International Journal of Molecular Sciences Article Insights into the Interaction of LVV-Hemorphin-7 with Angiotensin II Type 1 Receptor Amanat Ali 1,†, Elizabeth K. M. Johnstone 2,3,4,†, Bincy Baby 1, Heng B. See 2,3,4, Angela Song 5, K. Johan Rosengren 5 , Kevin D. G. Pfleger 2,3,4,6 , Mohammed Akli Ayoub 1,7,* and Ranjit Vijayan 1,* 1 Department of Biology, College of Science, United Arab Emirates University, Al Ain PO Box 15551, UAE; [email protected] (A.A.); [email protected] (B.B.) 2 Molecular Endocrinology and Pharmacology, Harry Perkins Institute of Medical Research, QEII Medical Centre, Nedlands, WA 6009, Australia; [email protected] (E.K.M.J.); [email protected] (H.B.S.); kevin.pfl[email protected] (K.D.G.P.) 3 Centre for Medical Research, The University of Western Australia, Crawley, WA 6009, Australia 4 Australian Research Council Centre for Personalised Therapeutics Technologies, Canberra, NSW 2609, Australia 5 School of Biomedical Sciences, Faculty of Medicine, The University of Queensland, St Lucia, QLD 4072, Australia; [email protected] (A.S.); [email protected] (K.J.R.) 6 Dimerix Limited, Nedlands, WA 6009, Australia 7 Zayed Center for Health Sciences, United Arab Emirates University, Al Ain PO Box 15551, UAE * Correspondence: [email protected] (M.A.A.); [email protected] (R.V.); Tel.: +971-3-713-6721 (M.A.A.); +971-3-713-6302 (R.V.) † These authors contributed equally to the work. Abstract: Hemorphins are known for their role in the control of blood pressure. Recently, we revealed the positive modulation of the angiotensin II (AngII) type 1 receptor (AT1R) by LVV-hemorphin-7 (LVV-H7) in human embryonic kidney (HEK293) cells. Here, we examined the molecular binding behavior of LVV-H7 on AT1R and its effect on AngII binding using a nanoluciferase-based biolu- minescence resonance energy transfer (NanoBRET) assay in HEK293FT cells, as well as molecular Citation: Ali, A.; Johnstone, E.K.M.; docking and molecular dynamics (MD) studies. Saturation and real-time kinetics supported the Baby, B.; See, H.B.; Song, A.; positive effect of LVV-H7 on the binding of AngII. While the competitive antagonist olmesartan Rosengren, K.J.; Pfleger, K.D.G.; competed with AngII binding, LVV-H7 slightly, but significantly, decreased AngII’s kD by 2.6 fold Ayoub, M.A.; Vijayan, R. Insights into with no effect on its Bmax. Molecular docking and MD simulations indicated that the binding of the Interaction of LVV-Hemorphin-7 LVV-H7 in the intracellular region of AT1R allosterically potentiates AngII binding. LVV-H7 targets with Angiotensin II Type 1 Receptor. residues on intracellular loops 2 and 3 of AT1R, which are known binding sites of allosteric mod- Int. J. Mol. Sci. 2021, 22, 209. ulators in other GPCRs. Our data demonstrate the allosteric effect of LVV-H7 on AngII binding, https://doi.org/10.3390/ijms22010209 which is consistent with the positive modulation of AT1R activity and signaling previously reported. This further supports the pharmacological targeting of AT1R by hemorphins, with implications in Received: 29 October 2020 vascular and renal physiology. Accepted: 24 December 2020 Published: 28 December 2020 Keywords: LVV-hemorphin-7; AngII; AT1R; NanoBRET; molecular docking; molecular dynam- ics; PAM Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional claims in published maps and institutional affiliations. 1. Introduction Hemorphins are endogenous hemoglobin-derived peptides implicated in numerous physiological and pathophysiological situations including spatial learning, inflammation, Copyright: © 2020 by the authors. Li- analgesia, and transient hypotension [1–7]. The molecular targets of hemorphins com- censee MDPI, Basel, Switzerland. This prise the cell-surface G protein-coupled receptor (GPCR) members such as the opioid article is an open access article distributed receptors [4,8–11] and also the membrane AngIV receptor [3,8]. Moreover, hemorphins under the terms and conditions of the are also known for their inhibitory effects on the angiotensin-converting enzyme (ACE), Creative Commons Attribution (CC BY) a key enzyme in the renin–angiotensin system (RAS). Inhibition of ACE leads to reduced license (https://creativecommons.org/ licenses/by/4.0/). angiotensin II (AngII) levels, which are associated with the well-known anti-hypertensive Int. J. Mol. Sci. 2021, 22, 209. https://doi.org/10.3390/ijms22010209 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 209 2 of 14 properties of hemorphins [6,12,13]. Recently, we described for the first time the AngII type Int. J. Mol. Sci. 2020, 21, x FOR 1PEER receptor REVIEW (AT1R) as a second GPCR to be pharmacologically targeted by2 the of 15decapep- tide LVV-hemorphin-7 (LVV-H7) in vitro with a positive modulation of its activity and downstreamtensin II (AngII) signaling levels, pathways which are associated in HEK293 with cells the well-known [14]. These anti-hypertensive effects were observed prop- on the canonicalerties of G hemorphinsαq/inositol [6,12,13]. phosphate Recently, pathway we described of AT1R, for the as wellfirst time as on the the AngII phosphorylation type 1 of thereceptor extracellular (AT1R) as signal-regulated-kinases a second GPCR to be pharmacologically (ERK1/2) [targeted14]. The by positive the decapeptide modulation of LVV-hemorphin-7 (LVV-H7) in vitro with a positive modulation of its activity and down- AT1Rstream implies signaling a hypertensive pathways in effectHEK293 and cells this [14]. was These surprising effects were and observed not consistent on the ca- with the anti-hypertensivenonical Gαq/inositol properties phosphate of hemorphins pathway of AT1R, through as well the as inhibition on the phosphorylation of ACE. This of further in- dicatesthe theextracellular complexity signal-regulated-kinases of the interplay between (ERK1/2) hemorphins [14]. The positive and modulation the physiological of AT1R systems suchimplies as the a vascular hypertensive system. effect and this was surprising and not consistent with the anti- hypertensiveIn this study, properties we provide of hemorphins early insightsthrough the into inhibition the binding of ACE. ofThis LVV-H7 further indi- to AT1R by usingcates the the NanoBRET complexity of technology the interplay recently between developedhemorphins and for the hormone-receptor physiological systems binding in such as the vascular system. real-time and live cells [15] as well as molecular docking and long molecular dynamics In this study, we provide early insights into the binding of LVV-H7 to AT1R by using (MD)the simulations. NanoBRET technology recently developed for hormone-receptor binding in real-time and live cells [15] as well as molecular docking and long molecular dynamics (MD) simu- 2. Resultslations. 2.1. LVV-H7 Positively Affected AngII Binding on AT1R in Live HEK293 Cells 2. Results We previously showed a positive allosteric modulation of AT1R activity and signaling by LVV-H72.1. LVV-H7 in HEK293Positively Affected cells [14 AngII]. In Binding this study, on AT1R we in examined Live HEK293 the Cells effect of LVV-H7 on the bindingWe of AngIIpreviously on AT1Rshowed using a positive NanoBRET allosteric modulation technology of inAT1R real-time activity and signal- intact cells as previouslying by LVV-H7 reported in [HEK29315]. First, cells we [14]. performed In this study, steady-state we examined saturation the effect of binding LVV-H7 experiments on the binding of AngII on AT1R using NanoBRET technology in real-time and intact cells as µ withpreviously BODIPY-labelled reported [15]. AngII First, (BODIPY-AngII)we performed steady-state in the saturation absence binding or presence experiments of 10 M of LVV-H7.with BODIPY-labelled As shown in AngII Figure (BODIPY-AngII)1A, LVV-H7 in did the not absence compete or presence for binding of 10 μM of with LVV- BODIPY- AngII,H7. in As contrast shown in toFigure the ~75%1A, LVV-H7 displacement did not compete of binding for binding observed with BODIPY-AngII, when treated in with the competitivecontrast to AT1R the ~75% antagonist displacement olmesartan of binding medoxomil observed when (Figure treated1B). with As the Figure competitive1A appeared to showAT1R a antagonist small increase olmesartan in binding medoxomil affinity (Figure of BODIPY-AngII1B). As Figure 1A inappeared the presence to show of a LVV-H7, we investigatedsmall increase thisin binding further affinity by generating of BODIPY-AngII BODIPY-AngII-specific in the presence of LVV-H7, binding we inves- curves in the tigated this further by generating BODIPY-AngII-specific binding curves in the presence presence of increasing concentrations of LVV-H7 (Figure2). Here we found that LVV-H7 of increasing concentrations of LVV-H7 (Figure 2). Here we found that LVV-H7 signifi- ± ± significantlycantly potentiated potentiated the binding the binding of BODIPY-AngII of BODIPY-AngII (mean K (meanD ± SEM: K D106.6 SEM:± 37.1 106.6nM at 0 37.1 nM at 0 µμM LVV-H7 vs. vs. 41.2 41.2 ± 14.7± 14.7 nM nMat 100 at μ 100M LVV-H7;µM LVV-H7; n = 6, np <= 0.01 6, p for< 0.01 paired for ANOVA) paired ANOVA) (Figure(Figure2A,B), 2A,B), without without altering altering thethe maximum binding binding (Figure (Figure 2C). 2SuchC). Suchan effect an suggests effect suggests a positivea positive allosteric allosteric interaction interaction between between LV LVV-H7V-H7 and and BODIPY-AngII BODIPY-AngII for binding for bindingto AT1R, to AT1R, whichwhich may may explain explain its its positive positive action on on AT1R AT1R activity activity and andsignaling signaling [14]. [14]. Figure 1. FigureSaturation 1. Saturation binding binding of BODIPY-angiotensin of BODIPY-angiotensin II II (AngII) (AngII) to AngII AngII type type 1 receptor 1 receptor (AT1R) (AT1R) using usingNanoBRET NanoBRET assay in assay in live HEK293FTlive HEK293FT cells.