (2011) in which the hM3Dq and hM4Di neuronal validation is reviewed in addition to the original technology development. The reader is also directed to other published reviews on DREADDs and RASSLs (Conklin et al., 2008, Dong et al., 2010, Pei et al., 2008). This review will focus on the uses of DREADDs in the intervening years and will also provide a novel perspective on this technology.
Section snippets
Gs-DREADD (rM3Ds) neuronal validation
The most recent of the DREADDs to be validated is the Gs-DREADD (rM3Ds). The rM3Ds was originally validated in pancreatic beta islet cells by Guettier et al. (2009). To determine whether the rM3Ds could modulate neuronal Gs-type signaling, Farrell et al. (In revision) created a transgenic mouse expressing the rM3Ds in the striatum. The striatum is a nucleus of the basal ganglia, an area of the brain responsible for volitional movements and reward processes (DeLong and Wichmann, 2009). The
Selective mimicry of endogenous receptors
One application of pharmacosynthetics is to use them as very selective pharmacological agents. In this manner, DREADDs can be expressed in a neuronal population that matches a pharmacologically intractable endogenous receptor. The Gs-DREADD validation study described above can be viewed as an example of this type of application, in which the DREADD enabled selective modulation of a distinct population of neurons residing in a nucleus of heterogenous neuronal composition. In this study, the
Considerations and implications of the above studies
Pharmacosynthesis requires consideration of multiple factors to be utilized effectively. The key elements to be considered are the expression of DREADD and the dose of CNO required for experimental manipulation. Here we provide a primer on the consideration of these elements.
Untapped utility of pharmacosynthetic cell-type specific modulation
Pharmacosynthetics has untapped potential. The utilities not yet applied are inherent in the nature of GPCR signaling in general and that of the pharmacosynthetic approach itself. With the advent of more specific cell-type expression and measurement systems, DREADD technology can be utilized to probe the mechanisms of pharmacotherapeutic efficacy and the nature of GPCR-induced neuronal modulation. Here we will discuss currently underutilized aspects of pharmacosynthetics.
A non CNO-based DREADD
First and foremost, the development of a second, non CNO-based DREADD would be the most advantageous development to further our neuropharmacological understanding of the brain. The availability of an additional DREADD could permit the mapping of functional neuronal circuits, for example, by placing an excitatory non CNO-based DREADD upstream of a nuclei modulated by the hM4Di. In this fashion, one could determine the functional involvement of a series of nuclei posited to be integral for a
Key difference between optogenetics and pharmacosynthetics
It is important to note the differences between the pharmacosynthetic approach and the optogenetic approach. Using optogenetics, one can gain precise spatio-temporal control of neuronal firing using a combination of light and transgenic expression of engineered receptors. Among the optogenetic tools available, one difference is the level of invasiveness required for experimental manipulation when compared to pharmacosynthetics. Using optogenetics, one must deliver light to neurons- a process
Acknowledgments
This work was funded by NIH Grant # 1F31MH091921 to MSF and RO1MH61887, U19MH82441, the NIMH Psychoactive Drug Screening Program and the Michael Hooker Chair in Pharmacology to BLR.
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