The surface of a polymeric nanoparticle (NP) is often functionalized with cell-interactive ligands and/or additional polymeric layers to control NP interaction with cells and proteins. However, such modification is not always straightforward when the surface is not chemically reactive. For this reason, most NP functionalization processes employ reactive linkers or coupling agents or involve pre-functionalization of the polymer, which are complicated and inefficient. Moreover, pre-functionalized polymers can lose the ability to encapsulate and retain a drug if the added ligands change chemical properties of the polymer. To overcome this challenge, we use dopamine polymerization as a way of functionalizing NP surfaces. This method includes brief incubation of the pre-formed NPs in a weak alkaline solution of dopamine, followed by secondary incubation with desired ligands. Using this method, we have functionalized poly(lactic-co-glycolic acid) (PLGA) NPs with three representative surface modifiers: a small molecule (folate), a peptide (Arg-Gly-Asp), and a polymer [poly(carboxybetaine methacrylate)]. We confirmed that the modified NPs showed the expected cellular interactions with no cytotoxicity or residual bioactivity of dopamine. The dopamine polymerization method is a simple and versatile surface modification method, applicable to a variety of NP drug carriers irrespective of their chemical reactivity and the types of ligands.

Agonists targeting the kappa opioid receptor (KOR) have been promising therapeutic candidates because of their efficacy for treating intractable itch and relieving pain. Unlike typical opioid narcotics, KOR agonists do not produce euphoria or lead to respiratory suppression or overdose. However, they do produce dysphoria and sedation, side effects that have precluded their clinical development as therapeutics. KOR signaling can be fine-tuned to preferentially activate certain pathways over others, such that agonists can bias signaling so that the receptor signals through G proteins rather than other effectors such as βarrestin2. We evaluated a newly developed G protein signaling–biased KOR agonist in preclinical models of pain, pruritis, sedation, dopamine regulation, and dysphoria. We found that triazole 1.1 retained the antinociceptive and antipruritic efficacies of a conventional KOR agonist, yet it did not induce sedation or reductions in dopamine release in mice, nor did it produce dysphoria as determined by intracranial self-stimulation in rats. These data demonstrated that biased agonists may be used to segregate physiological responses downstream of the receptor. Moreover, the findings suggest that biased KOR agonists may present a means to treat pain and intractable itch without the side effects of dysphoria and sedation and with reduced abuse potential.

G protein coupled receptors have historically been one of the most druggable classes of cellular proteins. The members of this large receptor gene family couple to primary effectors, G proteins, that have built in mechanisms for regeneration and amplification of signaling with each engagement of receptor and ligand, a kinetic event in itself. In recent years GPCRs, have been found to interact with arrestin proteins to initiate signal propagation in the absence of G protein interactions. This pinnacle observation has changed a previously held notion of the linear spectrum of GPCR efficacy and uncovered a new paradigm in GPCR research and drug discovery that relies on multidimensionality of GPCR signaling. Ligands were found that selectively confer activity in one pathway over another, and this phenomenon has been referred to as ‘biased agonism’ or ‘functional selectivity’. While great strides in the understanding of this phenomenon have been made in recent years, two critical questions still dominate the field: How can we rationally design biased GPCR ligands, and ultimately, which physiological responses are due to G protein versus arrestin interactions? This review will discuss the current understanding of some of the key aspects of biased signaling that are related to these questions, including mechanistic insights in the nature of biased signaling and methods for measuring ligand bias, as well as relevant examples of drug discovery applications and medicinal chemistry strategies that highlight the challenges and opportunities in this rapidly evolving field.

Adenylyl cyclase 1 (AC1) belongs to a group of adenylyl cyclases (ACs) that are stimulated by calcium in a calmodulin-dependent manner. Studies with AC1 knockout mice suggest that inhibitors of AC1 may be useful for treating pain and opioid dependence. However, non-selective inhibition of AC isoforms could result in substantial adverse effects. We used chemical library screening to identify a selective AC1 inhibitor with a chromone core structure that may represent a new analgesic agent. After demonstrating that the compound (ST034307) inhibited Ca2+-stimulated adenosine 5’,3’-monophosphate (cAMP) accumulation in HEK cells stably transfected with AC1 (HEK-AC1 cells), we confirmed selectivity for AC1 by testing against all isoforms of membrane-bound ACs. ST034307 also inhibited AC1 activity stimulated by forskolin- and Gαs-coupled receptors in HEK-AC1 cells and showed inhibitory activity in multiple AC1-containing membrane preparations and mouse hippocampal homogenates. ST034307 enhanced μ-opioid receptor (MOR)-mediated inhibition of AC1 in short-term inhibition assays in HEK-AC1 cells stably transfected with MOR; however, the compound blocked heterologous sensitization of AC1 caused by chronic MOR activation in those cells. ST034307 reduced pain responses in a mouse model of inflammatory pain. Our data indicated that ST034307 is a selective small molecule inhibitor of AC1 and suggest that selective AC1 inhibitors may be useful for managing pain.

BackgroundMany neglected tropical infectious diseases affecting humans are transmitted by arthropods such as mosquitoes and ticks. New mode-of-action chemistries are urgently sought to enhance vector management practices in countries where arthropod-borne diseases are endemic, especially where vector populations have acquired widespread resistance to insecticides.Methodology/Principal FindingsWe describe a “genome-to-lead” approach for insecticide discovery that incorporates the first reported chemical screen of a G protein-coupled receptor (GPCR) mined from a mosquito genome. A combination of molecular and pharmacological studies was used to functionally characterize two dopamine receptors (AaDOP1 and AaDOP2) from the yellow fever mosquito, Aedes aegypti. Sequence analyses indicated that these receptors are orthologous to arthropod D1-like (Gαs-coupled) receptors, but share less than 55% amino acid identity in conserved domains with mammalian dopamine receptors. Heterologous expression of AaDOP1 and AaDOP2 in HEK293 cells revealed dose-dependent responses to dopamine (EC50: AaDOP1 = 3.1±1.1 nM; AaDOP2 = 240±16 nM). Interestingly, only AaDOP1 exhibited sensitivity to epinephrine (EC50 = 5.8±1.5 nM) and norepinephrine (EC50 = 760±180 nM), while neither receptor was activated by other biogenic amines tested. Differential responses were observed between these receptors regarding their sensitivity to dopamine agonists and antagonists, level of maximal stimulation, and constitutive activity. Subsequently, a chemical library screen was implemented to discover lead chemistries active at AaDOP2. Fifty-one compounds were identified as “hits,” and follow-up validation assays confirmed the antagonistic effect of selected compounds at AaDOP2. In vitro comparison studies between AaDOP2 and the human D1 dopamine receptor (hD1) revealed markedly different pharmacological profiles and identified amitriptyline and doxepin as AaDOP2-selective compounds. In subsequent Ae. aegypti larval bioassays, significant mortality was observed for amitriptyline (93%) and doxepin (72%), confirming these chemistries as “leads” for insecticide discovery.Conclusions/SignificanceThis research provides a “proof-of-concept” for a novel approach toward insecticide discovery, in which genome sequence data are utilized for functional characterization and chemical compound screening of GPCRs. We provide a pipeline useful for future prioritization, pharmacological characterization, and expanded chemical screening of additional GPCRs in disease-vector arthropods. The differential molecular and pharmacological properties of the mosquito dopamine receptors highlight the potential for the identification of target-specific chemistries for vector-borne disease management, and we report the first study to identify dopamine receptor antagonists with in vivo toxicity toward mosquitoes.