This thesis is submitted in partial fulfillment of the requirements for the degree of Master of Science in Molecular Biology and Biotechnology. Elucidation of the interaction between hemorphins and targets in the renin-angiotensin system", hereby solemnly declare that this thesis is my own original research work done and prepared by me under the supervision of dr. hemorphins, short bioactive peptides produced by the enzymatic cleavage of the hemoglobin -chain, show anti-hypertensive effects through the inhibition of the angiotensin-1-converting enzyme (ACE1), a key component of the renin-angiotensin system (RAS) which controls blood pressure regulation.
ACE1 and its homolog ACE2, which is also involved in the RAS, share significant similarities in their catalytic domains. The study revealed interactions with equivalent conserved regions of the two ACE homologues and a similar interaction pattern with respect to ACE2 inhibitors.
Introduction
- Overview
- Statement of the Problem
- Research Objectives
- Relevant Literature
- Mammalian and Camel Hemorphin Variants
- Protein Targets of Hemorphins
- ACE1 and ACE2 in the Blood Regulation System
- ACE1 and ACE2 Structure
- Potential Contributions and Limitations of the Study
Several studies have revealed the various therapeutic effects of hemorphins in both human and animal models (Ali et al., 2020b). Recently, this aspect has been studied in a number of targets using in silico and in vitro techniques (Ali et al., 2019). Currently, identified targets of hemorphins include μ opioid receptors, angiotensin-converting enzyme-1 (ACE1), and insulin-regulated aminopeptidase (IRAP) (Ali et al., 2019).
Among these receptors is the Angiotensin II Type 1 receptor (AT1R) which is reported to be positively modulated by LVVHem7 (Ali et al., 2019b). This highlights the flexibility of ACE active site in terms of the wide range of potential inhibitors (Akif et al., 2011).
Methods
- In Vitro ACE Inhibition Assay
- Protein Structure Pre-Processing
- Active Site Identification and Grid Generation
- Peptide Docking
- Molecular Dynamics (MD) Simulations
The postures were ranked based on the GlideScore (GScore) scoring function (Friesner et al., 2006). Finally, the three lowest GScore values of the best docked poses were selected for further analysis. MD simulations of the protein-peptide complexes were performed using Desmond using the OPLS 2005 force field (Schrödinger Release, 2021d).
Sixteen simulation systems—ACE1 and ACE2 with each of the eight best peptides against ACE1—were prepared. RMSF) and root mean square deviation (RMSD) of the complexes were calculated from the trajectories.
Results and Discussions
In Vitro Inhibition Assay
- ACE Inhibitory Activity of Non-Camel and Camel
Molecular Docking
- Docking of the top-eight peptides to ACE1
- Docking of the top-eight peptides against ACE1
All peptides anchored in at least one of the subsites of the active site of ACE1 - S1, S2 and S1'. Camel LVVHem6 and LVVHem7 occupied all three subsites, LVVHem6, camel LVVHem5, camel LVVHem7 and LVVHem5 anchored in two subsites; while VVHem6 and camel Hem7 docked in just one of the three subsites. All peptides formed a hydrophobic contact with Ala354 at S1 and Tyr523 at S2, with the exception of Camel Hem7—the lowest of the top eight, which did not exhibit either a polar or hydrophobic contact with Ala354.
As the top peptide in terms of ACE1 inhibition, it exhibited hydrogen bonding contact with all three subsites of ACE1. The Arg8 residue of camel LVV-Hemorphin-6 exhibited a hydrogen bond and hydrophobic contact with Ala354 at the S1 subsite and formed an additional hydrogen bond and salt bridge with Asp415 ( Figure 4A ). The next residue, Arg9, made hydrogen bond contact with both S2 via Gln 81 and occupied the S1' subsite through an interaction with Glu16.
Furthermore, Arg9 is involved in a hydrogen bond and a salt bridge with Asp377 and Lys511 in S2 ( Figure 4A ). A hydrogen bond contact was mediated by Trp6 with Ala356 in S1, which also formed two - contacts with His410. The following Arg9 residue entered Glu16 in S1' through a hydrogen bond; It also made two hydrogen bonds and a salt bridge with Asp377.
This peptide failed to secure a polar interaction at the active site of ACE1, but made contact with Ala356 near the active site (Figure 4E). Arg9 formed a hydrogen bond and a salt bond with Glu16 at S1' and a hydrogen with His353 at S. The next Arg8 was involved in a single hydrogen bond interaction with Pro346 (ACE1 Ala354), two hydrogen bonds and one salt bond with Glu406.
The next Thr7 forms a hydrogen bond with Pro346 and Glu375, corresponding to Ala354 and Glu384, respectively, at the S1 subsite of ACE1. Thr7 contacts Tyr515 (ACE1 Tyr523) via hydrogen bonding, while Gln8 interacts with Arg273 (ACE1 Gln281) via both a hydrogen bond and a salt bridge.
Molecular Dynamics Simulations
- Simulations of the top-eight hemorphins bound
- Simulations of the top-eight hemorphins bound
In contrast, the C-terminal Phe10 of camel Hem7 failed to provide any interaction at the binding interface. During the simulation, several intermolecular polar and hydrophobic interactions were observed to form, break, and reform at the interaction interface. As the peptide reporting the lowest IC50 and the best binding free energy of MM-GBSA against ACE1, camel LVVHem6 showed the highest number of contacts in the active site of ACE1.
As illustrated in Figure 9A, Thr7 engaged both His513 at the S2 subsite and Tyr523 via a hydrogen bond in the side chain. Arg8 interacted with Ala354 at the S1 subsite through a backbone-to-spine hydrogen bond for nearly 50% of the simulation period. The C-terminal Arg9 mediates a hydrogen bond with His353 on the S2 subsite, and a salt bridge with Glu16 on S1'; along with both a hydrogen bond and a salt bridge interaction with Lys511 at S2.
As for the non-camel-LVVHem6 variant, the second leading peptide in terms of IC50 and MM-GBSA binding energy, the seven interactions in the active site are held by Gln8 and Arg9 at the C-terminal, as shown in Figure 9B; which together cover all three ACE1 subsites. As shown in Figure 6C, camel LVVHem5 provided three polar interactions on the ACE1 binding pocket through its C-terminal residues Thr7 and Arg8, which together occupy the S1 and S2 subsites. A single hydrophobic interaction with Tyr523 in the active site is mediated by Trp6 in the active site (Figure 10D).
As the peptide showing the second highest IC50, LVVHem5 reported a single interaction in the active site, namely a side chain–side chain hydrogen bond with His353 for slightly less than 50% of the simulation period (Figure 9G). Consistent with its reported highest IC50, camel Hem7 neither showed significant nor consistent contact with any residue in the ACE1 active site nor formed hydrophobic interactions in the active site (Figures 9H and 10H). All five contacts of LVVHem6 in the active site are individually held by its terminal arginine, which engages Arg273 (ACE1 Gln281) and Glu406 (ACE1 Asp415) a in the salt bridge (Figure 11B).
As for VVHem6, its terminal arginine forms a salt bridge and hydrogen bond with Arg273, while Gln8 contained a hydrogen bond with Tyr515 in the active site (Figure 11E). Unexpectedly, the terminal phenylalanine of camel Hem7 failed to make significant contact at the active ACE2, but instead forms a const.
Discussion
The N-terminal segment (LVV) of both ACE1 and ACE2 showed no active site interactions, instead making polar contacts and hydrophobic interactions with residues outside the active site, with the exception of Val3 from camel LVVHem7 which made hydrophobic interactions with Pro346 at the ACE2 active site as shown in Figure 12D. This implies that the LVV segment contributes to stable binding at the active site, as previously established (Liu et al., 2014; Kohmura et al., 1989; Moayedi et al., 2017). Camel LVVHem6 and camel LVVHem7 also showed greater hydrophobicity with ACE1 and ACE2, respectively, in the presence of arginine at the C-terminal segment (Figures 10A and 12D).
Camel LVVHem7 docked to two ACE1 subunits making only two hydrogen-bonding contacts and two hydrophobic interactions in the active site as reported in Table 4. MD simulations showed that the C-terminal Phe10 of camel LVVHem7 maintained only one significant hydrogen-bonding contact in ACE1 active site, respectively with Gln281 for 56% of the simulation length as illustrated in Figure 9F. Furthermore, Arg8 of camel LVVHem6 made two significant polar contacts, one of which was in the active site, while the equivalent arginine residue of camel LVVHem7 made two polar contacts with residues relatively distant from the catalytic region as shown in Figure 9A and D. respectively.
Regarding the persistence of hydrophobic interactions at the active site, Tyr4, Pro5, and Trp6 of camel LVVHem6 maintained strong contacts with Tyr523 as depicted in Figure 10A; while only Trp6 of camel LVVHem7 showed contact with Tyr523 as shown in Figure 10D. The molecular docking results report that camel Hem7 showed no contact at the active site except a single hydrophobic interaction with Tyr523 at S1 (Table 4). Similarly, the MD simulations showed no polar active site interaction of camel Hem7, but reported two hydrophobic interactions with Ala354 and Tyr523 at the S1 subsite of ACE1, mediated by its Pro5 residue ( Figure 10H ).
Camel LVVHem7 forms a third polar contact with Arg273 while LVVHem7 engages His345 in the ACE2 active region. Trp6 of camel LVVHem7 contains - stacking with His374 in the active and a hydrogen bond with Glu406 close to the zinc-binding motif, while the same residue of camel Hem7 maintains two active site contacts, through - stacking with His345 and a bond hydrogenated with Glu145. However, MD shows that the only polar interactions of LVVHem7 with Arg273 are maintained via a salt bridge and hydrogen bond contact via its C-terminal Phe10 throughout the simulation period as shown in ( Figure 11F ); an additional stable salt bridge with Glu145 in the active site was mediated by its Arg9 in the C-terminal segment.
Conclusion
Research Implications
A novel mechanism of inhibition of human angiotensin-I-converting enzyme (ACE) by a highly specific phosphine tripeptide. Camel hemomorphins exhibit stronger angiotensin-I converting enzyme inhibitory activity than other mammalian hemomorphins: an in silico and in vitro study. Crystal structure of the N domain of human somatic angiotensin I-converting enzyme provides a structural basis for the design of domain-specific inhibitors.
Substrate-based design of the first class of angiotensin-converting enzyme-related carboxypeptidase (ACE2) inhibitors. Identification of critical active site residues in angiotensin converting enzyme-2 (ACE2) by site-directed mutagenesis. ACE inhibitory peptides in standard and fermented deer velvet: an in silico and in vitro investigation.
Characterization of ACE Inhibitory Peptides from Mactra veneriformis Hydrolyzate by Nano-Liquid Electrospray Ionization Mass Spectrometry (Nano-LC-ESI-MS) and Molecular Docking. Three new ACE inhibitory peptides isolated from Ginkgo biloba seeds: purification, inhibitory kinetics and mechanism. Isolation and Characterization of Angiotensin I Converting Enzyme (ACE) Inhibitory Peptides from Saurida elongata Proteins Hydrolyzate by IMAC-Ni2+.
Characterization of the receptor binding domain (RBD) of the 2019 novel coronavirus: implication for the development of RBD protein as an inhibitor and vaccine for viral adhesion. Inhibition and inhibition kinetics of angiotensin-converting enzyme activity by haemorphins isolated from a peptic bovine hemoglobin hydrolysate. Of the eighteen peptides purchased, Hem5, LVVHem4 and camel Hem6 did not dissolve during sample preparation.
