Angiotensin II receptors

Chapter 20

Angiotensin II receptors: structurefunction and drug discovery Khuraijam Dhanachandra Singh and Sadashiva S. Karnik Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, United States

20.1 Introduction G protein-coupled receptors (GPCRs) are the largest class of membrane proteins in humans consisting of w826 receptors that include w400 olfactory receptors (Kolakowski, 1994; Hauser et al., 2017). Over 40% of the drugs marketed currently target the nonolfactory receptors which constitute w27% of the global market share of therapeutic drugs (Hauser et al., 2017; Luttrell, 2008; The IDG Knowledge Management Center, 2016). Approximately 20% of the 321 agents in clinical trials at present target GPCRs (Hauser et al., 2017). There may soon be a significant increase in the number of drugs targeting GPCRs due to recent breakthroughs in GPCR structural biology and advancement in GPCR pharmacology. GPCRs are responsible for normal physiology in our body. Dysfunction in the activity of GPCR0 s can lead to abnormal cell functions and are responsible for the etiology of numerous diseases and disorders. In general, drug development strategies target dysfunctional GPCRs to restore the normal physiology of the cells. Common functions of drug molecules are: (1) to compete with the endogenous ligand acting either as an agonist or an antagonist of the receptor (2) to act as a biased ligand (i.e., preferentially activate the desired intracellular signaling pathway while minimizing undesired effects of activating other pathways) and (3) to act as an allosteric ligand which bind to a site of the receptor other than the endogenous ligand binding site and modulate the receptor function. Receptors are classified into Class A to F. Class A receptors includes rhodopsin-like receptors. The angiotensin receptors are in Class A Subfamily A3. Angiotensin receptors are the receptors, which are activated by the peptide hormone Angiotensin II (AngII). The testing of nonpeptide compounds, DuP 753 and EXP655, against the angiotensin receptors led to the discovery of two different types of angiotensin receptors (i.e., angiotensin type 1 (AT1R) and angiotensin type 2 receptor (AT2R)) (Chiu et al., 1989a). These receptors are components of the renin-angiotensin system and are activated by the peptide hormone AngII. Most of the AngII actions, which include growth promotion, vasoconstriction, antinatriuresis, aldosterone secretion, inhibition of renin biosynthesis and release, salt appetite, thirst, and sympathetic outflow, are believed to occur through AT1R (Hein et al., 1995). The function of AT2R is less known and believed to oppose the AT1R mediated functions. For supporting this speculation, it is shown that AT2R inhibits growth and cell proliferation in most tissues opposing the cell dedifferentiation, proliferation, and growth effects mediated by AngII through AT1R signaling (Ichiki et al., 1995). Imbalance in AT1R signaling causes hypertension, cardiac arrhythmia, stroke, diabetic nephropathy, and metabolic disorders (Audoly et al., 2000; de Gasparo et al., 2000; Thomas and Mendelsohn, 2003), which are currently treated using AT1R-blockers effectively (Zaman et al., 2002; Billet et al., 2008; Akazawa et al., 2013; Michel et al., 2013; Seva Pessoa et al., 2013). Therefore, AT1R antagonists or AT1R-blockers (ARBs) are now an important class of drugs for the treatment of hypertension and heart failure including the protection from diabetic nephropathy (Zaman et al., 2002; Billet et al., 2008; Akazawa et al., 2013; Michel et al., 2013; Seva Pessoa et al., 2013). Until now, eight ARBs (azilsartan, candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan, valsartan) are approved by the FDA and are now in clinical use (Taylor et al., 2011). Fimasartan is another ARB, which is approved only in South Korea and presently seeking worldwide partnership (Fimasartan. Amardiova, 2011). Some ARBs, viz. Azilsartan, candesartan, and olmesartan are administered orally as prodrugs, and their metabolites are more active (Barreras and Gurk-Turner, 2003). Structural diversity within the ARBs is associated with differences in their bioavailability and insurmountable antagonism (Michel et al., 2013; Abraham et al., 2015). The differences in structures are also associated with differences in the pharmacokinetic profile,

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particularly the duration of action. Differences in the clinical outcome of each ARBs need to be addressed. Even though all the ARBs are highly specific to AT1R, telmisartan additionally is a partial agonist of peroxisome proliferator-activated receptor-Y (PPARY) and was shown to have a beneficial effect with insulin resistance (Yamagishi et al., 2007; Amano et al., 2012; Rios et al., 2012). In this chapter, we will be describing the recently solved crystal structures of both angiotensin receptors. This development opens up an avenue for further design of more potent drugs against the receptors. We will then discuss the time course of the development of drugs against angiotensin receptors.

20.2 Structure-function of angiotensin receptors 20.2.1 AT1R structure The AGT1R gene contains five exons and four introns which produce a coding transcript that translates into 359 amino acid residues, yielding a protein of w41 kDa (Sasaki et al., 1991; Murphy et al., 1991). AT1R belongs to the seven transmembranes (7TM) class of GPCRs, which has extracellular and intracellular domains. The extracellular domain consists of the N terminus and the extracellular loops (ECLs). ECLs contains three N-glycosylation sites, four cysteine residues forming two disulfide bonds, which are prone to inactivation by dithiothreitol and other reducing agents (Warnecke et al., 1999). The crystal structure of AT1R was solved with the antagonist ZD7155 at 2.9 Å resolution and olmesartan at 2.8 Å, which is a clinically used ARB (Zhang et al., 2015a). A thermostabilized apocytochrome, b562RIL (BRIL) (Chun et al., 2012), was fused to the N terminus of AT1R to facilitate the crystallization after truncating 11 residues. Forty residues were truncated from the C-terminus after the transmembrane helix VIII. The effects of the engineered protein were well studied, and its function was not altered. The final structure consists of 289 out of 359 residues in the ZD7155 bound structure (Zhang et al., 2015a). AT1R has the most homology structurally with the chemokine and opioid receptors (RMSDCa w1.8 Å) (Zhang et al., 2015a). The overall structures are highly similar but the tilts and extensions of the extracellular ends of helices I, V, VI, and VII are substantially different within the peptide receptors as well as helices IV and V in the intracellular side adopt the most diverse conformations (Zhang et al., 2015a). The conformation of helices II and III are nearly identical in all these peptide receptors. The extracellular part of AT1R consists of the N-terminal segment, the extracellular loops ECL1, ECL2, and ECL3, and two disulfide bonds, which help to shape the extracellular side of AT1R. Cys18-Cys274 connects the N terminus with ECL3 and Cys101-Cys180 connects helix III with ECL2. These disulfide bonds are similar to those found in the chemokine receptors CXCR4 and CCR5 (Wu et al., 2010; Tan et al., 2013). The ECL2 region exhibits a b-hairpin secondary structure in AT1R, which is also a common motif among peptide GPCRs (Zhang et al., 2015a). Regions of ECL2 serve as an epitope for pathogenic autoantibodies, which agonistically activate the AT1R and are found in preeclampsia and renal transplant rejection patients (Xia and Kellems, 2013; Herse and Lamarca, 2012; Lamarca et al., 2011; Dragun, 2007; Jobert et al., 2015). This epitope region is not conserved within the GPCR family and highly specific to AT1R. The intracellular domain consists of the intracellular loops ICL1, ICL2, and ICL3, helix VIII and a highly disordered long C-terminal region, which was truncated during the crystallization process. The highly conserved motifs in GPCRs, D(E)RY motif in helix III and the NPxxY motif in helix VII which are important for activation, are also present in AT1R in the intracellular region (Zhang et al., 2015a). However, the ionic lock between Arg3.50 (superscript indicates residue number as per the Ballesteros and Weinstein, 1995 [B&W] nomenclature) and Asp/Glu6.30 at the cytoplasmic end of helix VI is absent due to the lack of an acidic residue at position 6.30 (Zhang et al., 2015a). Helix VIII of AT1R is angled away from the membrane unlike other GPCRs, which run parallel to the membrane bilayer. It is reported to bind with the calcium-regulated effector proteins, calmodulin (Thomas et al., 1995) and filamin (Tirupula et al., 2015), and its integrity important for receptor internalization and coupling to G-protein activation and signaling (Thomas et al., 1995; Sano et al., 1997). Highly positive charged residues in helix VIII (306-KKFKR-312) are sensitive toward negatively charged lipids and lack putative palmitoylation sites like those found in other GPCRs (Zhang et al., 2015a).

20.2.2 Binding pocket of AT1R Eight ARBs have been approved by the FDA and are in clinical use (Taylor et al., 2011). Olmesartan, losartan, candesartan, irbesartan, valsartan, and azilsartan are the biphenyl tetrazole derivatives, and eprosartan and telmisartan are the nonbiphenyl tetrazole ARBs (Michel et al., 2013). Crystal structures of AT1R complex with ZD7155 and olmesartan reveal that the helices, I, II, III, and VII, as well as ECL2, are involved in the binding of a ligand (Zhang et al., 2015a,b). Comparison of the two AT1R crystal structures reveals that the conformation of pocket residues is very similar, but a large variation was observed at Ile2887.39, changing the rotamer toggle switch. A difference was also observed between the two structures with

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w1 Å shifts of Tyr351.39 in helix I and Gly1965.39 to Leu2015.44 in helix V toward the ligand pocket (Zhang et al., 2015a,b). These differences resulted in a slightly decreased size of the binding pocket in the olmesartan bound state of AT1R. Side chains Arg167ECL2 and Tyr351.39 form ionic and H-bond interactions with ZD7155 and olmesartan. The positively charged guanidine group Arg167ECL2 forms a strong interaction with the tetrazole group of ZD7155 and olmesartan, and the naphthyridine-2-one moieties of ZD7155 and carboxylic acid moieties of olmesartan (Zhang et al., 2015a,b). Binding pocket residues consists of Tyr351.39, Trp842.60, Tyr872.63, Tyr92ECL1, Arg167ECL2, Lys1995.42, Ile2887.39 and Tyr2927.34. Arg167ECL2, Tyr351.39, and Trp842.60, which are the critical amino acids for binding of biphenyl-tetrazole derivative ARBs (Zhang et al., 2015b; Singh et al., 2017). For the nonbiphenyl tetrazole derivative eprosartan, Ile2887.39 and Tyr2927.34 mutations significantly reduce binding affinity, and for telmisartan, Tyr92ECL1 and Ile2887.39 mutations significantly reduced binding affinity (Zhang et al., 2015b). All the clinically used biphenyl tetrazole derivative drugs also bind with the same pattern as olmesartan, as shown by molecular docking and simulation studies. Singh et al. (2017) performed extensive molecular dynamics simulation study of all ARBs and found that two acid moieties always project toward the side chain of Arg167ECL2 and form a strong bond with guanidine group (Singh et al., 2017). Mutagenesis reveals that mutation of Arg167ECL2 and Tyr351.39 to Ala completely abolishes the binding of peptide and nonpeptide ligands. Therefore, these residues are important for binding any nonpeptide and peptide ligands (Zhang et al., 2015b). An AngII (Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8) bound AT1R structure is not available as of yet, but mutagenesis and several molecular modeling studies reveal that it binds to the same site where olmesartan binds (Fillion et al., 2013; Matsoukas et al., 2013; Balakumar and Jagadeesh, 2014). Substitution of Asp1 in AngII to sarcosine enhances the binding affinity without any changes to its agonistic property (Wilkes et al., 2002; Fillion et al., 2010). Arg2 in AngII is important for binding and substitution to an uncharged residue significantly reduces its binding affinity and receptor desensitization (Feng et al., 1995). It forms a salt bridge with Asp2817.32 and Asp2787.29 and is important for ligand positioning and opening of the ligand binding pocket (Nikiforovich et al., 2006; Hjorth et al., 1994). Substitution of Tyr4 in AngII to nonaromatic residues reduces its agonistic property (Oliveira et al., 2007; Karnik et al., 2015). Substitution of His6 in AngII to Tyr causes loss of its binding affinity (Oliveira et al., 2007). The role of Pro7 is essential for the conformational geometry of AngII (Wilkes et al., 2002). Substitution of Phe8 to nonaromatic residues like Ala and Ile significantly reduces IP3 signaling, whereas substitution to Ala or Gly causes more selective b-arrestin-dependent signaling (Miura et al., 1999; Holloway et al., 2002; Zimmerman et al., 2012; Noda et al., 1996). If Phe8 is removed [des-Phe8-heptapeptide or Ang(1e7)], the agonistic property is completely lost. Molecular modeling reveals that the 3-amino group of Lys1995.42 forms an ionic bridge with the a-carboxyl of Phe8 in AngII. Mutagenesis studies further reveal that Tyr351.39, Trp842.60, Tyr872.63, Tyr92ECL1, Arg167ECL2, Lys1995.42, Ile2887.39, and Tyr2927.34 are the important residues for binding of AngII and substitution of any of these residues abolishes its binding (Balakumar and Jagadeesh, 2014; Karnik et al., 2015). Several studies have observed that the inactive conformation of AT1R is stabilized by the interaction between Asn1113.35 and Asn2957.46 and the mutation of either residue induces constitutive activation of the receptor (Balakumar and Jagadeesh, 2014; Karnik et al., 2015; Cabana et al., 2013). Binding of AngII to AT1R disrupts this interaction and allows interaction with the conserved Asp742.50 (Balakumar and Jagadeesh, 2014; Unal and Karnik, 2014). Interestingly, Asp742.50, Asn1113.35, and Asn2957.46, together with residue from WxP and NPxxY motif, viz. Trp2536.48 and Asn2987.49, respectively, belong to the putative sodium binding pocket of AT1R (Katritch et al., 2014), which was speculated from the superimposition of the high-resolution structure of the d-opioid receptor with the AT1R structure (Zhang et al., 2015a; Fenalti et al., 2014). All the residues in the putative sodium binding pocket are conserved except Asn2957.46, which is replaced by Ser in the d-opioid receptor. Ser7.46 at this position is conserved in most of the GPCRs and replaced with Asn7.46, a peculiar characteristic of AT1R. Of particular interest, Asn2957.46 forms hydrogen-bond (H-bond) with Asn1113.35 and maintains the receptor in the inactive state, and therefore, may impact sodium binding and functional properties of AT1R (Zhang et al., 2015a). Moreover, Phe772.53, which is in the binding pocket, and the neighboring residue, Asn1113.35, are important for inter-helical interactions (Miura et al., 2003). Mutating both these residues (i.e., Phe772.53 and Asn1113.35) to Ala result in a fully active receptor (Miura et al., 2008).

20.2.3 AT2R structure The AT2R structure was solved with a nonpeptide ligand (PDB id: 5unf, 5ung, 5unh) (Zhang et al., 2017) at a resolution of 2.8 Å and with the AngII analog s-AngII (PDB id: 5xjm) at a resolution of 3.2 Å. A crystal structure with an antibody that can activate AT2R has also been solved (PDB id: 5xli) (Asada et al., 2018). The AT2R crystal structure was solved by truncating N-terminal residues 1e34 and C-terminal residues 336e363, fusing a thermo-stabilized apo-cytochrome b562RIL in the truncated N-terminus via a four-residue linker (PDB id: 5unf, 5ung, 5unh). The apo-cytochrome b562RIL replaces 243 SYG245 in ICL3 in the s-AngII bound structure (PDB id: 5xjm) (Asada et al., 2018). AT2R exhibits a 7TM domain,

418 PART | III GPCRs in disease and targeted drug discovery

intracellular amphipathic helix VIII, b-hairpin conformation of the ECL2 and two pairs of disulfide bonds, which is similar to AT1R and other peptide binding GPCRs (Zhang et al., 2017). Unlike the AT1R structure, AT2R reveals a substantially different conformation and exhibits an active-like structure found in other active GPCRs (Zhang et al., 2017).

20.2.4 Binding pocket of AT2R Crystal structures reveal that Arg182ECL2, Tyr1.39, and Trp1002.60 are the key residues involved in the binding of nonpeptide compounds (Zhang et al., 2017). Mutagenesis further reveals that Arg182ECL2 to Ala and Lys2155.42 to Ala or Gln completely abolishes the binding of both nonpeptide compounds and AngII (Zhang et al., 2017; Asada et al., 2018). AT2R bound with s-AngII (Sar1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Ile8), an analog of AngII, reveals that the N-terminus of s-AngII (Sar1-Arg2-Val3) is extended into the extracellular domain and the C-terminus of s-AngII (Tyr4-Ile5-His6-Pro7-Ile8) is folded into a C-shape (Asada et al., 2018). Both crystal structures suggest that nonpeptide and peptide ligands binding lead to identical conformations in protein structure (Zhang et al., 2017; Asada et al., 2018). An intermolecular interaction between Tyr4 and Ile8 and the constraint imposed by Pro7 is responsible for the C-shape structure of s-AngII, as is also observed in AngII binding with AT1R in several modeling and NMR studies (Asada et al., 2018; Zhou et al., 1991). The carbonyl oxygens of His6 and Pro7 form H-bonds with guanidinium of Arg182ECL2 and the carboxyl group of Ile8 forms an ionic bond with Lys2155.42. Tyr4 of the peptide ligand forms an H-bond with Tyr1042.64 and Ile5 forms an H-bond with Tyr1082.68. Val3 and Ile5 form van der Waals interactions with the side chains of Ile187ECL2 (conserved in AT1R as Ile172ECL2), Leu190ECL2, Ala194ECL2, and Ile196ECL2. The C terminus of s-AngII is surrounded by hydrophobic residues (Trp1002.60, Leu1243.32, Met1283.36, Phe2726.51, Ile3047.39, and Phe3087.43). The indole ring of Pro7 stacks with Trp1002.60 (conserved in AT1R as Trp842.60) and the side chain of Ile8 interacts with Leu1243.32, Met1283.36, Phe2726.51, Ile3047.39 and Phe3087.43 (Asada et al., 2018). Radiolabeled ligand binding followed by mutagenesis experiments further confirms that Tyr1082.68, Met1283.36, Arg182ECL2, Lys2155.42, Phe2726.51, and Asp2977.32 play a critical role in s-AngII binding, which is also conserved in AT1R (Asada et al., 2018). The observed conformation of s-AngII may also be presumed to occur for AngII when bound to AT1R/AT2R; however, the aromatic side chain of Phe8 in AngII may have stronger interactions with the hydrophobic environment in the binding pocket compared to Ile8 in s-AngII. Arg2 in s-AngII forms salt bridges with Asp2796.58 and Asp2977.32 (which are conserved in AT1R as Asp2636.58 and Asp2817.32). Mutations of these residues strongly reduce the binding affinity of s-AngII, suggesting the N-terminus of s-AngII is important for binding, which is consistent with a previous study on AngII (Asada et al., 2018).

20.2.5 Comparison of AT1R and AT2R structures Superimposition of the backbone structures of AT1R and AT2R shows an RMSD less than 3 Å, but there are substantial differences in the conformation (Fig. 20.1) (Zhang et al., 2015a,b, 2017; Asada et al., 2018). The crystal structures solved for AT1R were in the inactive states (Zhang et al., 2015a,b) and for the AT2R were in active-like states (Zhang et al., 2017; Asada et al., 2018). The intracellular end of helix VI in AT2R shows an outward displacement by approximately 11.5 Å, and the intracellular end of helix VII in AT2R exhibits an inward displacement by 4.9 Å w.r.t. AT1R structure (Zhang et al., 2017). These types of large-scale shifts of helix VI and helix VII in the intracellular region are observed in G protein, and b-arrestin bound states of GPCRs (Zhang et al., 2017). Further comparison of the important motifs in GPCRs that are involved in activation viz. DR3.50Y, NP7.50xxY, and P5.50-I3.40-F6.44 show that AT2R is in a similar conformation as activated GPCRs, such as Gs-protein-bound b2AR and arrestin-bound rhodopsin (Rasmussen et al., 2011; Kang et al., 2015). The significant large-scale movement of helix VI in the activated GPCRs is enabled by rearrangements in the P5.503.40 6.44 I -F motif (Pro2235.50-Ile1323.40-Phe2656.44 in AT2R), as observed in GPCRs structures (Rasmussen et al., 2011; Kang et al., 2015). The side chain of Arg1423.50 of the DR3.50Y motif in AT2R is rotated by around 90 degrees as compared with Arg1263.50 in AT1R, allowing the adoption of the active state as observed in other GPCRs (Zhang et al., 2017). In addition, the side chain of Tyr3187.53 is shifted by 6.5 Å from the corresponding position of Tyr3027.53 in AT1R of the NP7.50xxY motif, which results in an inward movement of helix VII, a peculiar characteristic of GPCRs upon activation (Zhang et al., 2015a,b, 2017). Moreover, analysis of conserved residues in the G-protein binding pocket (L [M]3.46eI[A]6.37eY[Y]7.53) reveals the rearrangement of interactions as observed in active GPCRs (Zhang et al., 2017). A highly conserved residue at position 6.37 has been replaced with Ala and reduces the interhelical interaction (as observed in inactive states of GPCRs) of helix III and helix VI in AT2R (Zhang et al., 2017). The highly conserved sodium binding pocket in GPCRs is largely altered upon activation, and a similar change in the conformation was also observed in AT2R. It is collapsed and rearranged, hindering sodium ion binding, mainly owing to the inward shift of helix VII (Zhang et al., 2015a; Katritch et al., 2014; Tang et al., 2014). Out of 16 residues in the putative sodium binding pocket,

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FIGURE 20.1 Comparison of AT1R (PDB id: 4YAY) and AT2R (PDB id: 5UNF) crystal structures. AT1R is bound with an antagonist ZD7511 (magenta), and AT2R is bound with an agonist Compound 1 (green). Residues in the orthosteric site are shown in stick representation (gray for AT1R and cyan for AT2R).

only two residues (Ile1353.43 and Ser3117.46) in AT2R are different from AT1R (Leu1193.43 and Asn2957.46). Notably, Ser3117.46 cannot form an H-bond with Asn1273.35 in AT2R, potentially shifting the conformational equilibrium toward the active state and forming a highly constitutively active receptor (Miura and Karnik, 1999, 2000). Therefore, the conformation of AT2R adopts an active like GPCR structure. The orientation of helix-VIII in most GPCRs, including AT1R, lies parallel to the membrane pointing out from the 7TM bundle irrespective of its activation state (Zhang et al., 2015a). However, for AT2R, Zhang et al. (2017) found that helixVIII adopts a very different conformation and interacts with helices II, V, and VI. A similar conformation of helix VIII was observed in all three crystal structures solved, suggesting that it is not an artifact (Zhang et al., 2017). This conformation of helix VIII blocks the binding of G protein and b-arrestin, which is consistent with the absence of traditional downstream signaling by G protein and b-arrestin in assays of AT2R. To further study the role of helix VIII, molecular dynamics (MD) simulations were performed and showed that its position remained within the RMSD values of <4 Å and quickly returned to the crystallographic position. MD simulations that relocated helix VIII to a position similar to that in AT1R show that helix VI moves inwards to a position similar to the inactive state in AT1R (Zhang et al., 2017). These overall observations reveal that AT2R is a constitutively active GPCR, which has been auto activated by its own helix VIII.

20.3 Selectivity of ligands Olmesartan and ZD7511 are biphenyl tetrazole derivative compounds that selectively bind to AT1R. Two compounds, i.e., compound one and compound two reported by Zhang et al. (2017), are also biphenyl tetrazole derivative compounds that are dual ligands for both AT1R and AT2R (Zhang et al., 2017). Superimposition of backbone structures reveals that both AT1R and AT2R are highly similar (Zhang et al., 2015a,b, 2017; Asada et al., 2018). Important residues, Arg167ECL2, Tyr351.39 and Trp842.60, which are required for ligand binding in AT1R are also conserved in AT2R. Crystal structures reveal that the side chain of Trp2.60 is shifted about 3.1 Å in AT2R, which allows it to form hydrophobic interaction with the benzene ring of compound one and furan ring of compound 2 (Zhang et al., 2017). Moreover, these two compounds engaged in polar interaction with side chains of Lys2155.42, Thr1253.33, and Thr1784.60 of AT2R. The bottom part of AT2R is expanded owing to the smaller side chain of Leu932.53 compared to Phe772.53 in AT1R. Also, Tyr2927.43 in AT1R is changed to Phe3087.43 in AT2R, which prevents the H-bond with the backbone carboxyl group of helix III, resulting in a shift of 5.4 Å. This arrangement opens a subpocket in AT2R, which is slightly different than the binding pocket of AT1R (Zhang et al., 2017).

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20.4 Discovery and development of angiotensin II receptor blockers In 1898, Robert Tigerstedt and Per Bergman injected kidney extracts into rabbits and observed a rise in blood pressure (BP), which suggested that a protein is involved in this rise in BP and they later called it renin. In 1930, Goldblatt observed that a very potent substance is responsible for vasoconstriction and not very effective with renin. In 1939, Braun-Menendez and Page independently discovered this potent substance as “hypertension” or “angiotensin” (Tigerstedt and Bergman, 1898). Skeggs et al. (1953) attempted to isolate and purify hypertension, but in the process, they found two forms viz. hypertension I and hypertension II (Skeggs et al., 1954a,b). They found that hypertension I is the result of the action of renin from its substrate and hypertension II is formed from hypertension I by an enzyme that is activated by chloride ion (later calling it “angiotensin converting enzyme”) and found that hypertension II has very potent vaso-constriction activity. In 1955, Skeggs et al. purified and sequenced hypertension II and later it was renamed as “angiotensin II” (Skeggs et al., 1956a,b, 1957). In 1971, Pals et al., replaced Phe8 with Ala and found it to reduce the vasoconstriction activity of angiotensin II (Pals et al., 1971). Regoli et al. (1963) and Bumpus et al. (1964) had already shown that aminopeptidase recognizes the N-terminus and degrades AngII and its analogs (Regoli et al., 1963; Bumpus et al., 1964). This led to the replacement of Asp1 to sarcosine (SAR) and prevented the degradation by aminopeptidase. Subsequently, other independent studies also showed that SAR1 substitution enhanced the binding affinity and reduced the degradation of angiotensin II and its analogs. This led to the discovery of the first effective angiotensin II analog saralasin. Several other analogs of angiotensin were synthesized, and their pharmacological activities were also evaluated. However, these angiotensin II analogs were very useful as pharmacological tools but lacked therapeutic values due to poor bioavailability, short duration, and significant agonistic properties (Wexler et al., 1992). Further, this observation led to the discovery of new nonpeptide AngII-receptor antagonists. Moreover, the discovery of nonpeptide antagonists also added a new dimension for the treatment of hypertension through the inhibition of RAS (Wexler et al., 1992). Structural knowledge of AngII led to the discovery of the first competitive antagonist N-benzylimidazoles by Takeda Chemical Industries in Osaka, Japan (Furukawa et al., 1982a,b). These compounds are found to be a weak antagonist but selectively blocked AT1R. Du Pont Merck Pharmaceutical Company (Wilmington, Delaware) embarked on a program to design more potent and orally effective antagonists, which preserves AT1R selectivity. At Du Pont, two compounds were first synthesized, i.e., S-8307 and S-8308, and were very similar to the Takeda lead molecule (Wong et al., 1988; Chiu et al., 1988). These compounds were very small and had to compete with a bigger molecule AngII. Therefore, they hypothesized that a bigger molecule might effectively compete with AngII (Wexler et al., 1992). In order to modify Takeda0 s compound to make a bigger molecule, it was aligned with AngII. It was found that the carboxylic group on the benzylimidazole aligned with the C-terminal carboxylic acid of AngII. At physiologic pH, both these acidic groups were pointed toward the hypothetical positively charged regions of the receptor. The positive charge in this region was confirmed by Hsieh and Marshall (Balakumar and Jagadeesh, 2014), who found that substitution of positively charged groups, i.e., esterification or amidation in this carboxylic group, significantly reduces its biological activity. Second, the imidazole nitrogen in Takeda0 s benzylimidazole region was aligned with the side chain of His6 of AngII, and the lipophilic n-butyl chain was pointed toward the aliphatic side chain of Ile5 (Wexler et al., 1992). Finally, the benzyl group was pointed toward the N-terminus of AngII (Wexler et al., 1992). Structure of the molecule reveals that extension toward the N-terminus of AngII via elaboration at the para position of the benzyl ring may have a promising effect in enhancing the affinity of the compounds toward the receptor (Fig. 20.2). In addition, examining the functionality on the N-terminal residues of AngII, two acidic groups b-COOH of Asp1 and the acidic-phenolic eOH of Tyr4 are present. Therefore, the substitution of the acidic group will provide a negative charge at physiologic pH and mimic the acidic regions of b-COOH of Asp1 and eOH of Tyr4. A second acidic group at the para position of the benzyl ring was examined. This led to the discovery of first analogs dicarboxylic acid EXP6155 (Duncia et al., 1990). It was found that EXP6155 has a 10-fold increase in binding affinity (IC50 ¼ 1.2 mM) and potency (pA2) over Takeda0 s molecule, confirming that the hypothesis was promising. EXP6155 was also selective to AT1R and did not have an agonistic property like peptide AngII analogs. This molecule encouraged the enlarging of the molecule at the para position of the benzyl ring while retaining the terminal carboxylic acid group (Chiu et al., 1989b; Wong et al., 1989; Regoli and Park, 1972). Another milestone was the preparation of EXP6803, an amide-linked compound, which produced another 10-fold enhancement in affinity (IC50 ¼ 0.12 mM) (Wong et al., 1989; Regoli and Park, 1972). A substantial increase in affinity was the result of the introduction of the second phenyl ring (phthalamic acid), which locked the carboxyl group in a cisoid relationship with the amide bond. But the compound was not orally active, presumably due to high polarity, rapid metabolism to the parent diacid, and biliary secretion. A series of EXP6803 analogs in which the amide (eNHCOe) linkage was replaced by a variety of groups, zero to three atoms in length. The flexibility of the new analogs was well tolerated but binding affinity was optimal when the linker was either zero or one atom long. Moreover, all the compounds

Angiotensin II receptors: structure-function and drug discovery Chapter | 20

Cl N

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EXP7711; IC50=0.23 µM Orally active

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OH

O

EXP6155; IC50=1.2 µM Orally inactive O

1

O

O

O

HO

NH2

N

H3 C

R

O

421

HN

N N

O H 3C

N

CH3

Angiotensin II

N HO

FIGURE 20.2

N

Losartan (DuP 753); IC50=0.019 µM

Schematic representation of the development of the first nonpeptide AT1R-blockers.

were orally inactive. Synthesizing of the biphenyl derivative EXP7711 was a breakthrough in the discovery of orally active compounds (Wexler et al., 1992). However, it was slightly less potent than EXP6803 in inhibiting 3H-AngII binding and in antagonizing AngII induced contractions of the rabbit aorta (Timmermans et al., 1990). A series of acidic groups were evaluated as bioisosteric replacements of the carboxylic acid to improve the oral activity of this biphenyl compound (EXP 7711),. The key to success in the screening was the replacement of the carboxylic group to tetrazole ring. The newly synthesized biphenyl tetrazole compound was orally active and demonstrated a w300-fold increase in its binding affinity. This led to the discovery of the first AngII receptor blocker, losartan (DuP 753), with all the desired pharmacological properties. Thereafter, a series of biphenyl tetrazole derivatives were able to be synthesized, and a few are currently in clinical use. A nonbiphenyl ARB among the clinically used ARBs, eprosartan, was developed using a different hypothesis from losartan. Retaining the structure of the first synthesized nonpeptide AngII receptor blocker, S-8308, a thiophene substitution was made along with the carboxylic group, and nitrous acid at the ortho position of phenyl ring was substituted with a methyl group at the para position (Fig. 20.3). Substitution of thienyl rings mimics the phenyl side chain of Phe8 of AngII. The potency of the compound was also excellent (IC50 ¼ 0.0015 mM) (Carini et al., 1991; Wong and Timmermans, 1996; Timmermans et al., 1991).

FIGURE 20.3 Development of nonbiphenyl tetrazole derivative Angiotensin receptor blocker.

422 PART | III GPCRs in disease and targeted drug discovery

TABLE 20.1 Peptide/nonpeptide ligand that can bind to both AT1R and AT2R. Details of selective AT1R and AT2R ligands are available in IUPHAR (http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId [34; http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId[35). Peptide/nonpeptide ligand

AT1R

AT2R

Reference

AngII

7.92  109a

5.22  1010a

Wisler et al. (2007)

AngIII

2.11  108a

6.48  1010a

Wisler et al. (2007)

AngIV

5a

1.00  10

5a

8a

Wisler et al. (2007)

7a

4.86  10

Ang-(1e7)

1.00  10

2.46  10

Wisler et al. (2007)

Compound 1

180  109b

0.34  109b

Zhang et al. (2017)

9b

Zhang et al. (2017)

Compound 2

9b

3.07  10

0.35  10

a

IC50 (M). Ki (M).

b

During the initial discovery of the AngII receptor antagonist at Du Pont, they synthesized compounds such as EXP6155, EXP6803, EXP7711, and losartan (Chiu et al., 1989a,b). These nonpeptide compounds were not able to completely inhibit AngII binding. Therefore, they decided to test the compound, which was discovered by ParkeeDavis Pharmaceutical Co (Blankley et al., 1989). This compound can inhibit AngII binding and is structurally different from losartan; this compound was designated as PD123177. The compound was tested in Du Pont, and they found that it was inactive and able to inhibit only 20% of AngII binding. Their observation was contradictory to the result discovered by ParkeeDavis. Therefore, they examined the protocol used by the ParkeeDavis and found that dithiothreitol (DTT) was used in the binding buffer. In the presence of DTT, the inhibitory effect of losartan on AngII binding was completely abolished, but the inhibitory effect of PD123177 was enhanced. This result caused them to conclude that the binding site of losartan was sensitive to DTT, and the binding site of PD123177 was not. This led to the discovery of two types of AngII receptors (Chiu et al., 1989a). The standard nomenclature of AngII receptor subtypes AT1R and AT2R has been proposed and updated. Losartan was highly selective against AT1R with 10,000-fold higher binding affinity and PD123177 is highly selective against AT2R with 3000e4000-fold higher binding affinity. Finally, several studies reported that losartan could reduce blood pressure, and PD123177 could not. Until now, there is no suitable assay to study AT2R signaling. PD123177 could inhibit AngII binding, but it is not clearly understood whether it activates or inhibits the receptor (Karnik et al., 2015). AngII analogs and nonpeptide compounds that can bind to AT1R and AT2R are shown in Table 20.1.

20.4.1 b-Arrestin biased AT1R ligands Classical GPCR signaling involves G protein signaling and b-arrestin recruitment to terminate signaling. Agonists or antagonists, in general, will enhance or inhibit G protein signaling, respectively. Biased ligands are those ligands that specifically enhance or inhibit signaling pathways mediated by either G protein or b-arrestin (Karnik et al., 2015). Biased ligands for b-arrestin signaling for a few GPCRs have proven to be beneficial and are in clinical use (e.g., Carvedilol, a biased ligand for the b2 adrenergic receptor) (Bosnyak et al., 2011; Carr et al., 2016). For AT1R, biased ligand toward b-arrestin signaling is shown to be beneficial for acute heart failure in preclinical trails (Ikeda et al., 2015). The process of discovery of AT1R antagonists by modifying AngII led to the discovery of agonists, partial agonists, partial antagonists, and biased agonists. The AngII analog SII (Sar1Ile4Ile8AngII) was the first molecule to show biased signaling toward b-arrestin recruitment by increasing receptor phosphorylation (Wei et al., 2003; Takezako et al., 2017). Thereafter, Trevena synthesized an additional two analogs of AngII, i.e., TRV120023 and TRV027 (formerly known as TRV120027), which were developed based on the structure of SII-AngII (Fig. 20.4) (Takezako et al., 2017; Violin et al., 2010). TRV120023 increases cardiac performance in a transgenic mouse model of familial dilated cardiomyopathy (Tarigopula et al., 2015; Kim et al., 2012) and reduces myocyte apoptosis caused by mechanical stress and ischemia/reperfusion injury in mice (Monasky et al., 2013). TRV027 causes cardiac unloading action in a canine model of acute heart failure by preserving renal function (Boerrigter et al., 2011). TRV 027, can inhibit AngII stimulated G protein signaling and stimulate arrestin recruitment, and activate several kinase pathways, including ERK, Src, and endothelial NO synthase phosphorylation (Boerrigter et al., 2012). It also can reduce the blood pressure but unlike the clinically used ARBs, it increases cardiac performance (Violin et al., 2010; Tarigopula et al., 2015; Kim et al., 2012; Monasky et al., 2013; Boerrigter et al., 2011, 2012). In addition, this

Angiotensin II receptors: structure-function and drug discovery Chapter | 20

423

FIGURE 20.4 Process of development of TRV027, a biased ligand for AT1R which are in phase II clinical trial for acute heart failure (AHF).

compound has promising clinical applications in congestive heart failure, which was observed in a dog model (Pang et al., 2017). However, it failed in a phase II study (BLAST-AHF) due to e (1) baseline to death time in 30 days, (2) baseline to heart failure re-hospitalization through 30 days, (3) worsening heart failure from baseline through day 5, (4) the change from baseline over time from baseline through day 5 in dyspnea visual analog scale (VAS) score calculated as the area under the curve (AUC), and (5) baseline length of hospital stay (Michel and Charlton, 2018).

20.5 Summary In the 1950s, a series of experiments led to the discovery of AngII (first called hypertension II), which is highly potent in contracting the blood vessel. Radiolabeled AngII binding assays performed by injecting labeled AngII in mice by Bumpus and colleagues (Bumpus et al., 1964) led to the discovery of AngII receptors. During the discovery process of ARBs, subtypes of AngII receptors, i.e., AT1R and AT2R, were discovered. Several AngII analogs were synthesized, and none of them were clinically successful due to poor bioavailability. This led to the discovery of more potent orally active nonpeptide compounds that block AT1R. Losartan was the first AT1R blocker to be used clinically, and several other ARBs were developed thereafter. The AT2R inhibitor PD123177 was also discovered in the early process of development of ARBs. However, the function of AT2R is not clearly understood. Recent reports suggest that the AT2R inhibitors could be useful in treating neuropathic pain. A biased ligand for AT1R that can block AngII binding and prevent the harmful effect of overactivation of AT1R but retain the beneficial effect of AT1R signaling is in phase II clinical trials for acute heart failure (AHF). Recently available crystal structures of AngII receptors will further leverage the drug discovery process.

Acknowledgments This work was supported by National Institutes of Health Grants, HL132351 and R01HL142091 and LRI Chair0 s Innovative Research Award to S.S.K. We thank Russell Desnoyer and Abdo Boumitri for help with the English correction.

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