Inactive Conformation Research Articles

Proton Relay Network in the Bacterial P450s: CYP101A1 and CYP101D1.

Cytochrome P450s are among nature's most powerful catalysts. Their ability to activate molecular dioxygen to form high-valent ferryl intermediates (Compounds I and II) enables a wide array of chemistries ranging from simple epoxidations to more complicated C-H bond oxidations. Oxygen activation is achieved by reduction of the ferrous dioxygen complex, which requires the transfer of an electron from a redox partner and subsequent double protonation to yield a water molecule and a ferryl porphyrin π-cation radical (Compound I). Previous studies of the CYP101 family of cytochrome P450s demonstrated the importance of the conserved active site Asp25X residue in this protonation event, although its precise role is yet to be unraveled. To further explore the origin of protons in oxygen activation, we analyzed the effects of an Asp to Glu mutation at the 25X position in P450cam and in CYP101D1. This mutation inactivates P450cam but not CYP101D1. A series of mutagenic, crystallographic, kinetic, and molecular dynamics studies indicate that this mutation locks P450cam into a closed, inactive conformation. In CYP101D1, the D259E mutant changes the rate-limiting step to reduction of the P450-oxy complex, thus opening a window into the critical proton-coupled electron transfer step in P450 catalysis.

Stabilizing Inactive Conformations of MALT1 as an Effective Approach to Inhibit Its Protease Activity

AbstractThe paracaspase MALT1 (mucosa associated lymphoid tissue lymphoma translocated gene 1) plays an important role in various immune pathways and is proposed as a therapeutic target for autoimmune disorders as well as different types of cancer, such as diffuse large B‐cell lymphoma (DLBCL). Different mechanisms are explored to inhibit the protease activity of MALT1 and two unrelated chemical scaffolds are discovered. Biophysical and structural studies reveal that both scaffolds stabilize the protease in an inactive conformation. While one ligand binds to the allosteric site at the interface between the caspase and the Ig3 domain, the other ligand binds to the active site using a so far undescribed mechanism. Iterative structure‐based drug discovery on one scaffold results in the identification of a potent, selective, and orally bioavailable MALT1 inhibitor.

Hybrid Thermophilic/Mesophilic Enzymes Reveal a Role for Conformational Disorder in Regulation of Bacterial Enzyme I

Conformational disorder is emerging as an important feature of biopolymers, regulating a vast array of cellular functions, including signaling, phase separation, and enzyme catalysis. Here we combine NMR, crystallography, computer simulations, protein engineering, and functional assays to investigate the role played by conformational heterogeneity in determining the activity of the C-terminal domain of bacterial Enzyme I (EIC). In particular, we design chimeric proteins by hybridizing EIC from thermophilic and mesophilic organisms, and we characterize the resulting constructs for structure, dynamics, and biological function. We show that EIC exists as a mixture of active and inactive conformations and that functional regulation is achieved by tuning the thermodynamic balance between active and inactive states. Interestingly, we also present a hybrid thermophilic/mesophilic enzyme that is thermostable and more active than the wild-type thermophilic enzyme, suggesting that hybridizing thermophilic and mesophilic proteins is a valid strategy to engineer thermostable enzymes with significant low-temperature activity.

Computational and in vitro characterization of ICY-5: A potential candidate promoting mitochondrial apoptosis via the c-MET andSTAT3 pathways.

Targeted chemotherapy remains the primary choice in controlling various forms of breast cancer (BC) due to its heterogenous gene expressions in various subtypes. In silico and in vitro evaluation of ICY-5, a novel arylidene analogue against c-MET, was performed. ICY-5 exhibited a docking score of -9.6 kcal/mol in inactive conformation and, - 8.6 kcal/mol in active conformation for c-MET. ICY-5 inhibited c-MET enzyme with an IC50 of 34.34 nM. The compound effectively inhibited MDA-MB 231 and MCF-7 cell proliferation, with GI50 values of 62.61 and 75.31 nM, respectively, and hepatocyte growth factor (HGF)/R c-MET phosphorylation with IC50 s of 71.41 and 83.77 nM, respectively. ICY-5 dose-dependently inhibited HGF-induced transmigration, cell scattering, invasion and altered cell cycle. An increase in apoptotic populations of these cells, with a dose-dependent decease in phosphorylation of STAT3 protein was observed. Furthermore, ICY-5 upregulated the caspase-3, caspase-9, Bcl-2-associated X andsurvivin, and downregulated Bcl-2, vascular endothelial growth factor, matrix metalloproteinase-2 (MMP-2), and MMP-9 in both BC cell lines. In summary, ICY-5 exhibited excellent efficacy in BC cells, targeting c-MET/SAT-3-mediated mitochondrial apoptosis. Further research will be required to ascertain ICY-5 suitability as a targeted chemotherapeutic against multiple forms of BC.

An allosteric modulator binds to a conformational hub in the β2 adrenergic receptor.

Most drugs acting on G-protein-coupled receptors target the orthosteric binding pocket where the native hormone or neurotransmitter binds. There is much interest in finding allosteric ligands for these targets because they modulate physiologic signaling and promise to be more selective than orthosteric ligands. Here we describe a newly developed allosteric modulator of the β2-adrenergic receptor (β2AR), AS408, that binds to the membrane-facing surface of transmembrane segments 3 and 5, as revealed by X-ray crystallography. AS408 disrupts a water-mediated polar network involving E1223.41 and the backbone carbonyls of V2065.45 and S2075.46. The AS408 binding site is adjacent to a previously identified molecular switch for β2AR activation formed by I3.40, P5.50 and F6.44. The structure reveals how AS408 stabilizes the inactive conformation of this switch, thereby acting as a negative allosteric modulator for agonists and positive allosteric modulator for inverse agonists.

Scaffold hopping and redesign approaches for quinazoline based urea derivatives as potent VEGFR-2 inhibitors

In our attempt to discover effective anticancer agents, three series of novel quinazoline-based compounds have been designed, synthesized and tested as VEGFR-2 inhibitors. Five quinazoline −2-carboxamide derivatives (5d, 5e, 5 h, 5i, 5j) revealed potent nanomolar VEGFR-2 inhibition with IC50 values of 12.1, 40.3, 15.5, 13.1 and 57.4 nM, respectively, superior to that of the reference drug sorafenib (IC50 78.9 nM). Additionally, the quinazoline 2-carboxylate derivative bearing a fluorine substituent in middle ring (7a) showed IC50 values of 14.8 nM. Most of the new synthesized compounds are examined on NCI sixty cell lines of human cancer cells. Furthermore, molecular modeling study was administered to realize any clarification of the binding mode in the inactive VEGFR-2 conformation that demonstrates compatible binding modes similar to those of sorafenib and regorafenib. Finally, several ADME descriptors were calculated through a predictive kinetic study.

Discovery of Potent Small-Molecule Inhibitors of MLL Methyltransferase.

The mixed-lineage leukemia (MLL) protein, also known as MLL1, is a lysine methyltransferase specifically responsible for methylation of histone 3 lysine 4. MLL has been pursued as an attractive therapeutic target for the treatment of acute leukemia carrying the MLL fusion gene or MLL leukemia. Herein, we report the design, synthesis, and evaluation of an S-adenosylmethionine-based focused chemical library which led to the discovery of potent small-molecule inhibitors directly targeting the MLL SET domain. Determination of cocrystal structures for a number of these MLL inhibitors reveals that they adopt a unique binding mode that locks the MLL SET domain in an open, inactive conformation.

A Fast and Clean BTK Inhibitor

Bruton’s tyrosine kinase (BTK) is a major drug target for B-cell related malignancies; however, existing BTK inhibitors approved for cancer treatment have significant off-targets that limit their use for autoimmune and inflammatory diseases. Remibrutinib (LOU064) is a novel covalent BTK inhibitor that binds an inactive BTK conformation, which affords it unprecedented selectivity. Its optimization led to rapid BTK engagement in vivo and fast clearance, further limiting systemic exposure. Remibrutinib is currently in phase 2 clinical trials for treatment of chronic urticaria and Sjoegren’s syndrome.

Understanding thermal and organic solvent stability of thermoalkalophilic lipases: insights from computational predictions and experiments.

Bacillus thermocatenulatus lipase (BTL2), a member of the isolated lipase family known as thermoalkalophilic lipases, carries potential for industrial applications owing to its ability to catalyze versatile reactions under extreme conditions. This study investigates the molecular effects of distinct solvents on the stability of BTL2 at different temperatures, aiming to contribute to lipase use in industrial applications. Initially, molecular dynamic (MD) simulations were carried out to address for the molecular impacts of distinct solvents on the structural stability of BTL2 at different temperatures. Two lipase conformations representing the active and inactive forms were simulated in 5 solvents including water, ethanol, methanol, cyclohexane, and toluene. Low temperature simulations showed that polar solvents led to enhanced lid fluctuations compared with non-polar solvents reflecting a more dynamic equilibrium between active and inactive lipase conformations in polar solvents including water, while the overall structure of the lipase in both forms became more rigid in non-polar solvents than they were in polar solvent. Notably, the native lipase fold was maintained in non-polar solvents even at high temperatures, indicating an enhancement of lipase's thermostability in non-polar organic solvents. Next, we conducted experiments for which BTL2 was expressed in a heterologous host and purified to homogeneity, and its thermostability in different solvents was assessed. Parallel to the computational findings, experimental results suggested that non-polar organic solvents contributed to BTL2's thermostability at concentrations as high as 70% (v/v). Altogether, this study provides beneficial insights to the lipase use under extreme conditions. Graphical Abstract.

A high-resolution description of \u03b21-adrenergic receptor functional dynamics and allosteric coupling from backbone NMR

Signal transmission and regulation of G-protein-coupled receptors (GPCRs) by extra- and intracellular ligands occurs via modulation of complex conformational equilibria, but their exact kinetic details and underlying atomic mechanisms are unknown. Here we quantified these dynamic equilibria in the β1-adrenergic receptor in its apo form and seven ligand complexes using 1H/15N NMR spectroscopy. We observe three major exchanging conformations: an inactive conformation (Ci), a preactive conformation (Cp) and an active conformation (Ca), which becomes fully populated in a ternary complex with a G protein mimicking nanobody. The Ci ↔ Cp exchange occurs on the microsecond scale, the Cp ↔ Ca exchange is slower than ~5 ms and only occurs in the presence of two highly conserved tyrosines (Y5.58, Y7.53), which stabilize the active conformation of TM6. The Cp→Ca chemical shift changes indicate a pivoting motion of the entire TM6 that couples the effector site to the orthosteric ligand pocket.

Recruitment of kindlin-3 to plasma membrane through its PH domain precedes high affinity β2 integrin activation and neutrophil arrest

Abstract Leukocytes including neutrophils must adhere to the vascular endothelium in order to respond to injury or infection. Integrin-mediated neutrophil adhesion starts by arrest from rolling. Activation of integrins involves conformational changes from an inactive bent conformation to an extended conformation (E+) with high affinity for ligand binding (H+). The cytoplasmic protein kindlin-3 is required for leukocyte adhesion; mutations of kindlin-3 cause leukocyte adhesion deficiency type III syndrome. However, the molecular mechanism by which kindlin-3 regulates β2 integrin activation is unknown. Here we measured the spatiotemporal dynamics of kindlin-3 and β2 integrin conformation changes during neutrophil and HL-60 cell rolling and arrest under flow. Using high-resolution quantitative dynamic footprinting (qDF) microscopy and kindlin-3-fluorescent protein (FP) fusion proteins, we find that kindlin-3 is recruited to the plasma membrane prior to induction of the H+ β2 integrin conformation. Deletion of kindlin-3 or its lipid-binding pleckstrin homology (PH) domain completely abolished H+ β2 integrin induction. Intravital imaging of bone marrow chimeric mice revealed that EGFP-tagged kindlin-3-expressing neutrophils, but not control kindlin-3-deficient neutrophils, arrested in cremaster venules in vivo in response to CXCL1. Our data show that kindlin-3 is indispensable for the H+ β2 integrin conformation and crucial for integrin-mediated leukocyte adhesion during inflammation.

Abstract B45: Surveillance of RAS-RAF dynamics in vivo: Tracking activity conformations and drug-induced interactions

Abstract Live-cell recordings of protein conformations and interactions yield novel mechanistic insights into physiologic and pathologic protein functions. We have developed an adaptable and genetically encoded bioluminescence-based biosensor platform to precisely measure signaling dynamics of full-length RAS GTPases and RAF kinases upon physiologic pathway activation, post-translational modifications, mutagenesis, and drug binding. We have recorded the impact of both patient mutations and bioactive small-molecule binding on kinase conformations and binary interactions of GTP-loaded H, N, KRAS with kinases such as RAF. Systematic profiling of αC-helix-OUT BRAF inhibitors (BRAFi) vemurafenib, dabrafenib, encorafenib, and PLX8394 using the BRAF reporter reporter platform (displaying 10 different patient mutations) revealed differences in drug specificities and efficacies. Unexpectedly, the αC-helix-OUT BRAFi engagement with the catalytic pocket of mutated BRAF stabilized an inactive and intermediate full-length kinase conformation, which provoked the enhancement of binary RAS:RAF interactions. We showed that the interference with RAS interactions and nanoclustering has the potential to antagonize the sequential formation of drug-induced tetrameric RAS:RAF complexes. Recently, we have bioengineered a reporter toolbox for direct in vivo recordings of RAS activity conformations. First, we demonstrated that the wild-type RAS reporter—expressed at levels far below endogenous RAS proteins—responds to physiologic pathway activation. Second, we showed that hot-spot RAS mutations display a similar effect on the intramolecular dynamics. The systematic implementation, combination, and advancement of the GTPase and kinase reporter platform have the potential to be used to identify novel means for regulating or perturbing RAS-RAF-ERK signaling in vivo. Citation Format: Ruth Röck, Johanna Mayrhofer, Omar Torres-Quesada, Florian Enzler, Jakob Troppmair, Eduard Stefan. Surveillance of RAS-RAF dynamics in vivo: Tracking activity conformations and drug-induced interactions [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B45.

Nek7 conformational flexibility and inhibitor binding probed through protein engineering of the R-spine.

Nek7 is a serine/threonine-protein kinase required for proper spindle formation and cytokinesis. Elevated Nek7 levels have been observed in several cancers, and inhibition of Nek7 might provide a route to the development of cancer therapeutics. To date, no selective and potent Nek7 inhibitors have been identified. Nek7 crystal structures exhibit an improperly formed regulatory-spine (R-spine), characteristic of an inactive kinase. We reasoned that the preference of Nek7 to crystallise in this inactive conformation might hinder attempts to capture Nek7 in complex with Type I inhibitors. Here, we have introduced aromatic residues into the R-spine of Nek7 with the aim to stabilise the active conformation of the kinase through R-spine stacking. The strong R-spine mutant Nek7SRS retained catalytic activity and was crystallised in complex with compound 51, an ATP-competitive inhibitor of Nek2 and Nek7. Subsequently, we obtained the same crystal form for wild-type Nek7WT in apo form and bound to compound 51. The R-spines of the three well-ordered Nek7WT molecules exhibit variable conformations while the R-spines of the Nek7SRS molecules all have the same, partially stacked configuration. Compound 51 bound to Nek2 and Nek7 in similar modes, but differences in the precise orientation of a substituent highlights features that could be exploited in designing inhibitors that are selective for particular Nek family members. Although the SRS mutations are not required to obtain a Nek7–inhibitor structure, we conclude that it is a useful strategy for restraining the conformation of a kinase in order to promote crystallogenesis.

Insights into adenosine A2Areceptor activation through cooperative modulation of agonist and allosteric lipid interactions.

The activation process of G protein-coupled receptors (GPCRs) has been extensively studied, both experimentally and computationally. In particular, Molecular Dynamics (MD) simulations have proven useful in exploring GPCR conformational space. The typical behaviour of class A GPCRs, when subjected to unbiased MD simulations from their crystallized inactive state, is to fluctuate between inactive and intermediate(s) conformations, even with bound agonist. Fully active conformation(s) are rarely stabilized unless a G protein is also bound. Despite several crystal structures of the adenosine A2a receptor (A2aR) having been resolved in complex with co-crystallized agonists and Gs protein, its agonist-mediated activation process is still not completely understood. In order to thoroughly examine the conformational landscape of A2aR activation, we performed unbiased microsecond-length MD simulations in quadruplicate, starting from the inactive conformation either in apo or with bound agonists: endogenous adenosine or synthetic NECA, embedded in two homogeneous phospholipid membranes: 1,2-dioleoyl-sn-glycerol-3-phosphoglycerol (DOPG) or 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC). In DOPC with bound adenosine or NECA, we observe transition to an intermediate receptor conformation consistent with the known adenosine-bound crystal state. In apo state in DOPG, two different intermediate conformations are obtained. One is similar to that observed with bound adenosine in DOPC, while the other is closer to the active state but not yet fully active. Exclusively, in DOPG with bound adenosine or NECA, we reproducibly identify receptor conformations with fully active features, which are able to dock Gs protein. These different receptor conformations can be attributed to the action/absence of agonist and phospholipid-mediated allosteric effects on the intracellular side of the receptor.

Binding of agonist WAY-267,464 and antagonist WAY-methylated to oxytocin receptor probed by all-atom molecular dynamics simulations

AimsNon-peptide ligands of oxytocin receptor (OTR) have promising potentialities as therapeutic agents with improved pharmacological properties. WAY-267,464 is a non-peptide agonist which loses its agonist activity when its resorcinol moiety is methylated, yielding a partial antagonist (denoted here, WAY-Methylated). This study attempts to rationalize these opposing activities by comparative analyses of structural dynamicsof OTR in complex with these ligands. Main methodsGlide extra precision (XP) docking with and without positional constraints was employed to probe alternative binding poses of both WAY-267,464 and WAY-Methylated. The more preferred configuration of each system was subjected to an extended 2 μs MD simulation and the physics-based Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) binding energy was used to rank the complexes with improved accuracy, in addition to empirical-based Glide docking score. Network analysis was performed, and the identified critical residues were cross-referenced with the experimental mutagenesis data. Key findingsThe added methyl groups in the antagonist WAY-Methylated enhanced hydrophobicity, resulting in a flipped binding pose deeper in the binding pocket. Interestingly, OTR responded to the methylation by stabilizing the initial inactive conformation, decreasing fluctuations and increasing the overall secondary structural composition. Conversely, the agonist WAY-267,464 produced larger fluctuations to allow the receptor to change from the default inactive state to a state of partial activation. These transitions were further supported by the identified critical residues overlapping with experimental mutagenesis data. SignificanceThese findings provide insights into the activation mechanism of OTR by WAY-267.464 and its antagonism by WAY-Methylated.

Discovery of strong inhibitory properties of a monoclonal antibody of PKA and use of the antibody and a competitive photoluminescent orthosteric probe for analysis of the protein kinase

We show that the antibody, clone mAb(D38C6), of the α isoform of the catalytic subunit of PKA (PKAcα) inhibits the kinase-catalyzed phosphorylation with low-nanomolar inhibitory potency (Ki = 2.4 nM). This property of the antibody was established by its capacity to displace a synthetic small-molecule active site-binding (orthosteric) photoluminescent ARC-Lum(Fluo) probe from the complex with PKAcα. Likely, the competitiveness of association of the two binders with the protein is coming from two excluding conformations of PKAcα to which the binders bind. mAb(D38C6) possesses a linear peptide epitope and it binds to the disordered C-tail of unliganded inactive conformer of PKAcα. ARC-Lum(Fluo) probes bind to the ordered and active conformation of PKAcα with Phe327 residue from the C-tail taking part in the formation of the active core.Consecutive application of these competitive PKAcα binders was used to develop an immunoassay allowing the determination of PKAcα concentration in complex biological solutions. At first, PKAcα was captured from the solution by the isoform-specific antibody and thereafter a high-affinity ARC-Lum(Fluo) probe was used to displace PKAcα from the binary complex. The developed immunoassay could be used for quantification of small amounts (starting from 93 pg, 2.3 fmol) of PKAcα in cell lysates.

Recruitment of kindlin‐3 to plasma membrane through its PH domain precedes high affinity β2 integrin activation and neutrophil arrest

Leukocytes including neutrophils must adhere to the vascular endothelium in order to respond to injury or infection. Integrin‐mediated neutrophil adhesion starts by arrest from rolling. Activation of integrins involves conformational changes from an inactive bent conformation to an extended conformation (E+) with high affinity for ligand binding (H+). The cytoplasmic protein kindlin‐3 is required for leukocyte adhesion; mutations of kindlin‐3 cause leukocyte adhesion deficiency type III syndrome. However, the molecular mechanism by which kindlin‐3 regulates β2 integrin activation is unknown. Here we measured the spatiotemporal dynamics of kindlin‐3 and β2 integrin conformation changes during neutrophil and HL‐60 cell rolling and arrest under flow. Using high‐resolution quantitative dynamic footprinting (qDF) microscopy and kindlin‐3‐fluorescent protein (FP) fusion proteins, we find that kindlin‐3 is recruited to the plasma membrane prior to induction of the H+ β2 integrin conformation. Deletion of kindlin‐3 or its lipid‐binding pleckstrin homology (PH) domain completely abolished H+ β2 integrin induction. Intravital imaging of bone marrow chimeric mice revealed that EGFP‐tagged kindlin‐3‐expressing neutrophils, but not control kindlin‐3‐deficient neutrophils, arrested in cremaster venules in vivo in response to CXCL1. Our data show that kindlin‐3 is indispensable for the H+ β2 integrin conformation and crucial for integrin‐mediated leukocyte adhesion during inflammation.Support or Funding InformationThis work was supported by grants from the National Institutes of Health, USA (HL078784 to K.L., R01HL145454 to Z.F.), a Postdoctoral Fellowship (19POST34450228 to L.W.) and a Career Development Award (18CDA34110426 to Z.F.) from the American Heart Association, USA.

The role of mobility in flavivirus protease substrate selection

Flaviviruses, including Zika virus and dengue virus, continue to threaten public health. The associated viral proteases, known as NS2B‐NS3pro, are essential to the viral life cycles and can interfere with host responses, making the proteases drug targets of considerable interest. Although the viruses are highly homologous, Zika virus is capable of severely damaging developing fetuses as well as causing neurological abnormalities in adults whereas dengue causes a sometime fatal hemorrhagic fever. While the proteases are highly identical, the catalytic efficiency and the substrates selection between Zika and dengue proteases are highly divergent, leading us to hypothesize that unique properties in the viral proteases may contribute the disparate viral pathologies. This work has made significant progress towards uncovering the role of conformation and flexibility in NS2B‐NS3pro from the function of these two flaviviruses on multiple fronts. First, NS2B‐NS3 proteases transitions between two widely different conformational states: an ‘open’ (inactive) conformation and a ‘closed’ (active) conformation. We developed versions of NS2B‐NS3pro that allow us to trap the enzyme in distinct conformations. Our data suggests that the enzymatic activity is dependent not only on the movement of NS2B, but may also rely on the flexibility of the protease core. Locking the enzyme into the ‘closed’ conformation dramatically increased activity. Second, to elucidate how Zika virus protease affects host cellular pathways and consequent pathologies, we used unbiased N‐terminomics to identify 31 human proteins cleaved by the NS2B‐NS3 protease. In particular, autophagy‐related protein 16‐1 (ATG16L1) and eukaryotic translation initiation factor 4 gamma (eIF4G1) are dramatically depleted during Zika virus infection. ATG16L1 and eIF4G1 mediate type‐II interferon production and host‐cell translation, respectively, likely aiding immune system evasion and driving the Zika life cycle. Intriguingly, the NS2B co‐factor region from Zika virus protease is essential for recognition of host cell substrates. Replacing the NS2B region in another flavivirus protease enabled recognition of novel Zika‐specific substrates by hybrid proteases, suggesting that the co‐factor is the principal determinant in ZVP substrate selection.

Biased allosteric modulators as a novel approach to induce biased signalling in G‐protein coupled receptors: The case of beta 2 adrenergic receptor in cardiovascular diseases

G protein‐coupled receptors (GPCRs) are the therapeutic target of nearly half of the current drugs in the market. It has been now well established that GPCRs can signal through multiple transducers, including G proteins and beta‐arrestins. These signalling pathways can be activated or blocked by “balanced” agonists or antagonists, but also, they can also be selectively activated in a “biased” response. Until now, biased signalling has been induced by biased ligands and biased receptors, any of which can result in preferential signalling through G proteins or beta‐arrestins. However, the discovery and development of GPCR biased agonists has been quite challenging, since in most GPCRs there is no structure activity relationship studies available, moreover, different active conformations for each pathway are likely very similar. Traditionally, biased agonists have been developed to target only the orthosteric site which is the binding site of the endogenous ligands. Development of biased agonists targeting the orthosteric site might be extremely difficult, especially in the absence of structure‐activity relationship information. Biased agonism holds great promise as a mechanism to significantly reduce the side effects that current drugs in used in the clinic, as well as develop drugs for use on their own. Also, ligands that produce biased signalling will serve as valuable tools for elucidation of the molecular mechanisms underlying GPCR‐signalling. Allosteric modulators are ligands which bind to a receptor at a site distinct from that of the endogenous agonist and they do not activate the receptor rather than stabilize or induce intermediate active or inactive structural conformations modulating signalling of the orthosteric ligands. We tested the hypothesis that biased signalling of the receptor could be achieved by targeting allosteric domains which show high diversity in structure and amino acid sequence among the GPCRs. The rationale is that the development of a novel series of allosteric modulators will regulate GPCR signalling in such way that the endogenous ligand would produce biased signalling only when the target receptor is bound to an allosteric modulator. In this study, we targeted the intracellular domains of the beta 2 adrenergic receptor (b2‐AR) to produce beta‐arrestin biased signalling when activated by traditional orthosteric ligands. A highly selective b2‐AR biased allosteric modulator would be highly desirable to treat a number of cardiovascular diseases. Thus, we used a combination of state of art drug discovery platforms, in silico calculations and peptidomimetics to develop a beta‐arrestin biased allosteric modulator AR1981. This allosteric modulator increased the beta‐arrestin recruitment efficacy of isoproterenol by two‐fold. Also, AR1981 was able to increase the potency of isoproterenol in beta‐arrestin recruitment by more than ten‐fold. Thus, AR1981 is a promising tool to access a novel pharmacological profile stimulating cardioprotective signalling through the b2‐AR and can serve as a model for the next generation of cardiovascular drug development. The outcome of the proposed work will facilitate the development of a new generation of allosteric modulators for GPCRs.

Rational development of a high-affinity secretin receptor antagonist

The secretin receptor is a prototypic class B GPCR with substantial and broad pharmacologic importance. The aim of this project was to develop a high affinity selective antagonist as a new and important pharmacologic tool and to aid stabilization of this receptor in an inactive conformation for ultimate structural characterization. Amino-terminal truncation of the natural 27-residue ligand reduced biological activity, but also markedly reduced binding affinity. This was rationally and experimentally overcome with lactam stabilization of helical structure and with replacement of residues with natural and unnatural amino acids. A key new step in this effort was the replacement of peptide residue Leu22 with L-cyclohexylalanine (Cha) to enhance potential hydrophobic interactions with receptor residues Leu31, Val34, and Phe92 that were predicted from molecular modeling. Alanine-replacement mutagenesis of these residues markedly affected ligand binding and biological activity. The optimal antagonist ligand, (Y10,c[E16,K20],I17,Cha22,R25)sec(6–27), exhibited high binding affinity (4 nM), similar to natural secretin, and exhibited no demonstrable biological activity to stimulate cAMP accumulation, intracellular calcium mobilization, or β-arrestin-2 translocation. It acts as an orthosteric competitive antagonist, predicted to bind within the peptide-binding groove in the receptor extracellular domain. The analogous peptide that was one residue longer, retaining Thr5, exhibited partial agonist activity, while further truncation of even a single residue (Phe6) reduced binding affinity. This sec(6–27)-based peptide will be an important new tool for pharmacological and structural studies.