Natural Peptide Substrates Research Articles

Target identification for small-molecule discovery in the FOXO3a tumor-suppressor pathway using a biodiverse peptide library

SummaryGenetic screening technologies to identify and validate macromolecular interactions (MMIs) essential for complex pathways remain an important unmet need for systems biology and therapeutics development. Here, we use a library of peptides from diverse prokaryal genomes to screen MMIs promoting the nuclear relocalization of Forkhead Box O3 (FOXO3a), a tumor suppressor more frequently inactivated by post-translational modification than mutation. A hit peptide engages the 14-3-3 family of signal regulators through a phosphorylation-dependent interaction, modulates FOXO3a-mediated transcription, and suppresses cancer cell growth. In a crystal structure, the hit peptide occupies the phosphopeptide-binding groove of 14-3-3ε in a conformation distinct from its natural peptide substrates. A biophysical screen identifies drug-like small molecules that displace the hit peptide from 14-3-3ε, providing starting points for structure-guided development. Our findings exemplify “protein interference,” an approach using evolutionarily diverse, natural peptides to rapidly identify, validate, and develop chemical probes against MMIs essential for complex cellular phenotypes.

Multiscale Simulations of SARS-CoV-2 3CL Protease Inhibition with Aldehyde Derivatives. Role of Protein and Inhibitor Conformational Changes in the Reaction Mechanism.

We here investigate the mechanism of SARS-CoV-2 3CL protease inhibition by one of the most promising families of inhibitors, those containing an aldehyde group as a warhead. These compounds are covalent inhibitors that inactivate the protease, forming a stable hemithioacetal complex. Inhibitor 11a is a potent inhibitor that has been already tested in vitro and in animals. Using a combination of classical and QM/MM simulations, we determined the binding mode of the inhibitor into the active site and the preferred rotameric state of the catalytic histidine. In the noncovalent complex, the aldehyde group is accommodated into the oxyanion hole formed by the NH main-chain groups of residues 143 to 145. In this pose, P1–P3 groups of the inhibitor mimic the interactions established by the natural peptide substrate. The reaction is initiated with the formation of the catalytic dyad ion pair after a proton transfer from Cys145 to His41. From this activated state, covalent inhibition proceeds with the nucleophilic attack of the deprotonated Sγ atom of Cys145 to the aldehyde carbon atom and a water-mediated proton transfer from the Nε atom of His41 to the aldehyde oxygen atom. Our proposed reaction transition-state structure is validated by comparison with X-ray data of recently reported inhibitors, while the activation free energy obtained from our simulations agrees with the experimentally derived value, supporting the validity of our findings. Our study stresses the interplay between the conformational dynamics of the inhibitor and the protein with the inhibition mechanism and the importance of including conformational diversity for accurate predictions about the inhibition of the main protease of SARS-CoV-2. The conclusions derived from our work can also be used to rationalize the behavior of other recently proposed inhibitor compounds, including aldehydes and ketones with high inhibitory potency.

Histone H4-based peptoids are inhibitors of protein arginine methyltransferase 1 (PRMT1).

Methylation of arginine residues occurs on a number of protein substrates, most notably the N-terminal tails of histones, and is catalyzed by a family of enzymes called the protein arginine methyltransferases (PRMTs). This modification can lead to transcriptional activation or repression of cancer-related genes. To date, a number of inhibitors, based on natural peptide substrates, have been developed for the PRMT family of enzymes. However, because peptides are easily degraded in vivo, the utility of these inhibitors as potential therapeutics is limited. The use of peptoids, which are peptide mimetics where the amino acid side chain is attached to the nitrogen in the amide backbone instead of the α-carbon, may circumvent the problems associated with peptide degradation. Given the structural similarities, peptoid scaffolds may provide enhanced stability, while preserving the mechanism of action. Herein, we have identified that peptoids based on natural peptide substrates are not catalyzed to the product by PRMT1, but instead are inhibitors of this enzyme. Reducing the length of the peptoid reduces inhibition and suggest the residues distal from the site of modification are important for binding. Furthermore, a positive charge on the N-terminus helps promote binding and improves inhibition. Selectivity among family members is likely possible based on inhibition being moderately selective for PRMT1 over PRMT5 and provides a scaffold that can be used to develop pharmaceuticals against this class of enzymes.

Pan-NS3 protease inhibitors of hepatitis C virus based on an R3-elongated pyrazinone scaffold

Herein, we present the design and synthesis of 2(1H)-pyrazinone based HCV NS3 protease inhibitors and show that elongated R3 urea substituents were associated with increased inhibitory potencies over several NS3 protein variants. The inhibitors are believed to rely on β-sheet mimicking hydrogen bonds which are similar over different genotypes and current drug resistant variants and correspond to the β-sheet interactions of the natural peptide substrate. Inhibitor 36, for example, with a urea substituent including a cyclic imide showed balanced nanomolar inhibitory potencies against genotype 1a, both wild-type (Ki = 30 nM) and R155K (Ki = 2 nM), and genotype 3a (Ki = 5 nM).

Analysis of Angiotensin Metabolism in the Kidney Using Mass Spectrometry.

The renin angiotensin system (RAS) is a highly complex enzymatic system consisting of multiple peptide hormones, enzymes, and receptors. A thorough characterization of angiotensin peptide metabolism is crucial for understanding pathological states associated with an imbalanced RAS. Here, we describe two matrix-assisted laser desorption/ionization (MALDI) mass spectrometric (MS) approaches for the assessment of in vitro and in situ RAS enzymatic activities in the kidney using the natural angiotensin peptide substrates. These MS techniques demonstrate high specificity and are superior over conventional spectrophotometric or colorimetric assays since multiple proteolytic cleavage sites can be detected, thus unraveling the complexity of the RAS.

Multi-drug resistance profile of PR20 HIV-1 protease is attributed to distorted conformational and drug binding landscape: molecular dynamics insights

The PR20 HIV-1 protease, a variant with 20 mutations, exhibits high levels of multi-drug resistance; however, to date, there has been no report detailing the impact of these 20 mutations on the conformational and drug binding landscape at a molecular level. In this report, we demonstrate the first account of a comprehensive study designed to elaborate on the impact of these mutations on the dynamic features as well as drug binding and resistance profile, using extensive molecular dynamics analyses. Comparative MD simulations for the wild-type and PR20 HIV proteases, starting from bound and unbound conformations in each case, were performed. Results showed that the apo conformation of the PR20 variant of the HIV protease displayed a tendency to remain in the open conformation for a longer period of time when compared to the wild type. This led to a phenomena in which the inhibitor seated at the active site of PR20 tends to diffuse away from the binding site leading to a significant change in inhibitor–protein association. Calculating the per-residue fluctuation (RMSF) and radius of gyration, further validated these findings. MM/GBSA showed that the occurrence of 20 mutations led to a drop in the calculated binding free energies (ΔGbind) by ~25.17 kcal/mol and ~5 kcal/mol for p2-NC, a natural peptide substrate, and darunavir, respectively, when compared to wild type. Furthermore, the residue interaction network showed a diminished inter-residue hydrogen bond network and changes in inter-residue connections as a result of these mutations. The increased conformational flexibility in PR20 as a result of loss of intra- and inter-molecular hydrogen bond interactions and other prominent binding forces led to a loss of protease grip on ligand. It is interesting to note that the difference in conformational flexibility between PR20 and WT conformations was much higher in the case of substrate-bound conformation as compared to DRV. Thus, developing analogues of DRV by retaining its key pharmacophore features will be the way forward in the search for novel protease inhibitors against multi-drug resistant strains.

Abstract 169: Angiotensin-Converting Enzyme 2: Species-Specific Features and Pharmacologic Characterization by RAS Fingerprinting

The carboxypeptidase Angiotensin Converting Enzyme 2 (ACE2) is a crucial player of the Renin-Angiotensin-System (RAS) as it balances the ratio between vasoconstrictive and fibrotic Angiotensin II and vasodilative and anti-fibrotic Angiotensin 1-7. This balance is affected by ACE2 either by direct conversion of Angiotensin II to Angiotensin 1-7, or by cleavage of Angiotensin I to Angiotensin 1-9, which is further converted to Ang 1-7 by Angiotensin Converting Enzyme (ACE) or Neutral Endopeptidase (NEP). An increased activity of ACE2 leads to a shift of the RAS towards an Angiotensin 1-7 dominated pattern, which was shown to be protective in various models of cardiovascular and fibrotic diseases. We performed a comparative pharmacologic characterization of recombinant murine and human ACE2 using natural peptide substrates for the evaluation of the substrate specific ACE2 activity in whole blood. Furthermore, we investigated the impact of ACE2 activators and inhibitors on murine and human ACE2 in whole blood using an LC-MS/MS based approach allowing the simultaneous quantification of 10 different angiotensin peptides (RAS-Fingerprinting). The investigation of inhibitors and activators of ACE2 using our experimental setting revealed the presence of species-specific enzymatic features, which was indicated by differences between the human and murine enzyme regarding the susceptibility to pharmacologic manipulation and the specificity for different endogenous peptide substrates. Our studies demonstrate that sequence diversity observed between recombinant human and murine ACE2 significantly affects substrate specificity and pharmacologic enzyme properties. The utilization of Angiotensin I by ACE2 generates Angiotensin 1-7 via Angiotensin 1-9 and represents a pathway of alternative RAS activation, which might have been systematically underestimated in previous studies. The strongly reduced affinity of murine ACE2 for Angiotensin I results in a lack of production of Angiotensin 1-7 via this pathway, which might be of particular importance during anti-hypertensive treatments with ACE inhibitors, where the formation of Angiotensin II is prevented while increased levels of Angiotensin I are observed in circulation.

Recombinant Expression and Characterization of Human and Murine ACE2: Species-Specific Activation of the Alternative Renin-Angiotensin-System

Angiotensin-converting enzyme 2 (ACE2) is a monocarboxypeptidase of the renin-angiotensin-system (RAS) which is known to cleave several substrates among vasoactive peptides. Its preferred substrate is Angiotensin II, which is tightly involved in the regulation of important physiological functions including fluid homeostasis and blood pressure. Ang 1–7, the main enzymatic product of ACE2, became increasingly important in the literature in recent years, as it was reported to counteract hypertensive and fibrotic actions of Angiotensin II via the MAS receptor. The functional connection of ACE2, Ang 1–7, and the MAS receptor is also referred to as the alternative axis of the RAS. In the present paper, we describe the recombinant expression and purification of human and murine ACE2 (rhACE2 and rmACE2). Furthermore, we determined the conversion rates of rhACE2 and rmACE2 for different natural peptide substrates in plasma samples and discovered species-specific differences in substrate specificities, probably leading to functional differences in the alternative axis of the RAS. In particular, conversion rates of Ang 1–10 to Ang 1–9 were found to be substantially different when applying rhACE2 or rmACE2 in vitro. In contrast to rhACE2, rm ACE2 is substantially less potent in transformation of Ang 1–10 to Ang 1–9.

Angiotensin-(1-7)/Angiotensin-Converting Enzyme 2/Mas Receptor Axis and Related Mechanisms

Angiotensin-(1-7)/Angiotensin-Converting Enzyme 2/Mas Receptor Axis and Related Mechanisms

Achiral oligoamines as versatile tool for the development of aspartic protease inhibitors

Due to the important role that aspartic proteases play in many patho-physiological processes, they have intensively been targeted by modern drug development. However, up to now, only for two family members, renin and HIV protease, approved drugs are available. Inhibitor development, mostly guided by mimicking the natural peptide substrates, resulted in very potent inhibitors for several targets, but the pharmacokinetic properties of these compounds were often not optimal. Herein we report a novel approach for lead structure discovery of non-peptidic aspartic protease inhibitors using easily accessible achiral linear oligoamines as starting point. An initial library comprising 11 inhibitors was developed and screened against six selected aspartic proteases. Several hits could be identified, among them selective as well as rather promiscuous inhibitors. The design concept was confirmed by determination of the crystal structure of two derivatives in complex with the HIV-1 protease, and represents a promising basis for the further inhibitor development.

Insights from Atomic-Resolution X-Ray Structures of Chemically Synthesized HIV-1 Protease in Complex with Inhibitors

The human immunodeficiency virus 1 (HIV-1) protease (PR) is an aspartyl protease essential for HIV-1 viral infectivity. HIV-1 PR has one catalytic site formed by the homodimeric enzyme. We chemically synthesized fully active HIV-1 PR using modern ligation methods. When complexed with the classic substrate-derived inhibitors JG-365 and MVT-101, the synthetic HIV-1 PR formed crystals that diffracted to 1.04- and 1.2-Å resolution, respectively. These atomic-resolution structures revealed additional structural details of the HIV-1 PR's interactions with its active site ligands. Heptapeptide inhibitor JG-365, which has a hydroxyethylamine moiety in place of the scissile bond, binds in two equivalent antiparallel orientations within the catalytic groove, whereas the reduced isostere hexapeptide MVT-101 binds in a single orientation. When JG-365 was converted into the natural peptide substrate for molecular dynamic simulations, we found putative catalytically competent reactant states for both lytic water and direct nucleophilic attack mechanisms. Moreover, free energy perturbation calculations indicated that the insertion of catalytic water into the catalytic site is an energetically favorable process.

Assay for rapid analysis of the tri‐peptidase activity of LTA4 hydrolase

Leukotriene A4 hydrolase is a bifunctional zinc metalloenzyme with an epoxide hydrolase activity as well as an arginyl tri-peptidase activity. Detailed enzymological and mechanistic investigations of the latter activity have been hampered by the lack of a rapid and convenient enzyme assay. Here we have developed a new method allowing direct spectrophotometric assessment of the tri-peptide cleaving activity of leukotriene A4 hydrolase, as well as other peptidases. The method utilizes two competing substrates, one chromogenic reference substrate together with the tri-peptide substrate of interest, and relies on computer-assisted analysis of progress curves. The chromogenic reference substrate serves to disclose the "invisible" tri-peptide substrate for kinetic analysis. The method is fast and simple and will allow detailed kinetic studies and screening for natural peptide substrates of leukotriene A4 hydrolase as well as other members of the M1 family of aminopeptidases.

Thrombin-activable Fibrinolysis Inhibitor (TAFI) Zymogen Is an Active Carboxypeptidase

Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase found in human plasma, presumably as an inactive zymogen. The current dogma is that proteolytic activation by thrombin/thrombomodulin generates the active enzyme (TAFIa), which down-regulates fibrinolysis by removing C-terminal lysine residues from partially degraded fibrin. In this study, we have shown that the zymogen exhibits continuous and stable carboxypeptidase activity against large peptide substrates, and we suggest that the activity down-regulates fibrinolysis in vivo.

Synthesis, Crystal Structure, Structure−Activity Relationships, and Antiviral Activity of a Potent SARS Coronavirus 3CL Protease Inhibitor

A potent SARS coronavirus (CoV) 3CL protease inhibitor (TG-0205221, Ki = 53 nM) has been developed. TG-0205221 showed remarkable activity against SARS CoV and human coronavirus (HCoV) 229E replications by reducing the viral titer by 4.7 log (at 5 microM) for SARS CoV and 5.2 log (at 1.25 microM) for HCoV 229E. The crystal structure of TG-0205221 (resolution = 1.93 A) has revealed a unique binding mode comprising a covalent bond, hydrogen bonds, and numerous hydrophobic interactions. Structural comparisons between TG-0205221 and a natural peptide substrate were also discussed. This information may be applied toward the design of other 3CL protease inhibitors.

New Mass Spectrometric Assay for Angiotensin-Converting Enzyme 2 Activity

A novel assay was developed for evaluation of mouse angiotensin-converting enzyme (ACE) 2 and recombinant human ACE2 (rACE2) activity. Using surface-enhanced laser desorption/ionization time of flight mass spectrometry (MS) with ProteinChip Array technology, ACE1 and ACE2 activity could be measured using natural peptide substrates. Plasma from C57BL/6 mice, kidney from wild-type and ACE2 knockout mice, and rACE2 were used for assay validation. Plasma or tissue extracts were incubated with angiotensin I (Ang I; 1296 m/z) or angiotensin II (Ang II; 1045 m/z). Reaction mixtures were spotted onto the ProteinChips WCX2 and peptides detected using surface-enhanced laser desorption/ionization time of flight MS. MS peaks for the substrates, Ang I and Ang II, and the generated peptides, Ang (1-7) and Ang (1-9), were monitored. The ACE2 inhibitor MLN 4760 (0.01 to 100 micromol/L) significantly inhibited rACE2 activity (IC50=3 nmol/L). Ang II was preferably cleaved by rACE2 (km=5 mumol/L), whereas Ang I was not a good substrate for rACE2. There was no detectable ACE2 activity in plasma. Assay specificity was validated in a model of ACE2 gene deletion. In kidney extract from ACE2-deficient mice, there was no generation of Ang (1-7) from Ang II. However, Ang (1-7) was produced when Ang I was used as a substrate. In conclusion, we developed a specific and sensitive assay for ACE2 activity, which used the natural endogenous peptide substrate Ang II. This approach allows for the rapid screening for ACE2, which has applications in drug testing, high-throughput enzymatic assays, and identification of novel substrates/inhibitors of the renin-angiotensin system.

Hypotensive effects of hemopressin and bradykinin in rabbits, rats and mice: A comparative study

Hemopressin is a novel vasoactive nonapeptide derived from hemoglobin's α-chain as recently reported by Rioli et al. [Rioli V, Gozzo FC, Heimann AS, Linardi A, Krieger JE, Shida CS, et al. Novel natural peptide substrates for endopeptidase 24.15, neurolysin, and angiotensin-converting enzyme. J Biol Chem 2003;278(10):8547–55]. In anesthetized male Wistar rats, this peptide exhibited hypotensive actions similar to those of bradykinin (BK) when administered intravenously (i.v.), and was found to be metabolized both in vitro and in vivo by several peptidases, including the angiotensin-converting enzyme (ACE). In this study, these findings were expanded upon by examining: (i) the degradation kinetics following incubation with ACE purified from rabbit lung and (ii) the blood pressure lowering effects of HP and BK injected i.v. or intra-arterially (i.a.) in male rabbits, rats, and mice. Our findings demonstrate that, in vitro, HP and BK are both degraded by ACE, but at different velocity rates. Furthermore, both HP and BK induced transient hypotension in all animals tested, although the responses to HP relative to the administration sites were significantly lower (by 10–100-fold) on an equimolar basis compared to those of BK. In rabbits, the decrease of blood pressure induced by HP (10–100 nmol/kg) did not differ whether it was administered i.v. or i.a., suggesting an absence of pulmonary/cardiac inactivation in contrast to BK (0.1–1 nmol/kg). The in vivo effect of HP was significantly potentiated in rabbits immunostimulated with bacterial lipopolysaccharide (LPS), but was unaffected by both the B 2 receptor antagonist HOE 140 (0.1 μmol/kg) and captopril (100 μg/kg), contrary to BK. Therefore, HP acts as a weak hypotensive mediator, which does not activate kinin B 2 receptors, but uses a functional site and/or signaling paths appearing to be up-regulated by LPS.

Molecular cloning and functional characterization of crustapain: a distinct cysteine proteinase with unique substrate specificity from northern shrimp Pandalus borealis.

A cDNA clone encoding a cysteine proteinase of the papain superfamily has been isolated from the hepatopancreas of northern shrimp Pandalus borealis (NsCys). NsCys shares the highest identity of 64% with a cathepsin L-like cysteine proteinase from lobster, and its identity to the well-characterized mammalian cathepsins S, L, and K falls within a narrow range of 54-59%. However, it differs from each of these cathepsins in certain key residues including, for example, the unique occurrence of tryptophan and cysteine residues at the structurally important S2 subsite. Consequently, NsCys produced in Pichia pastoris appears to be distinct in various physicokinetic properties. The recombinant enzyme is active and stable over a wide range of pH values, and its substrate specificity is unusual, as demonstrated by its poor affinity for phenylalanine residues. Instead, it shows the highest specificity for proline residues, a property similar to cathepsin K. Unlike cathepsin K, however, NsCys cleaves valine residues more efficiently than leucine. Similar results were obtained with the natural peptide substrate glucagon. The shrimp proteinase is further distinguished by its potent collagenolytic activity, resulting in a cleavage pattern reminiscent of bacterial collagenase. To distinguish such unique structural and enzymatic properties, we propose the trivial name "crustapain" for the shrimp proteinase, indicating that it is a papain-like cysteine proteinase from a crustacean species.

Enteropeptidase: Structure, Function, and Application in Biotechnology

A preparative method for purification of enteropeptidase (enterokinase) (EC 3. 4. 21. 9) is developed. A highly purified form of this enzyme is stabilized by calcium ions and does not contain any other proteolytic enzyme contaminations. These enteropeptidase preparations were successfully used for cleavage of a variety of fusion proteins containing the tetraaspartyl-lysyl sequence in an arbitrary position on the polypeptide chain. A series of substrates was methodically studied, which resulted in the suggestion that the peptide and fusion protein substrates (K m = 200 μM and 125 μM, respectively) were bound to the enzyme through the linker (Asp) 4 Lys at the binding site on the light chain of enteropeptidase. Much more efficient hydrolysis of the natural substrate trypsinogen (K m = 2.4 μM) testifies to a significant contribution of other sites of the substrate and the enzyme in productive binding. A variation in the enzyme's unique specificity was shown to be a result of the autolysis caused by the loss of calcium ions; the cleavage sites were determined. The truncated enzyme containing the C-terminal fragment 466-800 of its heavy chain and the intact light chain does not distinguish the natural substrate trypsinogen, fusion protein, or peptide substrates. These results suggest that the N-terminal fragment 118-465 of the enteropeptidase heavy chain contains a secondary substrate-binding site that interacts directly with trypsinogen.

The structure and function of Escherichia coli penicillin-binding protein 3.

Escherichia coli penicillin-binding protein PBP3 is a key element in cell septation. It is presumed to catalyse a transpeptidation reaction during biosynthesis of the septum peptidoglycan but, in vitro, its enzymatic activity has only been demonstrated with thiolester analogues of the natural peptide substrate. It has no detectable transglycosylase activity with lipid II as substrate. This tripartite protein is constructed of an N-terminal membrane anchor-containing module that is essential for cell septation, a non-penicillin-binding (n-PB) module of unknown function and a C-terminal penicillin-binding (PB) module exhibiting all the characteristic motifs of penicilloyl serine transferases. The n-PB module, which is required for the folding and stability of the PB module, may provide recognition sites for other cell division proteins. Initiation of septum formation is not PBP3-dependent but rests on the appearance of the FtsZ ring, and is thus penicillin-insensitive. The control of PBP3 activity during the cell cycle is briefly discussed.

Mass spectrometry as a tool for studying the action of human aspartic proteases on peptides and proteins.

In the late eighties, two novel mass spectrometric methods with the sensitivity in the femtomole range were introduced, electrospray mass spectrometry (ESMS) and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF). These methods have been used for a variety of applications in protein chemistry (reviewed in [1] and [2]). In MALDI TOF the analyte is mixed with UV light-absorbing substance (matrix). The matrix and the compound to be analyzed are allowed to cocrys-tailize on the probe surface of the mass spectrometer. The sample is irradiated with UV light from a laser. The light is absorbed by the matrix causing the sample to be vaporized and ionized. The generated ions are accelerated in an electric field and are separated and detected by a time-of-flight detector. MALDI TOF was used for analysis of proteins up to 105 Da. ESMS is performed by evaporating droplets formed by the electrospraying of the protein solution followed by the transfer of the ions into the high vacuum. The evaporation generated highly charged ions are analyzed by the quadrupole mass spectrometer. We have applied the two techniques to the study of action of proteases on natural peptide and protein substrates. The present paper describes the advantages and limitations of the methods.