Impact of GPCR Structures on Drug Discovery.

Structures of 70 unique G protein-coupled receptors (GPCRs) have been determined, with over 370 structures in total bound to different ligands and the receptors in various conformational states. Structure-based drug design has been applied to an increasing number of GPCR targets over the past decade and now a few of these drug candidates have entered clinical trials. Given the length of time required for a drug to reach the market, there are no documented examples of licensed drugs being developed with the aid of a structure, but this is likely to change as current efforts come to fruition.

Activation mechanism of the class D fungal GPCR dimer Ste2.

The fungal class D1 G-protein-coupled receptor (GPCR) Ste2 has a different arrangement of transmembrane helices compared with mammalian GPCRs and a distinct mode of coupling to the heterotrimeric G protein Gpa1-Ste2-Ste181. In addition, Ste2 lacks conserved sequence motifs such as DRY, PIF and NPXXY, which are associated with the activation of class A GPCRs2. This suggested that the activation mechanism of Ste2 may also differ. Here we determined structures of Saccharomyces cerevisiae Ste2 in the absence of G protein in two different conformations bound to the native agonist α-factor, bound to an antagonist and without ligand. These structures revealed that Ste2 is indeed activated differently from other GPCRs. In the inactive state, the cytoplasmic end of transmembrane helix H7 is unstructured and packs between helices H1-H6, blocking the G protein coupling site. Agonist binding results in the outward movement of the extracellular ends of H6 and H7 by 6 Å. On the intracellular surface, the G protein coupling site is formed by a 20 Å outward movement of the unstructured region in H7 that unblocks the site, and a 12 Å inward movement of H6. This is a distinct mechanism in GPCRs, in which the movement of H6 and H7 upon agonist binding facilitates G protein coupling.

Structure of the class D GPCR Ste2 dimer coupled to two G proteins.

G-protein-coupled receptors (GPCRs) are divided phylogenetically into six classes1,2, denoted A to F. More than 370 structures of vertebrate GPCRs (belonging to classes A, B, C and F) have been determined, leading to a substantial understanding of their function3. By contrast, there are no structures of class D GPCRs, which are found exclusively in fungi where they regulate survival and reproduction. Here we determine the structure of a class D GPCR, the Saccharomyces cerevisiae pheromone receptor Ste2, in an active state coupled to the heterotrimeric G protein Gpa1-Ste4-Ste18. Ste2 was purified as a homodimer coupled to two G proteins. The dimer interface of Ste2 is formed by the N terminus, the transmembrane helices H1, H2 and H7, and the first extracellular loop ECL1. We establish a class D1 generic residue numbering system (CD1) to enable comparisons with orthologues and with other GPCR classes. The structure of Ste2 bears similarities in overall topology to class A GPCRs, but the transmembrane helix H4 is shifted by more than 20 Å and the G-protein-binding site is a shallow groove rather than a cleft. The structure provides a template for the design of novel drugs to target fungal GPCRs, which could be used to treat numerous intractable fungal diseases4.

Molecular basis of ß-arrestin coupling to formoterol-bound ß1-adrenoceptor.

The ß1-adrenoceptor (ß1AR) is a G-protein-coupled receptor (GPCR) that couples1 to the heterotrimeric G protein Gs. G-protein-mediated signalling is terminated by phosphorylation of the C terminus of the receptor by GPCR kinases (GRKs) and by coupling of ß-arrestin 1 (ßarr1, also known as arrestin 2), which displaces Gs and induces signalling through the MAP kinase pathway2. The ability of synthetic agonists to induce signalling preferentially through either G proteins or arrestins-known as biased agonism3-is important in drug development, because the therapeutic effect may arise from only one signalling cascade, whereas the other pathway may mediate undesirable side effects4. To understand the molecular basis for arrestin coupling, here we determined the cryo-electron microscopy structure of the ß1AR-ßarr1 complex in lipid nanodiscs bound to the biased agonist formoterol5, and the crystal structure of formoterol-bound ß1AR coupled to the G-protein-mimetic nanobody6 Nb80. ßarr1 couples to ß1AR in a manner distinct to that7 of Gs coupling to ß2AR-the finger loop of ßarr1 occupies a narrower cleft on the intracellular surface, and is closer to transmembrane helix H7 of the receptor when compared with the C-terminal α5 helix of Gs. The conformation of the finger loop in ßarr1 is different from that adopted by the finger loop of visual arrestin when it couples to rhodopsin8. ß1AR coupled to ßarr1 shows considerable differences in structure compared with ß1AR coupled to Nb80, including an inward movement of extracellular loop 3 and the cytoplasmic ends of H5 and H6. We observe weakened interactions between formoterol and two serine residues in H5 at the orthosteric binding site of ß1AR, and find that formoterol has a lower affinity for the ß1AR-ßarr1 complex than for the ß1AR-Gs complex. The structural differences between these complexes of ß1AR provide a foundation for the design of small molecules that could bias signalling in the ß-adrenoceptors.

VPS26 Moonlights as a ß-Arrestin-like Adapter for a 7-Transmembrane RGS Protein in Arabidopsis thaliana.

Extracellular signals perceived by 7-transmembrane (7TM)-spanning receptors initiate desensitization that involves the removal of these receptors from the plasma membrane. Agonist binding often evokes phosphorylation in the flexible C-terminal region and/or intracellular loop 3 of many 7TM G-protein-coupled receptors in animal cells, which consequently recruits a cytoplasmic intermediate adaptor, ß-arrestin, resulting in clathrin-mediated endocytosis (CME) and downstream signaling such as transcriptional changes. Some 7TM receptors undergo CME without recruiting ß-arrestin, but it is not clear how. Arrestins are not encoded in the Arabidopsis thaliana genome, yet Arabidopsis cells have a well-characterized signal-induced CME of a 7TM protein, designated Regulator of G Signaling 1 (AtRGS1). Here we show that a component of the retromer complex, Vacuolar Protein Sorting-Associated 26 (VPS26), binds the phosphorylated C-terminal region of AtRGS1 as a VPS26A/B heterodimer to form a complex that is required for downstream signaling. We propose that VPS26 moonlights as an arrestin-like adaptor in the CME of AtRGS1.

Cryo-EM structure of the serotonin 5-HT1B receptor coupled to heterotrimeric Go.

G-protein-coupled receptors (GPCRs) form the largest family of receptors encoded by the human genome (around 800 genes). They transduce signals by coupling to a small number of heterotrimeric G proteins (16 genes encoding different α-subunits). Each human cell contains several GPCRs and G proteins. The structural determinants of coupling of Gs to four different GPCRs have been elucidated1-4, but the molecular details of how the other G-protein classes couple to GPCRs are unknown. Here we present the cryo-electron microscopy structure of the serotonin 5-HT1B receptor (5-HT1BR) bound to the agonist donitriptan and coupled to an engineered Go heterotrimer. In this complex, 5-HT1BR is in an active state; the intracellular domain of the receptor is in a similar conformation to that observed for the ß2-adrenoceptor (ß2AR) 3 or the adenosine A2A receptor (A2AR) 1 in complex with Gs. In contrast to the complexes with Gs, the gap between the receptor and the Gß-subunit in the Go-5-HT1BR complex precludes molecular contacts, and the interface between the Gα-subunit of Go and the receptor is considerably smaller. These differences are likely to be caused by the differences in the interactions with the C terminus of the Go α-subunit. The molecular variations between the interfaces of Go and Gs in complex with GPCRs may contribute substantially to both the specificity of coupling and the kinetics of signalling.

PtdIns(4,5)P2 stabilizes active states of GPCRs and enhances selectivity of G-protein coupling.

G-protein-coupled receptors (GPCRs) are involved in many physiological processes and are therefore key drug targets1. Although detailed structural information is available for GPCRs, the effects of lipids on the receptors, and on downstream coupling of GPCRs to G proteins are largely unknown. Here we use native mass spectrometry to identify endogenous lipids bound to three class A GPCRs. We observed preferential binding of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) over related lipids and confirm that the intracellular surface of the receptors contain hotspots for PtdIns(4,5)P2 binding. Endogenous lipids were also observed bound directly to the trimeric Gαsßγ protein complex of the adenosine A2A receptor (A2AR) in the gas phase. Using engineered Gα subunits (mini-Gαs, mini-Gαi and mini-Gα12)2, we demonstrate that the complex of mini-Gαs with the ß1 adrenergic receptor (ß1AR) is stabilized by the binding of two PtdIns(4,5)P2 molecules. By contrast, PtdIns(4,5)P2 does not stabilize coupling between ß1AR and other Gα subunits (mini-Gαi or mini-Gα12) or a high-affinity nanobody. Other endogenous lipids that bind to these receptors have no effect on coupling, highlighting the specificity of PtdIns(4,5)P2. Calculations of potential of mean force and increased GTP turnover by the activated neurotensin receptor when coupled to trimeric Gαißγ complex in the presence of PtdIns(4,5)P2 provide further evidence for a specific effect of PtdIns(4,5)P2 on coupling. We identify key residues on cognate Gα subunits through which PtdIns(4,5)P2 forms bridging interactions with basic residues on class A GPCRs. These modulating effects of lipids on receptors suggest consequences for understanding function, G-protein selectivity and drug targeting of class A GPCRs.

High-mass MALDI-MS unravels ligand-mediated G protein-coupling selectivity to GPCRs.

G protein-coupled receptors (GPCRs) are important pharmaceutical targets for the treatment of a broad spectrum of diseases. Although there are structures of GPCRs in their active conformation with bound ligands and G proteins, the detailed molecular interplay between the receptors and their signaling partners remains challenging to decipher. To address this, we developed a high-sensitivity, high-throughput matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) method to interrogate the first stage of signal transduction. GPCR-G protein complex formation is detected as a proxy for the effect of ligands on GPCR conformation and on coupling selectivity. Over 70 ligand-GPCR-partner protein combinations were studied using as little as 1.25 pmol protein per sample. We determined the selectivity profile and binding affinities of three GPCRs (rhodopsin, beta-1 adrenergic receptor [ß1AR], and angiotensin II type 1 receptor) to engineered Gα-proteins (mGs, mGo, mGi, and mGq) and nanobody 80 (Nb80). We found that GPCRs in the absence of ligand can bind mGo, and that the role of the G protein C terminus in GPCR recognition is receptor-specific. We exemplified our quantification method using ß1AR and demonstrated the allosteric effect of Nb80 binding in assisting displacement of nadolol to isoprenaline. We also quantified complex formation with wild-type heterotrimeric Gαißγ and ß-arrestin-1 and showed that carvedilol induces an increase in coupling of ß-arrestin-1 and Gαißγ to ß1AR. A normalization strategy allows us to quantitatively measure the binding affinities of GPCRs to partner proteins. We anticipate that this methodology will find broad use in screening and characterization of GPCR-targeting drugs.

Cryo-Electron Microscopy: Moving Beyond X-Ray Crystal Structures for Drug Receptors and Drug Development.

Electron cryo-microscopy (cryo-EM) has revolutionized structure determination of membrane proteins and holds great potential for structure-based drug discovery. Here we discuss the potential of cryo-EM in the rational design of therapeutics for membrane proteins compared to X-ray crystallography. We also detail recent progress in the field of drug receptors, focusing on cryo-EM of two protein families with established therapeutic value, the γ-aminobutyric acid A receptors (GABAARs) and G protein-coupled receptors (GPCRs). GABAARs are pentameric ion channels, and cryo-EM structures of physiological heteromeric receptors in a lipid environment have uncovered the molecular basis of receptor modulation by drugs such as diazepam. The structures of ten GPCR-G protein complexes from three different classes of GPCRs have now been determined by cryo-EM. These structures give detailed insights into molecular interactions with drugs, GPCR-G protein selectivity, how accessory membrane proteins alter receptor-ligand pharmacology, and the mechanism by which HIV uses GPCRs to enter host cells.

Structures of Adrenoceptors.

The first structure of an adrenoceptor (AR), the human ß2-adrenoceptor (hß2AR) was published in 2007 and since then a total of 78 structures (up to June 2022) have been determined by X-ray crystallography and electron cryo-microscopy (cryo-EM) of all three ßARs (ß1, ß2 and ß3) and four out of six αARs (α1B, α2A, α2B, α2C). The structures are in a number of different conformational states, including the inactive state bound to an antagonist, an intermediate state bound to agonist and active states bound to agonist and an intracellular transducer (G protein or arrestin) or transducer mimetic (nanobody). The structures identify molecular details of how ligands bind in the orthosteric binding pocket (OBP; 19 antagonists, 18 agonists) and also how three different small molecule allosteric modulators bind. The structures have been used to define the molecular details of receptor activation and also the molecular determinants for transducer coupling. This chapter will give a brief overview of the structures, receptor activation, a comparison across the different subfamilies and commonalities of ligand-receptor interactions.

How Do Adolescent Smoking Prevention Interventions Work in Different Contextual Settings? A Qualitative Comparative Study Between the UK and Colombia.

BACKGROUND: Adolescent smoking is associated with significant health and social risks. Previous research has demonstrated the effectiveness of interventions based on behavior change theories in preventing adolescent smoking uptake. However, evidence from the theory-based perspective of evaluation is limited, especially for how such complex interventions work, and how they work when implemented in different contextual settings. METHOD: A comparative qualitative analysis was conducted to explore various influences on behavior change among participants taking part in two smoking prevention interventions in Northern Ireland and Bogotá. Twenty-seven focus groups were conducted in 12 schools (6 in Northern Ireland and 6 in Bogota, n = 195 pupils participated; aged 11-15 years). The Theoretical Domains Framework guided a content analysis of the data. RESULTS: We found similarities across settings in terms of knowledge, skills, and beliefs related to smoking or vaping behavior change, as well as differences in contextual resources and social influence. Different environmental resources included availability to purchase tobacco products in the neighborhoods and previous information about tobacco risk. Participants in both interventions perceived behavioral change outcomes related to personal skills and intention to not smoke or vape. CONCLUSION: These findings have highlighted how both individual factors and contextual resources influence behavior change for smoking prevention in practice. Local contextual factors and social influences affecting pupils should be taken into account in the implementation and evaluation of health behavior change interventions. In particular, this study supports using social and contextual influence strategies in interventions to reduce the onset of adolescent smoking and vaping.

Structure of the adenosine A(2A) receptor bound to an engineered G protein.

G-protein-coupled receptors (GPCRs) are essential components of the signalling network throughout the body. To understand the molecular mechanism of G-protein-mediated signalling, solved structures of receptors in inactive conformations and in the active conformation coupled to a G protein are necessary. Here we present the structure of the adenosine A(2A) receptor (A(2A)R) bound to an engineered G protein, mini-Gs, at 3.4 Å resolution. Mini-Gs binds to A(2A)R through an extensive interface (1,048 Å2) that is similar, but not identical, to the interface between Gs and the ß2-adrenergic receptor. The transition of the receptor from an agonist-bound active-intermediate state to an active G-protein-bound state is characterized by a 14 Å shift of the cytoplasmic end of transmembrane helix 6 (H6) away from the receptor core, slight changes in the positions of the cytoplasmic ends of H5 and H7 and rotamer changes of the amino acid side chains Arg3.50, Tyr5.58 and Tyr7.53. There are no substantial differences in the extracellular half of the receptor around the ligand binding pocket. The A(2A)R-mini-Gs structure highlights both the diversity and similarity in G-protein coupling to GPCRs and hints at the potential complexity of the molecular basis for G-protein specificity.

Diverse activation pathways in class A GPCRs converge near the G-protein-coupling region.

Class A G-protein-coupled receptors (GPCRs) are a large family of membrane proteins that mediate a wide variety of physiological functions, including vision, neurotransmission and immune responses. They are the targets of nearly one-third of all prescribed medicinal drugs such as beta blockers and antipsychotics. GPCR activation is facilitated by extracellular ligands and leads to the recruitment of intracellular G proteins. Structural rearrangements of residue contacts in the transmembrane domain serve as 'activation pathways' that connect the ligand-binding pocket to the G-protein-coupling region within the receptor. In order to investigate the similarities in activation pathways across class A GPCRs, we analysed 27 GPCRs from diverse subgroups for which structures of active, inactive or both states were available. Here we show that, despite the diversity in activation pathways between receptors, the pathways converge near the G-protein-coupling region. This convergence is mediated by a highly conserved structural rearrangement of residue contacts between transmembrane helices 3, 6 and 7 that releases G-protein-contacting residues. The convergence of activation pathways may explain how the activation steps initiated by diverse ligands enable GPCRs to bind a common repertoire of G proteins.

Structure determination of GPCRs: cryo-EM compared with X-ray crystallography.

G protein-coupled receptors (GPCRs) are the largest single family of cell surface receptors encoded by the human genome and they play pivotal roles in co-ordinating cellular systems throughout the human body, making them ideal drug targets. Structural biology has played a key role in defining how receptors are activated and signal through G proteins and ß-arrestins. The application of structure-based drug design (SBDD) is now yielding novel compounds targeting GPCRs. There is thus significant interest from both academia and the pharmaceutical industry in the structural biology of GPCRs as currently only about one quarter of human non-odorant receptors have had their structure determined. Initially, all the structures were determined by X-ray crystallography, but recent advances in electron cryo-microscopy (cryo-EM) now make GPCRs tractable targets for single-particle cryo-EM with comparable resolution to X-ray crystallography. So far this year, 78% of the 99 GPCR structures deposited in the PDB (Jan-Jul 2021) were determined by cryo-EM. Cryo-EM has also opened up new possibilities in GPCR structural biology, such as determining structures of GPCRs embedded in a lipid nanodisc and multiple GPCR conformations from a single preparation. However, X-ray crystallography still has a number of advantages, particularly in the speed of determining many structures of the same receptor bound to different ligands, an essential prerequisite for effective SBDD. We will discuss the relative merits of cryo-EM and X-ray crystallography for the structure determination of GPCRs and the future potential of both techniques.

Multiple complexation of CO and related ligands to a main-group element.

The ability of an atom or molecular fragment to bind multiple carbon monoxide (CO) molecules to form multicarbonyl adducts is a fundamental trait of transition metals. Transition-metal carbonyl complexes are vital to industry, appear naturally in the active sites of a number of enzymes (such as hydrogenases), are promising therapeutic agents, and have even been observed in interstellar dust clouds. Despite the wealth of established transition-metal multicarbonyl complexes, no elements outside groups 4 to 12 of the periodic table have yet been shown to react directly with two or more CO units to form stable multicarbonyl adducts. Here we present the synthesis of a borylene dicarbonyl complex, the first multicarbonyl complex of a main-group element prepared using CO. The compound is additionally stable towards ambient air and moisture. The synthetic strategy used--liberation of a borylene ligand from a transition metal using donor ligands--is broadly applicable, leading to a number of unprecedented monovalent boron species with different Lewis basic groups. The similarity of these compounds to conventional transition-metal carbonyl complexes is demonstrated by photolytic liberation of CO and subsequent intramolecular carbon-carbon bond activation.

Universal allosteric mechanism for Gα activation by GPCRs.

G protein-coupled receptors (GPCRs) allosterically activate heterotrimeric G proteins and trigger GDP release. Given that there are ∼800 human GPCRs and 16 different Gα genes, this raises the question of whether a universal allosteric mechanism governs Gα activation. Here we show that different GPCRs interact with and activate Gα proteins through a highly conserved mechanism. Comparison of Gα with the small G protein Ras reveals how the evolution of short segments that undergo disorder-to-order transitions can decouple regions important for allosteric activation from receptor binding specificity. This might explain how the GPCR-Gα system diversified rapidly, while conserving the allosteric activation mechanism.

Socio-environmental and psychosocial predictors of smoking susceptibility among adolescents with contrasting socio-cultural characteristics: a comparative analysis.

BACKGROUND: Despite a steady decline in adolescent smoking globally, it remains a prevalent risk factor for non-communicable disease. Previous research points to differences in socio-environmental and psychosocial risk factors for smoking and how they vary across different settings with disparate social and cultural characteristics. As a result, smoking rates have remained disproportionately higher in some settings while decreasing in others. This study explored the socio-environmental and psychosocial risk factors for smoking susceptibility in a high-income and upper-middle income setting. METHODS: Cross-sectional data were obtained from 1,573 male and female adolescents aged 11-15 years who completed self-administered questionnaires in schools in Northern Ireland and Bogotá, Colombia. Using logistic regression analysis, we examined how socio-environmental and psychosocial predictors of smoking susceptibility compared across the two countries. RESULTS: In Northern Ireland, reduced odds of smoking susceptibility were significantly associated with less family smoking (OR: 0.64, 95% CI: 0.41-1.00); having access to information about smoking in school (OR: 0.75, 95% CI: 0.59-0.96); negative attitudes towards smoking (OR: 0.35, 95% CI: 0.23-0.51); higher levels of openness (OR: 0.59, 95% CI: 0.50-0.69); and higher levels of self-reported wellbeing (OR: 0.57, 95% CI: 0.44-0.74). Increased odds of smoking susceptibility were associated with reporting less smoking of a mother (OR: 1.37, 95% CI: 1.06-1.76); higher levels of extraversion (OR: 1.40, 95% CI: 1.04-1.90); and receiving pocket money (OR: 1.20, 95% CI: 1.06-1.37). In Bogotá, reduced odds of smoking susceptibility were significantly associated with reporting less smoking among friends (OR: 0.86, 95% CI: 0.76-0.98); higher levels of self-efficacy (OR: 0.58, 95% CI: 0.40-0.83); greater perceived behavioural control to quit smoking (OR: 0.71, 95% CI: 0.56-0.90); and lower levels of truancy (OR: 0.69, 95% CI: 0.52-0.92). In Bogotá, no factors were associated with increased odds of smoking susceptibility in the final model. CONCLUSIONS: The findings illustrate that there were differences in predictors of adolescent smoking susceptibility across the two settings. By using a comparative approach we demonstrate that smoking interventions and policies must be sensitive to the cultural and normative context within which they are implemented.

How Do Branched Detergents Stabilize GPCRs in Micelles?

The structural and functional properties of G protein-coupled receptors (GPCRs) are often studied in a detergent micellar environment, but many GPCRs tend to denature or aggregate in short alkyl chain detergents. In our previous work [Lee, S., et al. (2016) J. Am. Chem. Soc. 138, 15425-15433], we showed that GPCRs in alkyl glucosides were highly dynamic, resulting in the penetration of detergent molecules between transmembrane α-helices, which is the initial step in receptor denaturation. Although this was not observed for GPCRs in dodecyl maltoside (DDM, also known as lauryl maltoside), even this detergent is not mild enough to preserve the integrity of many GPCRs during purification. Lauryl maltose neopentylglycol (LMNG) detergents have been found to have significant advantages for purifying GPCRs in a native state as they impart more stability to the receptor than DDM. To gain insights into how they stabilize GPCRs, we used atomistic molecular dynamics simulations of wild type adenosine A2A receptor (WT-A2AR), thermostabilized A2AR (tA2AR), and wild type ß2-adrenoceptor (ß2AR) in a variety of detergents (LMNG, DMNG, OGNG, and DDM). Analysis of molecular dynamics simulations of tA2AR in LMNG, DMNG, and OGNG showed that this series of detergents exhibited behavior very similar to that of an analogous series of detergents DDM, DM, and OG in our previous study. However, there was a striking difference upon comparison of the behavior of LMNG to that of DDM. LMNG showed considerably less motion than DDM, which resulted in the enhanced density of the aliphatic chains around the hydrophobic regions of the receptor and considerably more hydrogen bond formation between the head groups. This contributed to enhanced interaction energies between both detergent molecules and between the receptor and detergent, explaining the enhanced stability of GPCRs purified in this detergent. Branched detergents occlude between transmembrane helices and reduce their flexibility. Our results provide a rational foundation to develop detergent variants for stabilizing membrane proteins.

Evaluating Donor Effects in Isoindigo-Based Small Molecular Fluorophores.

Small molecular organic fluorophores have garnered significant interest because of their indispensable use in fluorescence imaging (FI) and optoelectronic devices. Herein, we designed triphenylamine (TPA)-capped donor-acceptor-donor (D-A-D)-based fluorophores having a variation at the heterocyclic donor (D) units, 3,4-ethylenedioxythiophene (EDOT), furan (FURAN), thiophene (THIO), and 1-methyl-1H-pyrrole (MePyr), with isoindigo as the core electron acceptor (A) unit. Synthesis of these fluorophores (II-X-TPA) resulted in four symmetrical dye molecules: II-EDOT-TPA, II-FURAN-TPA, II-THIO-TPA, and II-MePyr-TPA, where TPA functioned as a terminal unit and a secondary electron donor group. Photophysical, electrochemical, and computational analyses were conducted to investigate the effect of heterocyclic donor units on the II-X-TPA derivatives. Density functional theory (DFT) and time-dependent DFT (TD-DFT) calculations provided insightful features of structural and electronic properties of each fluorophore and correlated well with experimental observations. Electron density distribution maps, overlapping frontier molecular orbital diagrams, and highest occupied molecular orbital (HOMO) to lowest unoccupied molecular orbital (LUMO) electron transfer indicated intramolecular charge transfer (ICT). Theoretical studies confirmed the experimental HOMO energy trend and demonstrated its crucial importance in understanding each heterocycle's donor ability. Stokes shifts of up to ∼178 nm were observed, whereas absorptions and emissions were shifted deeper into the NIR region, resulting from ICT. Results suggest that this isoindigo fluorophore series has potential as a molecular scaffold for the development of efficient FI agents. The studied fluorophores can be further tuned with different donor fragments to enhance the ICT and facilitate in shifting the optical properties further into the NIR region.

Machine Learning for Prioritization of Thermostabilizing Mutations for G-Protein Coupled Receptors.

Although the three-dimensional structures of G-protein coupled receptors (GPCRs), the largest superfamily of drug targets, have enabled structure-based drug design, there are no structures available for 87% of GPCRs. This is due to the stiff challenge in purifying the inherently flexible GPCRs. Identifying thermostabilized mutant GPCRs via systematic alanine scanning mutations has been a successful strategy in stabilizing GPCRs, but it remains a daunting task for each GPCR. We developed a computational method that combines sequence-, structure-, and dynamics-based molecular properties of GPCRs that recapitulate GPCR stability, with four different machine learning methods to predict thermostable mutations ahead of experiments. This method has been trained on thermostability data for 1231 mutants, the largest publicly available data set. A blind prediction for thermostable mutations of the complement factor C5a receptor 1 retrieved 36% of the thermostable mutants in the top 50 prioritized mutants compared to 3% in the first 50 attempts using systematic alanine scanning.