Unnatural peptides targeting the scaffolding activity of pi3k gamma and therapeutic uses thereof

Abstract

Non-natural peptides having the ability to inhibit the A-kinase anchoring function of PI3K gamma, comprising an amino acid sequence as set forth in SEQ ID NO: 1, wherein each amino acid is a D-amino acid, and therapeutic uses and compositions thereof.

Description

Technical Field

[0001] This manual relates to non-natural peptides that target PI3Kγ scaffold activity and their use in the treatment of respiratory pathologies. Background Technology

[0002] 3'-5'-cyclic adenosine monophosphate (cAMP) second messengers control various cellular functions, including cell growth and differentiation, gene transcription, and protein expression. cAMP exerts its function by activating different effectors, among which protein kinase A (PKA) is the most widely characterized. cAMP directly binds to the regulatory subunit dimer of the PKA holoenzyme, prompting the release of two catalytic counterparts, which then freely phosphorylate various substrates. Although different G protein-coupled receptors (GPCRs) rely on the same second messenger cAMP to transmit signals intracellularly, strict temporal control of their concentrations ensures that activation of specific GPCRs leads to appropriate cellular responses (1). This local control of cAMP signaling is achieved through multi-protein complexes that isolate enzymes responsible for cAMP production (adenylate cyclase) and degradation (phosphodiesterase, PDE), as well as various signal transduction factors, in subcellular microdomains. Key organizers of these “signal islands” are kinase A-anchored proteins (AKAPs), which, as the name suggests, anchor PKA to substrates and their regulators in specific cellular compartments (2). This perturbation of the finely regulated compartmentalization of cAMP is the basis of a variety of pathologies, including cardiovascular and pulmonary diseases, cancer, neurological disorders, and inflammation. Based on this, there have been attempts to manipulate specific cAMP signaling molecules by blocking the interaction between AKAP and other components of the PKA or cAMP signaling pathway, and these efforts have been proven effective in preclinical models (2, 3).

[0003] Our previous research identified AKAP phosphatidylinositol 3-kinase γ (PI3Kγ) as a coordinator of a multi-protein complex that plays a central role in airway smooth muscle contraction, immune cell activation, and epithelial fluid secretion (4). In the lungs, PI3Kγ-bound PKA activates PDE4, ultimately limiting the cAMP response induced by β2-adrenergic receptors (the major GPCR mediating cAMP elevation in the airways). Displacing the PI3Kγ-anchored PKA pool with a cell-permeable PI3Kγ mimic peptide (PI3Kγ MP) inhibits PDE4 and promotes local cAMP elevation, ultimately leading to airway smooth muscle relaxation and reduced neutrophil infiltration in a mouse asthma model. In bronchial epithelial cells, PI3Kγ MP enhances cAMP near the cystic fibrosis transmembrane conduction regulator (CFTR, an ion channel controlling mucus hydration), thereby driving the opening of wild-type channels and synergistically restoring the function of the most common mutant in cystic fibrosis (F508del-CFTR) (4). Summary of the Invention

[0004] One object of the present invention is to provide a potent PI3Kγ / PKA disruptor for achieving therapeutic cAMP elevation in chronic respiratory diseases.

[0005] According to the present invention, the above-mentioned objectives are achieved by the products specified in the appended claims, which should be understood as forming an integral part of this specification.

[0006] This invention relates to a non-natural peptide that targets PI3Kγ, which has nanomolar affinity for PKA, high resistance to protease degradation, and high permeability to the lung mucus barrier, and can disrupt the PI3Kγ / PKA complex and induce an increase in cAMP in the airway.

[0007] In one embodiment, the present invention relates to a non-natural peptide having the ability to inhibit PI3Kγ A kinase anchoring function, comprising an amino acid sequence as shown in SEQ ID NO: 1, wherein each amino acid is a D-amino acid, and wherein the glutamine at position 3 and the glycine at position 4 may be replaced by any amino acid having similar hydrophobicity, hydrophilicity, charge and size, respectively.

[0008] In a further embodiment, the present invention relates to the use of the non-natural peptide as a medicine, combination products of the non-natural peptide, and pharmaceutical compositions comprising the non-natural peptide. Attached Figure Description

[0009] The invention will now be described by way of example only with reference to the accompanying drawings, in which: - Figure 1 The structure of DRI-Pep#20.

[0010] a) Chemical structure of DRI-Pep #20. DRI-Pep #20 contains the reversed sequence of the non-natural peptide RHQGK (SEQ ID NO: 1), the glycine (G) linker, and the cell-penetrating peptide penetratin 1 (P1), where each amino acid is a D-amino acid. b) Structural prediction of DRI-Pep #20 via PEP-FOLD3.5. The P1-G domain is shown as a black cartoon. Key residues R-1, H-2, Q-3, and K-5 of the RHQGK domain are labeled and shown as bars. c) Circular dichroism spectrum of DRI-Pep #20, showing peaks in the 190–240 nm range. The percentages of α-helices and β-sheets in the secondary structure calculated using K2D3 software are indicated. d) Molecular docking simulation of the interaction between DRI-Pep #20 and PKA via HADDOCK 2.4. The docking conformation of the complex of DRI-Pep#20 and PKA-RIIα residues 1-45 is shown. Key residues involved in the binding are labeled (DRI-Pep#20 residues are shown in bold) and displayed as bars. The hydrogen bonds between DRI-Pep#20 and PKA-RIIα are indicated by dashed lines. This structural model was constructed using PyMOL.

[0011] - Figure 2 The binding of DRI-Pep#20 / PKA-RIIα(1-45) was analyzed using the optimal hydrogen bond network.

[0012] a) The number of hydrogen bonds formed between the R-1, H-2, Q-3, G-4, and K-5 residues of DRI-Pep#20 and residues 1-45 of PKA-RIIα. b) The number of hydrogen bonds between residues 1-45 of PKA-RIIα and DRI-Pep #20. The interactions between PKA-RIIα and P1-G or RHQGK are shown in gray and black, respectively.

[0013] - Figure 3 DRI-Pep#20 binds PKA with high affinity.

[0014] a) Schematic diagram of fluorescence spectroscopy measurements used to characterize the interaction between DRI-Pep#20 and recombinant fluorescein 5-maleimide-labeled PKA-RIIα (PKA-F5M). b) Steady-state emission spectra of PKA-F5M in the presence of increasing concentrations of DRI-Pep#20 (0 to 20 µM). D : Dissociation constant. AU, arbitrary units. Inset: Nonlinear fitting used to monitor the maximum fluorescence intensity of the biomarker PKA at different DRI-Pep#20 concentrations. K A: Binding constant. c) For kinetic analysis, the fluorescence spectra of PKA-F5M in the presence of increasing concentrations of DRI-Pep#20 (0 to 5 µM) were analyzed and fitted to a single exponential function to obtain the observed rate constant (k obs The binding of DRI-Pep#20 to the biolabeled PKA was investigated under pseudo-first-order reaction conditions, and the kinetic constant k was determined. on and k off d) Schematic diagram of the substitution assay between DRI-Pep#20 and the PI3Kγ / PKA-F5M complex. e) Percentage of the PI3Kγ / PKA-RIIα complex replaced by DRI-Pep#20, calculated from the steady-state emission spectra of the PI3Kγ / PKA-F5M complex in the presence of increasing concentrations of DRI-Pep#20 (0 to 5 µM). The substitution efficiency is expressed as the percentage of PI3Kγ bound to PKA-F5M in the absence of DRI-Pep#20. f) From wild type (WT, black) and PI3Kγ - / - cAMP concentration in mouse (grey) peritoneal macrophages after treatment with 10–25 µM DRI-Pep#20 for 30 min. cAMP levels are expressed as untreated PI3Kγ. - / - Percentage of cAMP accumulation observed in cells. N≥6 technical replicates from N>3 independent experiments. P<0.001, WT and PI3Kγ - / - One-way ANOVA was used, followed by Bonferroni post-hoc test. Data are presented as mean ± SEM.

[0015] - Figure 4 Potency and tolerability of DRI-Pep#20 in 16HBE14o- cells.

[0016] a) In response to escalating concentrations of DRI-Pep#20 (range 31.6 nM to 316 µM) for 30 min, cAMP levels increased in 16HBE14o- cells. cAMP levels are expressed as the percentage of cAMP accumulation induced by 100 µM DRI-Pep#20. N ≥ 9 technical replicates from N = 3 independent experiments. b) Cell viability of 16HBE14o- cells treated with escalating concentrations of DRI-Pep#20 (0 to 1000 µM) for 24 h. N = 3 independent experiments. In (a) and (b), the half-maximal effective concentration (EC50) was... 50 ) and median lethal dose (LD50) 50 LD was obtained through nonlinear regression analysis. 50Data are calculated relative to untreated control cells, with viability set at 100%. All data are mean ± SEM.

[0017] - Figure 5 DRI-Pep#20 locally increases the level of cAMP in the airway of mice.

[0018] a) Schematic diagram of the treatment protocol. BALB / c mice received DRI-Pep#20 via intratracheal infusion (it). (bd) cAMP concentrations in the trachea (b), lungs (c), and heart (d) of BALB / c mice 24 hours after intratracheal infusion of different doses of DRI-Pep#20 (0 to 750 mg / kg). Values ​​in parentheses indicate the DRI-Pep#20 dose in mg / kg. The number of mice (n) was 3 to 6 per group. EC 50 The half-maximal effective concentration. In a) and b), P<0.05, P<0.01, P < 0.001, a one-way ANOVO test was used, followed by a Bonferroni post-hoc test. All data are presented as mean ± SEM.

[0019] - Figure 6 DRI-Pep#20 can penetrate pathological mucus and resist protease degradation.

[0020] a) Schematic diagram of parallel artificial membrane permeability assays (PAMPA) of cystic fibrosis (CF) sputum deposited and undeposited on artificial lipid membranes (MMs). b) Apparent permeability (P) of DRI-Pep#20 (2 mg / mL) in the absence and presence of CF sputum. app Measurement. The solid line represents the threshold P of high- to medium-permeability compounds. app (4×10) -6 cm s -1 The dashed line defines the limit for medium- to low-permeability molecules (1 × 10⁻⁶). -6 cm s -1). ns: Not significant by Student's t-test. c) Representative transmission electron microscopy (TEM) images of DRI-Pep#20 (0.1 mg / mL in water). d) Particle size distribution of DRI-Pep#20 obtained by dynamic light scattering (DLS) analysis. ef) cAMP concentration in 16HBE14o- cells treated with DRI-Pep#20 (25 µM, 30 min) in the absence (black) and presence (gray) of 3 µg / ml (e) or 20 µg / ml (f) of human neutrophil elastase (HNE). N ≥ 6 technical replicates from N > 3 independent experiments. P<0.05, P < 0.01, one-way ANOVA was used, followed by Bonferroni post-hoc test, ns: not significant. g) Cover with a layer of CF sputum, then treat with 25 µM DRI-Pep#20 for 30 min and 1 h to increase cAMP in 16HBE14o-cells. cAMP levels are expressed as the percentage of cAMP accumulation induced by DRI-Pep#20 in the sputum-free condition at 30 min. P<0.01, P < 0.001, compared with the sputum-free DRI-Pep #20, was determined using a two-way ANOVA followed by a Bonferroni post-hoc analysis. N ≥ 6 technical replicates were performed from N > 3 independent experiments. All data are presented as mean ± SEM.

[0021] - Figure 7 Predict the protease cleavage site within DRI-Pep #20.

[0022] a) Predict protease cleavage sites within the DRI-Pep#20 sequence using Expasy PeptideCutter software. b) Predict protease cleavage sites within the DRI-Pep#20 sequence using Expasy PeptideCutter software and the Protease Specificity Prediction Server (PROSPER). Cleavage sites within the P1-G and RHQGK regions of DRI-Pep#20 are indicated by underline and bold, respectively.

[0023] - Figure 8 DRI-Pep#20 restores wild-type and F508del-CFTR activity.

[0024] a) Schematic diagram of CFTR activity measurement using the Premo™ halide sensor assay. b) Mean fluorescence quenching curve of 16HBE14o- cells expressing halide-sensitive yellow fluorescent protein (HS-YFP) and treated with 25 μM DRI-Pep#20 or an equimolar amount of control peptide P1 for 30 min before the addition of Premo halide stimulation buffer. Fluorescence was continuously read (1 point per second) from 1 second before buffer addition to 120 seconds. CFTR inhibitor CFTR was used. inh -172 (10 μM, 5 min) was used to assess the selective activation of CFTR channels. c) CFTR activity (expressed as fluorescence change ΔF / F0) in HS-YFP-expressing 16HBE14o- cells was assessed in response to 30 min stimulation with increasing concentrations of DRI-Pep#20 (31.6 nM to 316 mM). The half-maximal effective concentration (EC50) was used to determine the activity. 50 d) The presence or absence of the CFTR inhibitor CFTR. inh CFTR activity (expressed as fluorescence change ΔF / F0) in 16HBE14o- cells expressing HS-YFP and treated with 10–25 μM DRI-Pep#20 for 30 min at -172 (10 μM, 5 min). Adenylate cyclase activator follicularin (FSK) was used as a positive control (100 nM, 5 min), while P1 was used as a negative control (25 μM, 30 min). UT: Untreated cells. e) CFTR activity in F508del-CFTR-CFBE41o- cells expressing HS-YFP and treated with elecapram / tazacaprate / ivacato (ETI) alone or with DRI-Pep#20. Cells were corrected for 24 h with elecapram (3 μM) and tazacaprate (10 μM) and then acutely exposed to ivacato (1 μM) for 30 min, alone (ETI) or with 25 μM DRI-Pep#20. As in (b), use the CFTR inhibitor CFTR inh -172. UT: Untreated cells. In (d) and (e), n ≥ 3 technique replicates from N > 3 independent experiments. P<0.01, P<0.001 compared to UT and ###P<0.001 compared to ETI plus DRI-Pep#20, were compared using one-way ANOVA followed by Bonferroni post-hoc test. All data are presented as mean ± SEM.

[0025] - Figure 9 RHQGK peptide binds to PKA.

[0026] a) Schematic diagram of fluorescence spectroscopy measurements used to characterize the interaction between RHQGK peptide and recombinant fluorescein 5-maleimide-labeled PKA-RIIα (PKA-F5M). b) Steady-state emission spectra of PKA-F5M in the presence of increasing concentrations of RHQGK peptide (0 to 1000 μM). KD: dissociation constant. AU, arbitrary units. Inset: Nonlinear fitting of the maximum fluorescence intensity obtained at different RHQGK peptide concentrations for monitoring biolabeled PKA. KA: binding constant. Detailed Implementation

[0027] The present invention will now be described in detail by way of non-limiting embodiments.

[0028] The following description provides numerous specific details to offer a thorough understanding of the implementation scheme. The implementation scheme may be carried out without one or more of these specific details, or using other methods, components, materials, etc. In other instances, well-known structures, materials, or operations have not been shown or described in detail so as not to obscure aspects of the implementation scheme.

[0029] Throughout this specification, references to "an embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. Therefore, the phrases "in one embodiment" or "in an embodiment" appearing throughout this specification do not necessarily refer to the same embodiment. Furthermore, a particular feature, structure, or characteristic may be combined in any suitable manner in one or more embodiments.

[0030] The titles provided in this document are for convenience only and do not explain the scope or meaning of the implementation plan.

[0031] In one embodiment, the present invention relates to a non-natural peptide having the ability to inhibit the A-kinase anchoring function of PI3Kγ, comprising an amino acid sequence as shown in SEQ ID NO: 1 (RHQGK), wherein each amino acid is a D-amino acid, and wherein the glutamine at position 3 and the glycine at position 4 may be replaced by any amino acid having similar hydrophobicity, hydrophilicity, charge and size, respectively.

[0032] In one implementation, the glutamine at position 3 may be replaced by asparagine, histidine, and serine.

[0033] In one embodiment, the glycine at position 4 may be replaced by asparagine, serine, and cysteine.

[0034] In one embodiment, the non-natural peptide also comprises a cell-penetrating peptide.

[0035] In one embodiment, the cell-penetrating peptide is selected from Penetratin (pAntp-RQIKIWFQNRRMKWKK - SEQ ID NO: 3), HIV TAT peptide (YGRKKRRQRRR - SEQ ID NO: 4), R7 peptide (RRRRRRR - SEQ ID NO: 5), KALA peptide (WEAKLAKALAKALAKHLAKALAKALKACEA - SEQ ID NO: 6), Buforin 2 (TRSSRAGLQFPVGRVHRLLRK - SEQ ID NO: 7), MAP (KLALKLALKALKAALKLA-amide - SEQ ID NO: 8), Transportan (GWTLNSAGYLLGKINLKALAALAKKIL-amide - SEQ ID NO: 9), Transportan 10 (AGYLLGKINLKALAALAKKIL-amide - SEQ ID NO: 10), Pvec (LLIILRRRIRKQAHAHSK-amide - SEQ ID NO: 10), and Pvec (LLIILRRRIRKQAHAHSK-amide - SEQ ID NO: 10). 11) MPG peptide (GALFLGWLGAAGSTMGAPKKKRKV-amide-SEQ ID NO: 12).

[0036] In one embodiment, the cell-penetrating peptide sequence is in reverse order relative to the native sequence. Indeed, it is well known that amino acid sequences can be presented in reverse order to preserve the biological function of peptides synthesized using D-amino acids.

[0037] In one embodiment, the non-natural peptide further comprises a connector linking the amino acid sequence of SEQ ID NO: 1 to the cell-penetrating peptide sequence.

[0038] In one embodiment, the adapter is an amino acid adapter.

[0039] In one embodiment, the connector comprises one or more glycines or one or more amino acids having similar hydrophobicity, hydrophilicity, charge, and size to glycine, such as alanine, proline, and serine.

[0040] In one embodiment, the connector is selected from the following sequence: GG, GAG, GPA, GGGS (SEQ ID NO:13).

[0041] In one embodiment, the non-natural peptide comprises, from the N-terminus to the C-terminus, the amino acid sequence of SEQ ID NO: 1, a linker, and a cell-penetrating peptide.

[0042] In one embodiment, the connector and / or the cell-penetrating peptide are composed of D-amino acids.

[0043] In one embodiment, the cell-penetrating peptide is composed of D-amino acids and is in reverse order compared to the native sequence.

[0044] In one embodiment, the non-natural peptide has the amino acid sequence shown in SEQ ID NO: 2 (d(RHQGKGGKKWKMRRNQFWIKIQR) - hereinafter referred to as DRI-Pep#20). SEQ ID NO: 2 consists entirely of D-amino acids and contains the non-natural sequence SEQ ID NO: 1 (i.e., RHQGK) fused to a glycine (G) residue in a reversed form of Penetratin 1.

[0045] In one embodiment, the non-natural peptide, preferably the non-natural peptide of SEQ ID NO: 2, comprises one or more modifications that do not alter the primary sequence, including chemical derivatization, such as acetylation, deuteration, or Fmocization.

[0046] In one embodiment, the present invention relates to the non-natural peptides disclosed herein, which are used as pharmaceuticals.

[0047] In one embodiment, the present invention relates to the non-natural peptides disclosed herein for the treatment of respiratory diseases, preferably bronchial obstructive diseases.

[0048] In one embodiment, the respiratory disease is selected from allergic asthma, cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), non-CF bronchiectasis, pulmonary hypertension (PH), and idiopathic pulmonary fibrosis (IPF).

[0049] In one embodiment, the non-natural peptide is suitable for administration by inhalation.

[0050] In one embodiment, the present invention relates to a product comprising: i) at least one non-natural peptide disclosed herein, and ii) At least one cystic fibrosis transmembrane conductance modulator (CFTR) enhancer and / or at least one cystic fibrosis transmembrane conductance modulator (CFTR) corrector, as a combination formulation, for use sequentially, simultaneously or separately to treat respiratory diseases.

[0051] In one implementation, the respiratory disease is a bronchial obstructive disease.

[0052] In one implementation, the respiratory disease is cystic fibrosis.

[0053] In one embodiment, the cystic fibrosis transmembrane conductance modulator (CFTR) enhancer is selected from ivacato or VX-770 (N-(2,4-di-tert-butyl-5-hydroxyphenyl)-1,4-dihydro-4-oxoquinoline-3-carboxamide), Navocaftor or ABBV-3067 ([5-[3-amino-5-[4-(trifluoromethoxy)phenyl]sulfonylpyridin-2-yl]-1,3,4-oxadiazol-2-yl]methanol) and deuterated ivacato or D9-ivacato (N-[2-tert-butyl-4-[1,1,1,3,3,3-hexadeuterated-2-(trideuteratedmethyl)propyl-2-yl]-5-hydroxyphenyl]-4-oxo-1H-quinoline-3-carboxamide).

[0054] In one embodiment, the cystic fibrosis transmembrane conductance modulator (CFTR) corrector is selected from Lumacaftor or VX-809 (3-(6-((2,2-difluorobenzo[d][1,3]dioxacyclopenten-5-yl)cyclopropanecarbamate-3-methylpyridin-2-yl)benzoic acid), Tezacaftor or VX-661 (1-(2,2-difluoro-1,3-benzodioxacyclopenten-5-yl)cyclopropanecarbamate-3-methylpyridin-2-yl)benzoic acid). 5-yl)-N-[(1-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2-(1-hydroxy-2-methylpropyl-2-yl)indol-5-yl]cyclopropane-1-carboxamide), Elaxacaftor or VX-445 (N-(1,3-dimethylpyrazol-4-yl)sulfonyl-6-[3-(3,3,3-trifluoro-2,2-dimethylpropoxy)pyrazol-1-yl]-2-[(4S) -2,2,4-trimethylpyrrolidin-1-yl]pyridine-3-carboxamide), Galicaftor or ABBV-2222 (4-[(2R,4R)-4-[[1-(2,2-difluoro-1,3-benzodioxacyclopenten-5-yl)cyclopropanecarbonyl]amino]-7-(difluoromethoxy)-3,4-dihydro-2H-chromen-2-yl]benzoic acid) and Vanzacaftor ((3Z ,14S)-8-[3-(2-dispiro[2.0.24.13]hept-7-ylethoxy)pyrazol-1-yl]-12,12-dimethyl-2,2-dioxo-2λ6-thia-3,9,11,18,23-pentazatetracyclo[17.3.1.111,14.05,10]tetracosyl-1(22),3,5(10),6,8,19(23),20-hepten-4-ol).

[0055] In one embodiment, the present invention relates to a pharmaceutical composition comprising at least one non-natural peptide disclosed herein and a pharmaceutically acceptable mediator.

[0056] In one embodiment, the pharmaceutically acceptable medium is selected from phosphate-buffered saline, saline, hypertonic saline, and water.

[0057] In one embodiment, the pharmaceutical composition comprises at least one excipient.

[0058] In one embodiment, the pharmaceutical composition comprises at least one excipient selected from the following: NaCl, HCl, NaOH, EDTA, polysorbate 20, polysorbate 80, citric acid, sodium citrate, potassium phosphate, sodium phosphate, sodium bicarbonate, ascorbic acid, lactose, glucose, mannitol, trehalose, sucrose, magnesium stearate, glutathione, vitamin E, cyclodextrin, DPPC, DSPC, DMPC, leucine, isoleucine, methionine, histidine, glycine, poloxamer, chitosan, trimethyl chitosan, PLGA, PEG.

[0059] The unexpectedly high resistance to degradation and potency of DRI-Pep#20 make it an ideal candidate for therapeutic cAMP modulation, particularly for the treatment of chronic respiratory diseases, as the PI3Kγ-PKA signaling hub is crucial for multiple functions of airway cells, such as smooth muscle relaxation, epithelial ion transport, and neutrophil infiltration (4). We observed that DRI-Pep#20 can be efficiently delivered locally to the airways, and its bioactivity is fully preserved in the presence of human neutrophil elastase, supporting the possibility of using this peptide to ensure therapeutically relevant cAMP elevations in highly inflamed lungs. This is relevant for patients with COPD, non-CF bronchiectasis, and certain forms of asthma, as well as patients with the fatal genetic disease CF, whose airway inflammation has not been resolved despite the introduction of highly potent modulatory therapies (HEMTs) targeting the underlying genetic defects of the disease (5, 6).

[0060] Besides inflammation-associated proteases, another potential barrier to the bioavailability of inhaled therapy applied to diseased lungs is the thick mucus layer covering the respiratory epithelium (7), particularly in individuals with CF and in patients with COPD, non-CF bronchiectasis, and asthma. Cell-based assays using patient-derived sputum as a substitute for CF mucus revealed good mucus penetration of DRI-Pep#20, likely due to its molecular size being compatible with the pore size of pathological mucus. TEM and DLS analyses showed that the peptide can form aggregates up to 200 nm in size, which can freely diffuse through the 100–1000 nm pores of the bundled fibrous network that is typically formed by CF mucus biopolymers and filled with low-viscosity fluids (8).

[0061] With its ability to effectively penetrate the mucus layer, DRI-Pep#20 can be used to achieve therapeutic cAMP elevation in bronchial epithelial cells of CF, particularly in PI3Kγ-guided subcellular compartments, which we have previously shown to positively influence CFTR activity and maximize the efficacy of HEMT (4). Our data support this, showing that DRI-Pep#20 doubles the efficacy of the gold standard CFTR modulator combination (elecapram / terzacaffore / ivacato) in rescuing the activity of the most common CFTR mutant, F508del. Given that recent studies have shown that CFTR potentiators and correctors can only partially restore the function of mutant channels, up to 60% of wild-type CFTR levels (9-11), resulting in residual disease in CF patients treated with HEMT (5, 6, 12), these findings have significant clinical implications. Therefore, DRI-Pep#20 offers the potential to significantly improve the efficacy of standard treatment regimens for CF patients.

[0062] In summary, DRI-Pep#20 can be used to achieve therapeutic cAMP elevation locally in the lungs in chronic respiratory diseases with high unmet medical needs, such as fatal genetic diseases like CF.

[0063] result Structural and biological characteristics of DRI-Pep#20.

[0064] The chemical structure of DRI-Pep#20 is as follows: Figure 1 As shown in figure a (d(RHQGKGGKKWKMRRNQFWIKIQR) - SEQ ID NO: 2). The predicted three-dimensional structure of this peptide indicates the presence of an α-helix, flanked by two non-coiled regions ( Figure 1 b). Circular dichroism analysis confirmed the existence of the α-helical structure (18.51%), which showed a bimodal signal with a maximum value at 200 nm (a typical feature of the α-helical structure) and a minimum value in the 220-240 nm region (a typical feature of the irregularly coiled region) (13). Figure 1 c). Computer simulations of the binding of DRI-Pep#20 to amino acids 1-45 of PKA-RIIα (the typical binding surface of AKAP) indicate that this kinase can form hydrogen bonds with the RHQGK sequence, primarily involving arginine at position 1 and lysine at position 5, followed by histidine 2 and glutamine 3. Figure 1 d, Figure 2 (and Table 1).

[0065] Table 1. Energetics and kinetics of DRI-Pep#20 obtained by molecular docking analysis.

[0066] The high affinity of DRI-Pep#20 for PKA was further confirmed by in vitro experiments, which showed that the binding dissociation constant (Ki) of this peptide to recombinant PKA-RIIα was... D In the nanomolar range (76 nM; Figure 3 ab). Further fluorescence spectroscopy experiments confirmed that the RHQGK sequence of DRI-Pep#20 can directly bind to PKA (ab). Figure 9 ab). Furthermore, stopped fluorescence assays revealed that DRI-Pep#20 binds extremely rapidly to PKA-RIIα, K on 10 -6 s, while the dissociation process is significantly slower, K Off 10 -2 s ( Figure 3 c and Table 2).

[0067] Table 2. Binding kinetics of the interaction between DRI-Pep#20 and PKA-RIIα.

[0068] Consistent with the high affinity of DRI-Pep#20 for PKA-RIIα, this peptide can displace the binding between recombinant PI3Kγ and PKA-RIIα with an efficiency of up to 74%. Figure 3 Notably, despite its increased binding affinity for PKA-RIIα, DRI-Pep#20 retained its selectivity for PI3Kγ-binding PKA pools, as the peptide failed to increase cAMP in PI3Kγ-deficient cells. Figure 3 f).

[0069] Dose-response experiments showed that DRI-Pep#20 increased cAMP levels in 16HBE14o- cells, including EC14. 50 15 μM ( Figure 4 a) and is well tolerated, its LD50 50 Compared to EC 50 10 times higher ( Figure 4 b).

[0070] Overall, these results identify DRI-Pep#20 as a selective PI3Kγ / PKA-RIIα peptide disruptor with high affinity for PKA-RIIα.

[0071] DRI-Pep#20 exhibits good mucus penetration and protease resistance. Next, we aimed to determine the extent to which DRI-Pep#20 could be used for therapeutically targeted modulation of cAMP in the lungs. First, we assessed its suitability for local delivery to the airway. Following endotracheal instillation ( Figure 5 a) DRI-Pep#20 induced a dose-dependent increase in cAMP levels in the trachea and lungs, including EC20. 50 The concentrations were 8.06 μg / kg and 11.78 μg / kg, respectively. Figure 5 bc). Notably, cardiac cAMP metabolism remained unchanged ( Figure 5 (d) indicates that the peptide locally increased cAMP in the lungs without any systemic effect.

[0072] Since the efficacy of inhaled therapy may be hampered by extracellular barriers imposed by diseased lungs (such as a thick mucus layer rich in proteases) (7), we next aimed to determine the extent to which DRI-Pep#20 could penetrate the mucus layer and resist protease degradation. DRI-Pep#20 penetrated the phospholipid membrane of the PAMPA system ( Figure 6 a), of which apparent permeability (P) app ) is 1.88 x 10 -6 cm s -1 ( Figure 6 b). It is worth noting that pathological CF sputum was added on top of the phospholipid layer ( Figure 6 a) The P of this peptide was not significantly affected. app (P) app 2.55 x 10 -6 cm s -1 () Figure 6 b). To verify whether the good mucus penetration of DRI-Pep#20 could be attributed to its molecular size being compatible with the pore size of CF mucus (14), transmission electron microscopy (TEM) and dynamic light scattering (DLS) measurements were performed. TEM images showed that DRI-Pep#20 formed irregular aggregates with a size of 5–40 nm ( Figure 6 c), which is consistent with the 10-20 nm particle diameter obtained by DLS analysis. Figure 6 d).

[0073] Next, we tested whether DRI-Pep#20 retained its ability to increase cAMP in lung cells in the presence of neutrophil elastase (the most abundant protease in the lungs of patients with neutrophilic airway diseases such as COPD and CF (15)). The peptide's ability to increase cAMP in 16HBE41o- cells was completely unaffected by the presence of 3 μg / ml recombinant human neutrophil elastase (HNE) ( Figure 6e), this dose has previously been shown to inactivate other therapeutic peptides (16). Notably, even in the presence of HNE at concentrations up to 10 times higher (a level typically detected in the lungs of patients with severe bronchiectasis (15)), the activity of DRI-Pep#20 was completely preserved ( Figure 6 f), which is consistent with the absence of any predicted cleavage sites via HNE ( Figure 7 a). The good resistance of DRI-Pep#20 to degradation was demonstrated in the presence of more complex biological matrices (containing other proteases that may cleave the peptide). Figure 7 b), that is, in CF sputum, DRI-Pep#20 retains 72% of its biological activity ( Figure 6 g).

[0074] In summary, these data demonstrate that DRI-Pep#20 enhances the ability of lung cells to generate cAMP in the presence of an unfavorable extracellular environment characterized by a protease-rich mucus barrier (a hallmark of diseased lungs).

[0075] DRI-Pep#20 promotes cAMP-dependent activation of wild-type and F508del-CFTR in human bronchial epithelial cells. Next, we aimed to assess the extent to which DRI-Pep#20 could effectively restore cAMP levels, thereby reactivating the activity of CFTR (a cAMP-dependent chloride channel (17) impaired in a range of respiratory diseases, primarily CF). First, we evaluated the peptide's ability to stimulate wild-type channel activity in 16HBE141o-cells expressing halogen-sensitive yellow fluorescent protein (HS-YFP). Figure 8 (b) HS-YFP allows for the quantification of CFTR activity based on the fluorescence quenching rate induced by iodine ion influx (18). DRI-Pep#20 induced a 60% decrease in YFP fluorescence, while co-application of the CFTR inhibitor CFTR... inh -172 can completely prevent ( Figure 8 (b) demonstrates the selective activation of the CFTR channel. Dose-response experiments reveal EC 50 20 μM ( Figure 8 c), and demonstrated that 25 μM DRI-Pep#20 was as effective as 10 μM trichomoniasis (an adenylate cyclase activator) in triggering CFTR gating in 16HBE141o- cells. Figure 8 d).

[0076] Next, we evaluated the extent to which DRI-Pep#20 could be used in combination with standard treatments for CF (including two CFTR correctors (elecapram / tazacaprate) and one CFTR enhancer (ivacato), which partially rescued the transport and gating defects of the mutant channel, respectively (19)) to restore F508del-CFTR activity. In CF bronchial epithelial cells overexpressing the F508del-CFTR mutant and HS-YFP, elecapram / tazacaprate / ivacato (ETI) reduced YFP fluorescence by 50%, while when DRI-Pep#20 was added in conjunction with ETI, fluorescence was further reduced to 25% ( Figure 8 e).

[0077] Therefore, these data support the use of DRI-Pep#20 as a single agent or as an adjunct to CFTR modulators for therapeutic stimulation of wild-type and F508del CFTR activity.

[0078] Materials and Methods Peptides and reagents The peptides were synthesized by GenScript (Piscataway, NJ) with a purity >95%.

[0079] The recombinant human PKA regulatory subunit RIIα (PKA-RIIα) and PI3Kγ catalytic subunit (p110γ) were purchased from Biaffin GmbH & Co KG (product code: PK-PKA-R2A025, Kassel, DE) and Origene Technologies (TP307790, Rockville, US), respectively.

[0080] Human neutrophil elastase was purchased from Sigma-Aldrich (CAS 9004-06-2, Sigma-Aldrich, Saint Louis, MO) and reconstituted in 50 mM sodium acetate (pH 5.5, containing 200 mM NaCl). VX-809 (Rumakafto), VX-770 (Ivakafto), VX-661 (Tzakafto), and VX-445 (Elekapor) were purchased from MedChemExpress LLC (Princeton, USA). Trichodin and CFTR were also used. inh -172 was purchased from Sigma-Aldrich (CAS 66575-29-9, Sigma-Aldrich, Saint Louis, MO).

[0081] cell lines Immortalized human bronchial epithelial cells expressing wild-type CFTR (16HBE14o-) or F508del-CFTR (CFBE41o-) were purchased from Sigma-Aldrich (16HBE14o-, product code: CAS SCC150; CFBE41o-, product code: CASSCC151, Sigma-Aldrich, Saint Louis, MO). Cells were grown in minimum essential medium (MEM) (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FBS, 5 mM L-glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin. Culture dishes were pre-coated with human fibronectin (1 mg / ml; Sigma-Aldrich, Saint Louis, MO), bovine collagen I (3 mg / ml; Sigma-Aldrich, Saint Louis, MO), and bovine serum albumin (0.1%; Sigma-Aldrich, Saint Louis, MO) diluted in LHC-8 basal medium (Invitrogen, Waltham, MA). Cells were passaged no more than 15 times. All cells were cultured at 37°C under a 5% CO2 atmosphere.

[0082] animal PI3Kγ-deficient mice (PI3Kγ) - / - (4) The mutant mice were backcrossed with C57Bl / 6j mice for 15 generations to purify the genetic background, with C57Bl / 6j mice used as a control (WT). The age of the mice used in all experiments ranged from 8 to 12 weeks. The mice were housed in groups in a controlled facility with free access to standard feed and water, and were used in accordance with the institution’s animal welfare guidelines and regulations and with the approval of the local animal ethics committee. All animal experiments were approved by the Animal Ethics Committee of the University of Turin and the Italian Ministry of Health (authorization number: 757 / 2016-PR) and have met the obligations of Legislative Decree No. 206 of April 12, 2001 and Legislative Decree No. 224 of July 8, 2003.

[0083] Isolation of mouse peritoneal macrophages Following the previously described method (4), wild-type (WT) and PI3Kγ mice aged 8 to 12 weeks were subjected to... - / -Peritoneal macrophages were prepared from mice. Briefly, cells were collected from euthanized animals by peritoneal lavage with 5 mL of PBS supplemented with 5 mM EDTA. Cells were centrifuged at 300 g for 3 min, and the pellet was resuspended in medium containing Roswell Park Memorial Institute (RPMI) medium, 100 U / ml penicillin, 100 μg / ml streptomycin, and 10% heat-inactivated FBS (Thermo Fisher Scientific, Waltham, MA). Macrophages were seeded in 96-well plates (l 10 6 (1 cell / well), and maintained at 37°C and 5% CO2 for at least 16 / 18 hours, followed by peptide treatment and cAMP quantification.

[0084] cAMP measurement Cell source: Following the manufacturer’s instructions, the cAMP content in 16HBE14o- cells was measured at specified time points after treatment with the specified dose of peptide using the Promega cAMP-Glo™ assay kit (Promega, Milano, IT).

[0085] Tissue source: Lungs, trachea, and heart were collected from euthanized mice 24 hours after intratracheal instillation of varying doses of peptide (0 to 750 mg / kg in 50 µL PBS). The frozen tissues were powdered in liquid nitrogen and extracted with cold 6% trichloroacetic acid. Samples were sonicated for 10 seconds, incubated with gentle shaking at 4°C for 10 minutes, and then centrifuged at 13,000 rpm for 10 minutes at 4°C. The supernatant was washed four times with five volumes of ether-saturated water and then lyophilized. cAMP levels were detected using a cyclic adenosine monophosphate ELISA kit (Cayman Chemical, Michigan, USA) according to the manufacturer's protocol.

[0086] Cell viability assay Human bronchial epithelial cells (16HBE14o-) were seeded in 96-well plates (2 10 4 Cells per well were incubated at 37°C and 5% CO2 for at least 16 / 18 hours prior to the experiment. Unattached cells were washed with PBS to remove non-adherent cells, and then stimulated for 24 hours with eight different doses of the specified peptide (0 μM–1 mM range). ATP levels were assessed as an indicator of cell viability using the Cell Titer-Glo® Luminescent Cell Viability Assay Kit (Promega, Milan, Italy) according to the manufacturer's protocol. The median lethal dose (LD50) was calculated relative to untreated control cells (with viability set at 100%).50 ).

[0087] CFTR activity measurement CFTR-mediated anion transport was measured using the Premo™ halide sensor (Thermo Fisher Scientific, Waltham, MA), which assesses CFTR activity by measuring the rate of YFP fluorescence quenching induced by iodide / chloride ion transmembrane exchange. In short, halide-sensitive yellow fluorescent protein (HS-YFP) was expressed in 16HBE14o- and F508del-CFTR-CFBE41o- cells using BacMam technology, following the manufacturer's protocol. Cells expressing HS-YFP were cultured in 96-well plates and treated with the specified peptide / compound for the specified time. Fluorescence was assessed immediately in a microplate reader after adding 150 µL of halide stimulation buffer (a solution containing NaI) to bring the final NaI concentration in the wells to 75 mM. Fluorescence was continuously read (1 point per second) from 1 second before the addition of halide stimulation buffer until 120 seconds later. CFTR activity was expressed as ΔF / F0, where ΔF was obtained by subtracting background fluorescence (fluorescence of cells not expressing HS-YFP) from fluorescence measured at specific time points after NaI addition. ΔF was then normalized to the initial fluorescence F0 (fluorescence of cells expressing HS-YFP measured immediately after NaI addition) to obtain the relative fluorescence ΔF / F0 measurement.

[0088] CF sputum Spontaneously coughed sputum samples were collected from CF patients in stable clinical condition from the Department of Bronchiectasis and Cystic Fibrosis at the Department of Respiratory Medicine, Fondazione IRCCS Ca' Granda Ospedale MaggiorePoliclinico, Milan (Italy), and processed as previously described (20). Patients provided explicit, free, and informed consent for the collection and use of their biological samples. Briefly, saliva was removed first, and sputum plugs were then selected and weighed during sample processing. Samples were diluted 8-fold in PBS, vortexed until sputum dissolved, and then centrifuged at 3000g for 15 minutes. The supernatant was recovered and stored at -80°C, thawed overnight at 4°C, and all subsequent experiments were performed within 24 hours after thawing. Neutrophil elastase was quantified as previously described (20), and sputum samples containing 20 µg / mL of active neutrophil elastase were used to assess the activity of the peptide in 16HBE14o- cells in the presence of CF sputum. Briefly, cells were seeded in 96-well plates (20-10 ... 10 4Cells / well were incubated at 37°C and 5% CO2 for at least 16 / 18 hours prior to the experiment. Subsequently, the peptide was diluted in PBS to a final concentration of 25 µM, and a PBS:sputum mixture (1:1) was added to the adherent cells (100 µL / well). cAMP levels were quantified at specified time points using the Promega cAMP-Glo™ assay kit (Promega, Milano, IT) according to the manufacturer's protocol.

[0089] PAMPA assay To assess the permeability of the peptide through the CF sputum layer, a parallel artificial membrane permeation system (PAMPA) as described above (Corning Gentest pre-coated PAMPA, 353015, USA plates) was used. This system allows for the measurement of the ability of a drug to diffuse across an artificial membrane from the donor compartment to the recipient compartment (21). The bottom well of the PAMPA system (“recipient” well) was filled with 300 µL of PBS (10 mM, 150 mM NaCl, pH 7.4), while the “donor” well was filled with 200 µL of peptide solution (2 mg / mL, in 10 mM PBS, 150 mM NaCl, pH 7.4), with or without CF sputum. In the latter case, 40 µL of CF sputum was first deposited onto the PAMPA membrane, followed by the addition of the peptide solution to the CF sputum layer. The two wells were then coupled and incubated at room temperature for 5 hours. After incubation, the plates were separated, and the amount of peptide diffused into the acceptor pores was quantified by fluorescence spectroscopy using a Horiba Jobin Yvon Fluorolog 3 TCSPC fluorescence spectrophotometer (Horiba, Kyoto, Japan) equipped with a 450-W xenon lamp and an R928 photomultiplier tube (Hamamatsu Photonics, Hamamatsu, Japan). The excitation wavelength was 280 nm, while emission was recorded in the 290–500 nm wavelength region (maximum emission wavelength 362 nm). The excitation and emission slits were set to 4 nm and 5 nm, respectively. Peptide concentration was calculated using a 6-point calibration curve. Apparent permeability (P0.05) was also measured. app This can be expressed by the following relation: P app It originates from Fick's law (22) under steady-state conditions, where dQ is the amount of peptide (in moles) that permeates into the acceptor chamber at time t (18000 seconds), C0 is the initial concentration of the peptide in the donor pore, and A is the area of ​​the pore membrane (0.3 cm²). 2 ).

[0090] PKA-RIIα bioconjugation and fluorescence spectroscopy As previously described (23), recombinant PKA-RIIα was bioconjugated with fluorescein 5-maleimide (F5M) using 75 µg PKA-RIIα and a 50-fold excess of F5M. Immediately after bioconjugation, the derivative was purified using a Sephadex® G-25 desalting column and phosphate-buffered saline (PBS) (20 mM, 150 mM NaCl, pH 7.2) as the elution buffer. To assess F5M labeling efficiency, the dye / protein ratio (D / P) of the conjugate was determined by the absorption spectrum of the labeled protein in PBS (20 mM, 150 mM NaCl, pH 7.2) according to the following equation: Where A 280 It is the absorbance of the conjugate at 280 nm; A max It is the absorbance of the conjugate at the corresponding F5M maximum absorption wavelength; c is the correction factor (which must be used to normalize A). 280 The signal is due to the fact that the fluorescent dye (F5M) also absorbs at 280 nm, which is equal to the dye's A. 280 Divided by the dye's A max (c=0.29); e Prot (25,169 M) -1 cm -1 ) and e dye (63,096 M) -1 cm -1 ( ) are the molar extinction coefficients of PKA and F5M, respectively. PKA-RIIα exhibits six cysteine ​​residues, with a final D / P value of 0.2.

[0091] The UV-Vis absorption spectra were measured at room temperature using a UH5300 spectrophotometer (Hitachi, Tokyo, Japan) with a 1 cm optical path quartz cuvette. The fluorescence emission spectra in steady-state mode were acquired at room temperature using a Jobin Yvon Fluorolog 3TCSPC fluorescence spectrophotometer (Horiba, Kyoto, Japan) equipped with a 450-W xenon lamp and an R928 photomultiplier tube (Hamamatsu Photonics, Hamamatsu, Japan). Steady-state fluorescence spectra were recorded in the 500–600 nm range. The excitation wavelength was set to 490 nm, and the excitation and emission slits were set to 2 and 4, respectively. The equilibrium binding constant (K0) was determined. D and K A (Obtained from steady-state data)

[0092] Fluorescence kinetics were measured using an Applied Photophysics SX20 stop-flow spectrophotometer (Applied Photophysics, North Carolina, US), which has a 495 nm cutoff filter between the cell and the fluorescence detector and is equipped with a thermostatic bath set to 25 ± 0.2 °C. Binding and dissociation rate constants (kJ / kb) were calculated based on the stop-flow kinetic data. on and k off Data acquisition, visualization, and analysis were performed using Pro-Data software from Applied Photophysics Ltd (Applied Photophysics, North Carolina, US).

[0093] To evaluate the ability of DRI-Pep#20 to displace PI3Kγ bound to PKA-F5M, steady-state emission spectra of the PI3Kγ / PKA-F5M complex were acquired in the presence of incremental peptide concentrations. Briefly, 50 nM of recombinant PI3Kγ was added to 100 nM of F5M-bound PKA-RIIα in a total volume of 100 µL PBS. The concentration of the PI3Kγ / PKA-F5M complex was kept constant while titrating stepwise with peptides at concentrations increasing from 0 to 5 µM. As described above, the complex was excited at 490 nm, and emission spectra were recorded in the 500–600 nm spectral range. The degree of displacement of the PKA-RIIα-PI3Kγ complex was expressed as the percentage of fluorescence quenching after peptide addition.

[0094] Circular dichroism Circular dichroism (CD) measurements were performed on a Jasco-810 circular dichroism spectrometer equipped with a Peltier thermoelectric controller (Jasco Inc., Easton, US). Using 0.1 cm path length quartz cuvettes (Hellma GmbH, Müllheim, DE), the spectra of peptides were recorded in continuous mode from 260 to 180 nm at 25 °C, with a total peptide concentration of 0.2 mg / mL, dissolved in 2 mM PBS (0.6 mM KH₂PO₄, 1.6 mM K₂HPO₄), pH 7.4. CD spectra in the 190–240 nm range were used to predict the secondary structure content of the peptides using a K2D3 web server (24).

[0095] Transmission electron microscopy (TEM) and dynamic light scattering (DLS) Self-assembled peptide nanostructures were analyzed by transmission electron microscopy (TEM). TEM images were obtained using a JEOL 3010-UHR TEM (JEOL, Tokyo, Japan) at an accelerating voltage of 300.00 kV. TEM samples were prepared by dissolving the peptides in water at 0.1 mg / mL and drying them on a carbon-coated copper grid. The nominal magnifications used for recording the nanostructures were ×500,000 and ×800,000.

[0096] The particle size distribution of self-assembled peptides was determined by dynamic light scattering (DLS, Malvern Zetasizer, Worcestershire, UK). Samples were prepared at a concentration of 4 mg / mL in 2 mM PBS (0.6 mM KH₂PO₄, 1.6 mM K₂HPO₄) at pH 7.4. Measurements were taken after a 60-second equilibration time, which allowed the samples to reach a temperature of 25 °C.

[0097] PKA-RIIα structure prediction The 3D structure of residues 1-45 of PKA-RIIα (SEQ ID NO: 14-MSHIQIPΜGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA) was predicted using the Iterative Threading ASSEmbly (I-TASSER) web server (an online platform for predicting protein structure and function based on the I-TASSER algorithm) (25). In short, starting with the FASTA amino acid sequence, I-TASSER runs a three-step simulation: First, using a spectro-spectrum alignment-based threading algorithm, the sequence is threaded through a representative PDB structure library to search for possible template folds or supersecondary structure fragments. Second, continuous fragments cut from the PDB template are reassembled into a full-length model, while unaligned regions are constructed through de novo modeling. Finally, the structural trajectories are clustered, the lowest-energy structures are selected, and a full-atom model is constructed using REMO41 by optimizing the hydrogen bond network. Subsequently, the five best models obtained by I-TASSER are evaluated based on the threaded template and the predicted C-score. The model with the highest C score (-0.22) and using the NMR structure of PKA-RIIα as the threading template (PDB ID 2KYG) (26) was selected for prediction.

[0098] DRI-Pep#20 Structural Prediction The structure of DRI-Pep#20 was predicted using PEP-FOLD3.5 (a de novo method for predicting peptide structure from amino acid sequences) (27). In short, starting from the amino acid sequence, a series of 200 simulations were run, each sampling different regions of the conformational space using Generator taboo sampling 5 (ts5), recommended for peptides longer than 10 amino acids. The output was an archive of all model clusters, ranked using TM scores, and then model quality was evaluated using Apollo (28). The top five models representing the five best conformations in each cluster were selected based on the lowest sOPEP energy and the highest TM score, and further supported by RMSD. Finally, the optimal peptide structure was validated by visual analysis on PYMOL.

[0099] PKA-RIIα-DRI-Pep#20 docking The PKA-RIIα-DRI-Pep#20 docking was studied using the High Fuzziness-Driven Biomolecular Docking (HADDOCK) software. In the HADDOCK process, residues 1-45 of PKA-RIIα were selected as active residues and forced to become part of the interface by applying fuzzy interaction constraints. In short, starting with the PKA-RIIα and DRI-Pep#20 structures, HADDOCK docking ran in three consecutive steps: First, the molecules were randomly oriented and a rigid body search was performed (it0). The output was an archive of 1000 models, from which the top 200 structures were selected based on energy functions and entered a semi-flexible simulated annealing stage in torsion angle space (it1). In the third stage, the structures were refined in Cartesian space with an explicit solvent layer (water) and short molecular dynamics simulations were performed at 300 K. During the refinement process, the side chains and main chains of the interface residues were gradually allowed to move.

[0100] The final model was automatically clustered based on positional interface ligand RMSD (iL-RMSD) by fitting conformational changes at the receptor (PKA-RIIα) interface and the smaller chaperone (peptide) interface. Finally, the protein-peptide binding conformation was evaluated using HADDOCK Reports, and binding affinity was assessed using the optimal hydrogen bond network. The optimal binding conformation was validated using visual analysis on PYMOL.

[0101] Statistical analysis Data are presented as scatter plots with error bars (mean ± SEM). Prism software (GraphPad Software Inc.) was used for statistical analysis. The raw data were first analyzed to confirm normality via the Shapiro-Wilk test, followed by unpaired Student's t-test, one-way ANOVA, or two-way ANOVA. Bonferroni correction (one-way and two-way ANOVA) was applied to correct for multiple comparisons. P < 0.05 was considered significant.

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Claims

1. A non-natural peptide having the ability to inhibit the A-kinase anchoring function of PI3Kγ, comprising an amino acid sequence as shown in SEQ ID NO: 1, wherein each amino acid is a D-amino acid, and wherein the glutamine at position 3 and the glycine at position 4 may be replaced by any amino acid having similar hydrophobicity, hydrophilicity, charge and size, respectively.

2. The non-natural peptide according to claim 1, wherein the peptide further comprises a cell-penetrating peptide.

3. The non-natural peptide according to claim 2, wherein the cell-penetrating peptide is selected from Penetratin (pAntp), HIV TAT peptide, R7 peptide, KALA peptide, Buforin 2, MAP, Transportan, Transportan 10, pVEC, and MPG peptide.

4. The non-natural peptide according to claim 2 or claim 3, wherein the peptide further comprises a connector linking the amino acid sequence of SEQ ID NO: 1 to the cell-penetrating peptide.

5. The non-natural peptide according to any one of the preceding claims, wherein the cell-penetrating peptide sequence is reversed relative to the natural sequence.

6. The non-natural peptide according to claim 4 or claim 5, wherein the linker is an amino acid linker comprising one or more glycine amino acids and / or one or more amino acids similar to glycine in hydrophobicity, hydrophilicity, charge and size.

7. The non-natural peptide according to any one of the preceding claims, having an amino acid sequence as shown in SEQ ID NO:

2.

8. The non-natural peptide according to any one of the preceding claims, used as a medicine.

9. The non-natural peptide according to any one of the preceding claims, for the treatment of respiratory diseases, preferably bronchial obstructive diseases.

10. The non-natural peptide for use according to claim 9, wherein the respiratory disease is selected from allergic asthma, cystic fibrosis, chronic obstructive pulmonary disease, non-cystic fibrotic bronchiectasis, pulmonary hypertension, and idiopathic pulmonary fibrosis.

11. The non-natural peptide for use according to any one of claims 8 to 10, wherein the non-natural peptide is suitable for administration by inhalation.

12. Products, which include: i) at least one non-natural peptide according to any one of claims 1 to 7, and ii) At least one cystic fibrosis transmembrane conductance modulator (CFTR) enhancer and / or at least one cystic fibrosis transmembrane conductance modulator (CFTR) corrector, as a combination formulation for the sequential, simultaneous or separate treatment of respiratory diseases.

13. The product of claim 12, wherein the cystic fibrosis transmembrane conduction modulator (CFTR) synergist is selected from ivacarto, navocaftor, and deuterated ivacarto.

14. The product of claim 12 or claim 13, wherein the cystic fibrosis transmembrane conduction modulator (CFTR) corrector is selected from Lumacaftor, Tezacaftor, Elaxacaftor, Galicaftor, and Vanzacaftor.

15. A pharmaceutical composition comprising at least one non-natural peptide according to any one of claims 1 to 7 and a pharmaceutically acceptable mediator.

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