EP4731198A1

EP4731198A1 - Compounds for use in the treatment of nape-pld related diseases and methods of identification thereof

Info

Publication number: EP4731198A1
Application number: EP24734858.4A
Authority: EP
Inventors: Gianpiero GARAU
Original assignee: Fondazione Istituto Italiano di Tecnologia
Current assignee: Fondazione Istituto Italiano di Tecnologia
Priority date: 2023-06-20
Filing date: 2024-06-17
Publication date: 2026-04-29
Also published as: IT202300012672A1; WO2024260920A1

Abstract

It forms an object of the present invention chemical entities that bind to the homodimer interface of NAPE-PLD protein ("thiazide-binding site"), or homologues thereof. It forms a second object of the present invention crystal structure coordinates of NAPE-PLD and methods of using these crystal structure coordinates to identify and design compounds that bind to the dimer interface of the protein NAPE-PLD, or homologues thereof.

Description

COMPOUNDS FOR USE IN THE TREATMENT OF NAPE-PLD RELATED DISEASES AND METHODS OF IDENTIFICATION THEREOF

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Background

The membrane-associated enzyme N-acyl phosphatidylethanolaminespecific phospholipase D (NAPE-PLD) hydrolyses aa family of glycerophospholipids (N-acyl phosphatidylethanolamine, NAPE) having a rare structure of three fatty acyl chains embedded in the lipid bilayer, which confers rigidity to membranes (Lafrance et al. 1997 Biophys J 72: 2559-2568). The protein NAPE-PLD is widely expressed in all body tissues. It belongs to the metallo-[3-lactamase protein superfamily, and its activity generates bioactive lipid signalling molecules that have key protective roles in several physiological pathways, including stress and pain response, homeostasis, metabolism, inflammation, and lifespan (Calignano et al. 1998 Nature 394: 277-281 ; Berghuis et al. 2007 Science 316: 1212-1216; Kotas & Medzhutov 2015 Cell 160: 816-827; Everard et al. 2018 Nature Comm 10: 457; Di Marzo 2018 Nature Rev Drug Discov 17: 623-639).

The crystal structure of human NAPE-PLD, coordinates deposited at the Protein Data Bank (PDB) under accession number PDB ID: 4QN9 (Deposited 17/06/2014, Deposition Author: Garau, G.) shows that the membrane protein has a binuclear zinc active site, and forms homodimers with an internal ~9 A- wide channel.

NAPE-PLD has specific binding sites for bile acid molecules, which act as cofactors to promote enzymatic catalysis and enhance protein dimer assembly (Magotti et al. 2015 Structure 3: 598-604; Margheritis et al. 2016 ACS Chem Biol 10: 2908-2914).

Active-site directed inhibitors of NAPE-PLD have been identified (Castellani 2017 Chem Comm 53: 12814-12817; Aggarwal et al 2020 J Biol Chem 295: 7289-7300).

Studies have shown that, in the adipose tissue, NAPE-PLD modulates fat mass development by altering the browning process and gut microbiota (Geurts et al. 2015 Nature Comm 6: 6495). In the intestine, NAPE-PLD is a key sensor in nutritional adaptation to fat intake (Evard et al. 2019 Nature Comm 10: 457). Centrally, the expression of NAPE-PLD responds to proinflammatory stimuli (Zhu et al. 2011 Molecular Pharm 79: 786-792) and shows altered expression in autopsy brain samples from patients with multiple sclerosis (Zhang et al. 2011 Brain Pathol 21 : 544-557). The identification of different gene variants in dog breeds affected by leukoencephalopathies has suggested that NAPE-PLD might be a target for myelination disorders (Minor et al. 2018 Scientific Rep 8: 5818).

Hypertension is a major cause of cardiovascular disease and deaths worldwide, and a leading risk factor for several other diseases, including stroke, heart disease, chronic kidney disease, and cognitive decline (Al-Makki et al. 2022 Hypertension 79: 293-301 ). Lowering blood pressure can reduce cardiovascular morbidity and mortality rates and slow the progression of renal disease and overall mortality. Despite the availability of different preventive and therapeutic approaches, patients treated with antihypertensive drugs continue to raise in the world (NCD Risk Factor Collaboration 2021 Lancet 398: 957-980). Partial or total non-adherence has been recognized as a major issue in the long-term management of hypertension (Burnier et al. 2020 Fron Cardiov Med 7: 1 -9). These aspects demand the development of novel and rationale drug treatments for hypertension and neuroendocrine modulation, and interventional and knowledge-based treatments to improve cardiovascular and other hypertension-associated outcomes. Moreover, among patients with incident hypertension in whom treatment was begun, 2% of patients developed resistant hypertension (Daugherty et al 2012 Circulation 1635-1642), requiring innovative and rational pharmacological approaches.

Thiazides are drugs listed in hypertension guidelines as one of the preferred options for essential hypertension, as monotherapy or multidrug regimens, and are also used to manage cardiovascular disease, edema associated with chronic heart failure, renal dysfunction and failure, and liver diseases (Ernest and Moser 2009 N Engl J Med 361 : 2153-2164; Burnier et al. 2019 J Hypertens 37:1574-1586; Duarte & Cooper-DeHoff 2010 Expert Rev Cardiovasc Ther 8: 793-802).

The technical problem solved by the present invention is the identification of more specific and more efficient molecules able to interact with the subunits interface of NAPE-PLD, wherein said molecules have a biological activity comparable or even superior to that of thiazide diuretics in the management of (i) hypertension and related diseases, including cardiovascular disease, hypervolemia, electrolyte disorders, kidney disease, diabetes, stone prevention, hypercalciuria and (ii) NAPE-PLD related diseases and vitamin B6- related disorders, including metabolic disorder, neuromuscular disease, neurodegeneration, multiple sclerosis, myelination disorders and white matter disease progression, epilepsy and autism spectrum disorders.

Description

The present invention solves the technical problem by stabilizing the structure of NAPE-PLD to enhance its membrane function during time using a novel and original approach, where the target structure is stabilised with allosteric compounds. Surprisingly, contrary to the action of inhibitors identified so far, the here described solution is useful to preserve the protective role of NAPE-PLD in the organism.

In an embodiment, the present invention refers to chemical entities that bind to the homodimer interface of NAPE-PLD protein (“thiazide-binding site”) (Figure 1 ), or homologues thereof.

In an embodiment, the invention relates to molecular complexes which comprise binding pockets of the homodimer interface NAPE-PLD, or homologues thereof.

In an embodiment, the invention relates to crystal structure coordinates of NAPE-PLD and methods of using these crystal structure coordinates to identify and design compounds that bind to the dimer interface of the protein NAPE- PLD, or homologues thereof.

In an embodiment, the invention relates to crystallizable compositions and crystals comprising complexes involving the dimer interface of NAPE-PLD protein and chemical entities.

In an embodiment, the present invention refers to a method to select chemical entities that bind to the homodimer interface of NAPE-PLD protein, as well as methods for evaluating the ability of a chemical entity to associate with the homodimer interface of NAPE-PLD protein.

The invention allows to identify, search for, evaluate, design, or modify chemical compounds having a biological activity comparable or even superior to that of thiazide diuretics in the management of (i) hypertension and related diseases, including cardiovascular disease, hypervolemia, electrolyte disorders, kidney disease, diabetes, stone prevention, hypercalciuria; and (ii) NAPE-PLD related diseases and vitamin B6-related disorders, including metabolic disorder, neuromuscular disease, neurodegeneration, multiple sclerosis, myelination disorders and white matter disease progression, epilepsy and autism spectrum disorders.

Drawings

Figure 1 : Schematic 3D representation of the human NAPE-PLD structure in complex with different stabilizing allosteric compounds that bind to its homodimer interface (“thiazide-binding site”, depicted as oval dotted line): (A) hydrochlorothiazide (HCTZ, PDB ID: 8PC4), (B) pyridoxal phosphate (PLP, PDB ID: 8P96) and (C) (1 , 3, 7)-naphthalene trisulfonic acid (N3SA, PDB ID: 8PC4) (compound 3, according to the invention). On the right to each structure, expanded view of the binding site showing interactions with involved protein residues of the homodimer interface and solvent molecules (dashed lines with distances in A indicate hydrogen bonding; continuous lines with distances in A indicate hydrophobic interactions). Hydrogen bond distances from an acceptor/donor atom of the interacting compound and a donor/acceptor atom the protein homodimer interface can span from 2.2 A to 3.3 A. Hydrophobic interaction distances can span from 3.3 A to 4.0 A.

Figure 2: Representative SPR-based in vitro assays (A, B, C) with examples of “classical injections” at different concentrations of HCTZ (A), and “one-step gradient injections” using the compound (3), N3SA (B). In (C) is shown the profile of one-step injections using N3SA and deoxycholic acid (DCA or DC), simultaneously, to test their synergic interaction. In (D) is illustrated the thermal shift profile of the stabilization of the protein NAPE-PLD by N3SA (compound (3), according to the invention).

Figure 3: Anti-hypertensive effect of N3SA (compound (3), according to the invention) in animal model of SHRs. (A) Time-course of the effect in SHRs at 8 weeks of age. (B) Time-course of the effect of N3SA, of the bile acid DCA, and of their combination in SHRs at 14 weeks of age. After about 2 weeks of treatment, administration was interrupted to follow the duration of the effect.

Figure 4: Compounds that bind to the homodimer interface NAPE-PLD, including structural analogues of N3SA (compound (3), according to the invention).

Figure 5: Graph histogram shows binding energies of compounds in Figure 4, obtained by in silico approach (AutoDock Vina). The effective capacity of these compounds to bind NAPE-PLD has been validated experimentally by SPR using the relationship [AG = -RT In(K_D)] and the compounds (1 ) HCTZ: KD = 8 (±2) pM, AG₍i) = -7.0 kcal/mol; (2) PLP: K_D = 32.7 (±0.1 ) μM, ΔG₍₂) = -6.1 kcal/mol; and (3) N3SA: K_D = 0.73 (±0.02) pM, AG₍₃₎ = -8.4 kcal/mol. These experimental values correspond in the graph histograms to calculated binding energies of -5.2 kcal/mol, -4.0 kcal/mol, and -6.8 kcal/mol, respectively. The correlation between measured and calculated values provides an evaluation of the ranking power of the applied in silico approach.

Definitions

In the context of the present description, the term “thiazide-binding site” corresponds to the entire homodimer interface of NAPE-PLD protein, comprising all the residues facing the internal channel of the dimer, as shown in Figure 1 .

The term "associating with" refers to a condition of proximity between a chemical entity or compound, or portions thereof, and a ligand-binding pocket or binding site on a protein. The association may be non-covalent wherein the juxtaposition is energetically favoured by hydrogen bonding or by van der Waals or electrostatic interactions or it may be covalent.

The term "to quantify" refers in the present context to measure values by experiments or to compile calculated values from the computational calculations of said fitting operation between the chemical entity and the homodimer interface NAPE-PLD or its binding pockets, or the “thiazide-binding site” of NAPE-PLD, which refers to the binding pocket where hydrochlorothiazide associates, defined by the structure coordinates of a certain set of amino acid residues present in the NAPE-PLD structure. Examples of ligands that bind the homodimer interface NAPE-PLD or its binding pockets, or the “thiazide-binding site” of NAPE-PLD are hydrochlorothiazide, pyridoxal phosphate, and (1 ,3,7)-naphthalene trisulfonic acid, as defined by the structure coordinates of the homodimer interface of NAPE-PLD protein in complex with these molecules.

The term "domain" refers to a portion of the NAPE-PLD protein or NAPE- PLD like protein that can be separated based on its biological function (e.g., ligand binding site, zinc active site, dimer interface). The domain may comprise a binding pocket, a sequence, or a structural motif.

The term “thiazide” refers to the benzothiadiazides, compounds having a scaffold of 3,4-dihydro-2H-1 ,2,4-benzothiadiazine 1 ,1 -dioxide (IUPAC name: 1 ,1 -dioxo-3,4-dihydro-2H-1λ6,2,4-benzothiadiazine), which can have different substituents, for example a chloro at position 6 and a sulphonamide at 7, the hydrochlorothiazide.

The term “pyridoxal phosphate” is the active form of vitamin B6 and refers to a pyridine carbaldehyde (IUPAC name: (4-formyl-5-hydroxy-6- methylpyridin-3-yl) methyl dihydrogen phosphate).

The atomic structure coordinates of the two crystallographic independent molecules of the NAPE-PLD homodimer, chain A and chain B, respectively, were derived by X-ray diffraction and deposited in the Protein Data Bank (PDB ID: 8PC4, 8P90, 8P96). The “ATOM” records present the atomic coordinates for atoms of standard amino acids (“_atom_site” record in the crystallographic Information File “cif” format). They also present the occupancy and temperature factor for each atom. The element symbol is always present on each ATOM record. Non-polymer chemical coordinates use the “HETATM” record type. Atom type refers to the element whose coordinates are measured. The first letter in the column defines the element. The "#" sign is an abbreviation for "number" in a numbering of atoms and residues. Residues further have a chain name indicated by a letter in front of the digits as part of the residue numbering. "Resid" refers to the amino acid residue identity in the molecular model. "X, Y, Z" define the atomic position of the element measured (“_atom_site.Cartn_x”, _”atom_site.Cartn_y”, _”atom_site.Cartn_z” record, respectively, in the crystallographic Information File “cif” format). "B" is a thermal factor that measures movement of the atom around its atomic center (“_atom_site.B_iso_or_equiv”, in the crystallographic Information File “cif” format). "Occ" is an occupancy factor that refers to the fraction of the molecules in which each atom occupies the position specified by the coordinates (“_atom_site. occupancy”, in the crystallographic Information File “cif” format). A value of "1" indicates that the atom has the same position in all symmetry related molecules of the crystal.

The following abbreviations of amino acid residues are used throughout the application: A = Ala = Alanine; R = Arg = Arginine; N = Asn = Asparagine; D = Asp = Aspartic Acid; C = Cys = Cysteine; Q = Gin = Glutamine; E = Glu = Glutamic Acid; G = Gly = Glycine; H = His = Histidine; l= lie = Isoleucine; L = Leu = Leucine; K = Lys = Lysine; M = Met = Methionine; F = Phe = Phenylalanine; P = Pro = Proline; S = Ser =Serine; T = Thr =Threonine; W = Trp = Tryptophan; Y = Tyr = Tyrosine; V = Vai = Valine.

A residue type of “ZN” indicates a zinc ion of the enzyme active site.

A residue type of “3PE” indicates molecule of phosphatidylethanolamine.

A residue type of “DXC” indicates molecules of deoxycholic acid.

A residue type of “HOH” indicates a zinc ion of the enzyme active site.

A residue type of “PLP” indicates molecules of pyridoxal phosphate.

A residue type of “HCZ” indicates molecules of hydrochlorothiazide.

A residue type of “NSA” indicates molecules of 1 ,3,7-naphthalene trisulfonic acid.

Methods for identifying a corresponding amino acid are known in the art and are based upon sequence, structural alignment, its functional position, or a combination thereof as compared to the NAPE-PLD protein molecule. For example, corresponding amino acids may be identified by superimposing the backbone atoms of the amino acids in human NAPE-PLD and the NAPE-PLD homologues (e.g., mouse NAPE-PLD or mutants of NAPE-PLD) using well known software applications, such as COOT (Emsley et al. 2010 Acta Cryst D 66: 486-501 ). The corresponding amino acids may also be identified using sequence alignment programs such as the "BLAST" program available online (Johnson et al. 2008 Nucleic Acids Res 36: W5-W9).

The term "fitting operation" refers to an operation that utilizes the structure coordinates of a chemical entity, binding pocket, molecule or molecular complex, or portion thereof, to associate the chemical entity with the binding pocket, molecule or molecular complex, or portion thereof. This may be achieved by positioning, rotating, or translating the chemical entity in the binding pocket to match the shape and electrostatic complementarity of the binding pocket. Covalent interactions, non-covalent interactions such as hydrogen bond, electrostatic, hydrophobic, van der Waals interactions, and non-complementary electrostatic interactions such as repulsive charge- charge, dipole-dipole and charge-dipole interactions may be optimized. Alternatively, one may minimize the deformation energy of binding of the chemical entity to the binding pocket.

The terms "generating a three-dimensional structure" or "generating a three- dimensional representation" refer to converting the lists of structure coordinates into structural models or graphical representation in three- dimensional space. This can be achieved through commercially or publicly available software (e.g., PYMOL Molecular Graphic System, Schrodinger, LLC). The three-dimensional structure may be displayed or used to perform computer modelling or fitting operations. In addition, the structure coordinates themselves may be used to perform computer and fitting operations.

The term "homology model" refers to a structural model derived from known three-dimensional structure(s). Generation of the homology model, termed "homology modelling", can include sequence alignment, residue replacement, residue conformation adjustment through energy minimization, or a combination thereof.

The term "homologue of NAPE-PLD" or "NAPE-PLD homologue" refers to a molecule that is homologous to NAPE-PLD by structure or sequence. Examples of homologues comprise but are not limited to human NAPE-PLD and NAPE-PLD from another species with conservative substitutions, additions, deletions, or a combination thereof; or another member of the NAPE-PLD family including, but not limited to members of the Metallo Beta- Lactamase Superfamily (Garau et al. 2004 Antimicrob Agents Chemother 48: 2347-2349), with conservative substitutions, additions, deletions, or a combination thereof.

The term "sufficiently homologous to NAPE-PLD" refers to a protein that has a sequence homology of at least 20% compared to NAPE-PLD protein. In one aspect, the sequence homology is at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%.

The term "part of a binding pocket" refers to less than all the amino acid residues that define the binding pocket. For example, the structure coordinates of residues that constitute part of a binding pocket may be specific for defining the chemical environment of the binding pocket, or useful in designing fragments of an inhibitor that may interact with those residues. For example, the portion of residues may be key residues that play a role in ligand binding or may be residues that are spatially related and define a three-dimensional compartment of the binding pocket. The residues may be contiguous or non- contiguous in primary sequence.

The term "molecular complex" or "complex" refers to a molecule associated with at least one chemical entity.

The term "structure coordinates" refers to Cartesian coordinates derived from mathematical equations related to the patterns obtained on diffraction of a monochromatic beam of X-rays by the atoms (scattering centers) of a protein or protein complex in crystal form. The diffraction data are used to calculate an electron density map of the repeating unit of the crystal. The electron density maps are then used to establish the positions of the individual atoms of the molecule or molecular complex.

The term "three-dimensional structural information" refers to information obtained from the structure coordinates. Structural information generated can include the three-dimensional structure or graphical representation of the structure. Structural information can also be generated when subtracting distances between atoms in the structure coordinates, calculating chemical energies for a NAPE-PLD molecule or molecular complex or homologues thereof, calculating or minimizing energies for an association of a NAPE-PLD molecule or molecular complex or homologues thereof to a chemical entity.

Detailed description

In an embodiment, compounds that bind to homodimer interface of NAPE- PLD are described.

In an embodiment, said compounds are compounds of Formula (I), or pharmacologically acceptable derivatives thereof, wherein

A is C, N or S;

R¹ is SO₃H, PO₃H₂, SO₂NH₂ or SO₂NHR⁶ wherein R⁶ is an aromatic or heteroaromatic ring, saturated or unsaturated COHN cyclic;

R² is SO₃H, SO₂NH₂, PO₃H₂, halogen, COOH, CONH₂, C1 -C4 alkyl, C1 - C4 alkyl halogen substituted, or optionally substituted NHAr;

R³ is H, S, CH₃;

R⁴ is H,

R⁵ is SO₃H, PO₃H₂, SO₂NH₂, SO₂CH₂OH, or R⁴ and R⁵ close to form a cycle which is

In an embodiment, said compounds are compounds of Formula (I), or pharmacologically acceptable derivatives thereof, wherein A is C, N or S;

R³ is H, S;

R⁴ is H;

R⁵ is SO₃H, PO₃H₂, SO₃H, PO₃H₂, SO₂NH₂, SO₂CH₂OH; or R⁴ and R⁵ close to form a cycle which is

In an embodiment, the compounds of the present invention have the general Formula (I) or they are a pharmacologically acceptable derivative thereof, wherein

A is C, N or S;

R¹ is SO₃H, SO₂NH₂, or PO₃H₂;

R² is SO₃H, SO₂NH₂, PO₃H₂, CF₃, or Cl;

R³ is H, S;

R⁴ is H;

R⁵ is SO₃H, SO₂NH₂, PO₃H₂.

In an embodiment, the bicyclic structure of the general Formula (I) is a naphthalene.

In an embodiment, the compounds are selected in the group comprising or consisting of:

In an embodiment, said group additionally comprises:

Surprisingly, said compounds demonstrated allosteric properties for the stabilization of the target.

For example, the compound (3) binds to NAPE-PLD with an equilibrium dissociation constant measured in KD = 8 (±2) pM, stabilizing the homodimeric structure.

In an embodiment, it is here described the first crystal structure with atomic coordinates of the human membrane NAPE-PLD in complex with an exogenous chemical entity which is hydrochlorothiazide (6-Chloro-3,4- dihydro-2H-1 ,2,4-benzothiadiazine-7-sulfonamide 1 ,1 -dioxide), the representative member of the class of thiazide diuretics (Figure 1A). The coordinates at 2.8 A of resolution obtained by X-ray diffraction (PDB ID: 8PC4) confirm that hydrochlorothiazide binds to the homodimer interface of NAPE- PLD protein, forming a stable form of NAPE-PLD homodimer (Figure 1 A).

The thiazide hydrochlorothiazide binds to NAPE-PLD with an equilibrium dissociation constant measured in KD = 8 (±2) pM, stabilizing the homodimeric structure. The binding ligand efficiency of hydrochlorothiazide for NAPE-PLD results LE = (-AG/Nha) = 0.46 kcal/mol, with a residence time kinetics (dissociation constant = k_off) of K_d = 4.22 ±0.09) e^-3 s^-1.

The NAPE-PLD subunit interface comprises resides PHE150, SER151 , SER152, ARG153, ALA154, SER155, PRO156, SER157, GLN158, TYR159, MET160, GLY161 , PRO162, LYS163, ARG164, PHE165, ARG166, ARG167, SER168, PRO169, CYS170, THR171 , ILE172, SER173, GLU174, ASP192, TYR193, ASN194, SER195, VAL196, ILE197, ALA198, and GLU201. This amino acid numbering scheme corresponds to that of the crystal structure PDB ID: 4QN9 (cited) and defines all the NAPE-PLD structure resides reported and described in the present description. The homodimer interface forms an internal (~9 A) channel containing different binding pockets. The complex coordinates revealed useful in designing novel chemical entities targeting the association of the two NAPE-PLD protein subunits, using methodologies reported herein.

The resulting discovered binding site of the drug, never targeted before, is located at the interface of the two protein subunits of NAPE-PLD protein (named “thiazide-binding site” or “thiazide-binding pocket”). Crystal structure coordinates show for the first time that hydrochlorothiazide contribute to stabilize the association in the structure of the two protein subunits. Of note, the thiazide-binding site is a distinct and innovative allosteric site, apart from the bile acid binding site, and apart from the binuclear zinc active site of NAPE- PLD where enzyme inhibitors bind (Figure 1A).

The complex structure also contains molecules of bounded deoxycholate, a bile acid that associates to a different and specific binding site of the homodimer interface of NAPE-PLD (named “bile acid-binding site” or “bile acid-binding pocket”) (Figure 1A).

In the absence of other chemical entities, bound bile acids contribute to stabilize the association of the two protein subunits and shows a dissociation constant measured in KD = 40 (±2) pM.

Increasing the concentration of deoxycholate from 0 pM to 250 pM, the dissociation constant of hydrochlorothiazide from a value of KD = 8 (±2) pM gets to KD = 0.9 (±0.4) pM, thus resulting about one order of magnitude more potent, with a LE = 0.55 kcal/mol.

When the two chemical entities hydrochlorothiazide and deoxycholate are used together at the same time in a mixture, solution, or composition, they potentiate reciprocally the affinity for NAPE-PLD, with an advantage in term of residence time and thus pharmacokinetics for the target protein. The advantage of the combined use of a chemical entity (e.g., a compound that targets NAPE-PLD) and a bile acid can be applied to further strength the homodimer assembly of the target protein, and thus to enhance its pharmacological effect.

Therefore, it forms a further object of the present invention a composition comprising at least a bile acid and a chemical entity targeting NAPE-PLD, wherein said chemical entity is selected from the compounds of general Formula (I), as above described, a thiazide diuretic (e.g., hydrochlorothiazide, or HCTZ, or 6-chloro-1 ,1 -dioxo-3,4-dihydro-2H-1λ⁶,2,4-benzothiadiazine-7- sulfonamide), or a vitamin B6 derivative (e.g., pyridoxal 5’-phosphate, or PLP, or (4-Formyl-5-hydroxy-6-methylpyridin-3-yl)methyl dihydrogen phosphate), or a naphthalene trisulfonic acid (e.g., (1 ,3,7)-naphthalene trisulfonic acid, N3SA, or analogue compounds for use in the in the treatment of (i) hypertension and related diseases, including cardiovascular disease, hypervolemia, electrolyte disorders, kidney disease, diabetes, stone prevention, hypercalciuria and/or (ii) NAPE-PLD related diseases and vitamin B6-related disorders, including metabolic disorder, neuromuscular disease, neurodegeneration, multiple sclerosis, myelination disorders and white matter disease progression, epilepsy and autism spectrum disorders.

Surprisingly, the here described thiazide-binding site on NAPE-PLD paves the way to identify chemical entities targeting said homodimeric interface of NAPE-PLD. Said chemical entities that bind to the homodimer interface of NAPE-PLD, are useful for preventing, treating and in the management of (i) hypertension and related disorders, including cardiovascular disease, hypervolemia, electrolyte disorders, kidney disease, diabetes, stone prevention, hypercalciuria, and (ii) NAPE-PLD related diseases and vitamin B6-related disorders, including metabolic disorder, neuromuscular disease, neurodegeneration, multiple sclerosis, myelination disorders and white matter disease progression, epilepsy and autism spectrum disorders.

In an embodiment, the structure coordinates of the homodimeric interface of NAPE-PLD in complex with hydrochlorothiazide (PDB ID: 8PC4) allowed to select pyridoxal phosphate as a novel chemical entity targeting said homodimeric interface, Figure 1 B. The chemical entity is the active form of vitamin B6, pyridoxal phosphate (pyridoxal-5’-phosphate, 3-Hydroxy-2-methyl- 5-([phosphonooxy] methyl)-4-pyridinecarboxaldehyde). The crystal structure coordinates of the complex showed that pyridoxal phosphate binds to the interfacial allosteric interface of NAPE-PLD, stabilizing the protein homodimer similarly to the mode of binding of hydrochlorothiazide (PDB ID: 8P90).

This chemical entity was selected among natural compounds, and then validated experimentally by approaches of X-ray crystallographic screening for ligand discovery (Blundell et al. 2002 Nature Rev Drug Discovery 1 : 45-54), with an approach that targets specifically the homodimer interface of NAPE- PLD. The methodology started from classes of chemical molecules, including classes of natural compounds present in eukaryotic cells. The low micromolar affinity of pyridoxal phosphate for NAPE-PLD in our approach was measured by surface plasmon resonance. Other methodologies of binding assays to quantify the binding affinity can be similarly applied, including approaches and methodologies of fragment-based and high-throughput screening (e.g., nuclear magnetic resonance, microcalorimetry, scintillation proximity assay, fluorescence detection techniques).

In a third embodiment, the same approach leads to the selection of a novel ligand, Figure 1 C. The chemical entity is (1 ,3,7)-naphthalene trisulfonic acid, compound (3). The complex structure (PDB ID: 8P96) shows interactions common to the complex of hydrochlorothiazide (Figure 1 A) and pyridoxal phosphate (Figure 1 B) (e.g., hydrogen bond involving Ser152), and few compound-specific features (Figure 1 C) (e.g., hydrogen bond involving Arg167). This result represents the proof-of-concept for the application of the structure coordinates (PDB ID codes 8PC4, 8P90, 8P96) to identify and characterize novel chemical entities that binds to the interfacial allosteric interface of NAPE-PLD, promoting a desired therapeutic profile (Figure 3).

Compound 3 has been initially selected and probed by methodologies of virtual screenings and compute-added drug discovery and design, starting from the coordinates of NAPE-PLD in complex with hydrochlorothiazide and pyridoxal phosphate, dedicated in silico software packages (e.g., AutoDock Vina) and classes of small organic molecules (e.g., containing groups of sulfone, sulphonamide, sulfoxide, and phosphate).

The nanomolar affinity of compound (3), (1 ,3,7)-naphthalene trisulfonic acid, KD = 0.73 (±0.02) pM, has then been experimentally measured by the label-free methodology of surface plasmon resonance (Figure 2B), as well as its kinetics parameters, the association constant Ka = Kon = 2.45 (±0.04) x 10³ (M^-1 s^-1) and the dissociation constant Kd = Koff = 1 .79 (±0.03) x 10^-3 s^-1.

Compared with the affinity of the hydrochlorothiazide (KD = 8 (±2) pM), use of complex structure coordinates reported herein have allowed: (i) the discovery of a novel compound, (ii) having an increment of one order of magnitude in potency.

The ability of compound (3) to enhance the thermal stability of NAPE-PLD (increase of melting temperature (ATm « 3-4°C) has been measured by thermal shift assays, in presence and absence of the ligand (Figure 2D). The structure of the complex has been characterized at atomic details by X-ray crystallography (PDB ID: 8P96, Figure 1 C).

The crystal structure of the complex has been achieved experimentally using a mixture of isomers of (1 ,3,6) naphthalene trisulfonic acid trisodium salt and (1 ,3,7) naphthalene trisulfonic acid trisodium salt (isomeric mixture = 80:20, respectively). The final crystal structure coordinates of the complex (PDB ID: 8P96) unveil that only the (1 ,3,7) isomer - and not the (1 ,3,6) -, binds to NAPE-PLD (Figure 1 C). This finding demonstrates that the methodology allows to distinguish and select different ligands for their predisposition to bind the target homodimer interface.

Therefore, it forms a further object of the present invention the use of crystal structures coordinates, images and representations as herein reported to identify, select, design, compounds that bind to the interfacial surface of NAPE-PLD.

As a proof-of-concept, described for the first time is also the discovery that the chemical entity (1 ,3,7)-naphthalene trisulfonic acid (N3SA), which binds to the “thiazide-bind site” of NAPE-PLD, proves to attenuate hypertension in animal model of spontaneous hypertensive rats (Figure 3), a pharmacological in vivo profile never shown before for the compound.

Therefore, it forms a further object of the present invention a composition comprising compound 3, or analogues compounds (Figure 4, compounds 4- 42), for use in the treatment of hypertension and related disorders. In a preferred embodiment, said analogues compounds are compounds 4, 6, 7, 17, 19, 20, 24, 25, 26, 30, 32, 34, 35, 40. In a preferred embodiment, said analogues compounds are compounds 4, 17, 34, 40.

Here, it has been demonstrated that effective NAPE-PLD ligand for the homodimer interface must preferably demonstrate a relatively small difference in energy between its bound and free states (e.g., a small deformation energy of binding). Thus, most efficient ligands or chemical entities should preferably be designed with a deformation energy of binding of not greater than about 10 kcal/mole, more preferably, not greater than 7 kcal/mol. NAPE-PLD homodimer interface ligands may interact with a binding pocket in more than one conformation, which is similar in overall binding free energy (e.g., from 4 to 12 kcal/mol). In those cases, the deformation energy of binding is taken to be the difference between the energy of the free chemical entity and the average energy of the conformations observed when the ligand or chemical entity binds to the protein.

The NAPE-PLD protein or its homologue may be produced by any well- known method, including synthetic methods, such as solid phase, liquid phase, and combination solid phase/liquid phase syntheses; recombinant DNA methods, including cloning, optionally combined with site directed mutagenesis; and/or purification of the natural products. In a preferred aspect, the protein is overexpressed in an E. coli system or mammalian cells.

In a further embodiment, it is here claimed a method to identify compounds capable to stabilise NAPE-PLD, wherein said method comprises, but it is not limited to:

- Making available the subunit interface on NAPE-PLD, wherein said subunit interface is defined as the volume of NAPE-PLD structure (Figure 1A) delimited by residues PHE150, SER151 , SER152, ARG153, ALA154, SER155, PRO156, SER157, GLN158, TYR159, MET160, GLY161 , PRO162, LYS163, ARG164, PHE165, ARG166, ARG167, SER168, PRO169, CYS170, THR171 , ILE172, SER173, GLU174, ASP192, TYR193, ASN194, SER195, VAL196, ILE197, ALA198, and GLU201 , wherein said numbering is referred to the crystal structure PDB ID: 4QN9.

- Selecting, within the subunit interface of NAPE-PLD, at least one residue in the group comprising SER151 , SER152, LYS163, ARG167, ILE172, whose interactions with selected atoms and compounds are useful to stabilize, promote, enhance, protract, the association of its protein subunits (Figure 1 ).

- Identifying compounds that bind to said subunit interface of NAPE-PLD joining structurally two NAPE-PLD subunits or reinforcing their association (Figure 1 ).

In an embodiment, said residues selected within the subunit interface of NAPE-PLD are PHE150, SER151 , SER152, ASN158, LYS163, ARG167, THR171 , ILE172, ASN194. These residues may belong to at least one of the dime subunits.

In an embodiment, said identified compounds are for use in the treatment of (i) hypertension and related diseases, including cardiovascular disease, hypervolemia, electrolyte disorders, kidney disease, diabetes, stone prevention, hypercalciuria and (ii) NAPE-PLD related diseases and vitamin B6- related disorders, including metabolic disorder, neuromuscular disease, neurodegeneration, multiple sclerosis, myelination disorders and white matter disease progression, epilepsy and autism spectrum disorders.

Advantageously, said method has proven useful in the identification of pyridoxal phosphate (PLP), 1 ,3,7-naphtalene trisulfonic acid (N3SA) as compounds that bind the interfacial surface of NAPE-PLD and stabilize, promote, enhance, protract, the association of its protein subunits, as reported herein. In an embodiment, the interactions of the compounds identified with the method according to the present invention are validated and quantified with methodologies known in the state of the art, comprising crystallography, cryo- electron microscopy, surface plasmon resonance, thermal shift assay, nuclear magnetic resonance, microcalorimetry, fluorescence spectroscopy, scintillation proximity assay, mutagenesis, enzymatic assays, and computational approaches, as reported herein.

In an embodiment, the compound-mediated stabilization of NAPE-PLD is validated and quantified with methodologies known in the state of the art, comprising crystallography, cryo-electron microscopy, surface plasmon resonance, thermal shift assay, nuclear magnetic resonance, microcalorimetry, fluorescence spectroscopy, scintillation proximity assay, mutagenesis, enzymatic assays, and computational approaches, as reported herein. In an embodiment, the pharmacological effect of the identified compounds is validated and quantified by means of appropriate in vivo methodologies (e.g., measurement of blood pressure) on animal models (e.g., spontaneous hypertensive rat, SHR), as reported herein.

Finally, the complex coordinates of the stabilised NAPE-PLD are claimed, said coordinates (PDB ID codes 8PC4, 8P90, 8P96) defining: (i) the atomic interactions involved between the NAPE-PLD subunit interface and the compounds HCTZ, PLP, N3SA, respectively, that promote the stabilization of NAPE-PLD (Figure 1 ); (ii) how the compound-mediated stabilization process of NAPE-PLD takes place and can be replicated by different compounds; (iii) the structure-activity relationships at the basis of the compound-mediated stabilization of NAPE-PLD, and how they can be optimized by different compounds to stabilize, promote, enhance, protract, the association of its protein subunits for therapeutic intervention, as reported herein.

The herein claimed coordinates of the complexes define at the same time the subunit interface of NAPE-PLD and the volume of the protein structure that can be occupied by a compound to stabilize efficaciously NAPE-PLD (Figure 1A-C).

The herein claimed coordinates of the complexes are immediately useful to identify, design, generate, different and novel compounds (e.g., by different approaches of structure-based drug discovery and design) that can replicate the stabilization of NAPE-PLD as HCTZ, PLP, N3SA, and are useful to stabilize, promote, enhance, protract, the association of its protein subunits for therapeutic intervention, as reported herein.

Using structural information of the complex coordinates (PDB ID codes 8PC4, 8P90, 8P96), structural analogues of compound (3) have been designed and evaluated in silico, estimating, and quantifying their potential ranking binding affinities for the thiazide-binding site of NAPE-PLD. Results for compounds listed in Figure 4 are reported in Figure 5, allowing to define the general Formula (I) above described.

Advantages

For the first time, described are allosteric chemical entities that are useful in stabilizing the structure of NAPE-PLD.

The stabilization of the structure of the protein target NAPE-PLD has been demonstrated using specific chemical entities (hydrochlorothiazide, pyridoxal phosphate, (1 ,3,7)-naphthalene trisulfonic acid) that bind to the interfacial surface of NAPE-PLD. Together with the use in clinic of hydrochlorothiazide, and the known antihypertensive effect in vivo of pyridoxal phosphate, the proof of concept of (1 ,3,7)-naphthalene trisulfonic acid confirms that targeting the interfacial surface of NAPE-PLD has therapeutic potential for thiazide-treated diseases, including hypertension, and associated cardiovascular diseases, stroke, heart disease, chronic kidney disease, and cognitive decline.

The shown stabilization of the NAPE-PLD protein by chemical entities through targeting its homodimeric interfacial surface indicates also that disfunction of the NAPE-PLD protein target and vitamin B6-related disorders can be evaluated in the treatment, as suggested by recently reported references. Among these, metabolic disorders, obesity, gastrointestinal disease, liver disease, epilepsy, neuromuscular disease, and neurodegeneration, multiple sclerosis, myelination disorders and white matter disease progression, can be mentioned.

The use of bile acid molecules to enhance the potency, action and pharmacological profile of chemical entities that bind to the homodimeric interface of NAPE-PLD has been validated and described in detail (Figure 2C, Figure 3B).

In addition, validated is a reproducible and useful approach to identify, search for, evaluate, design, and develop chemical entities that bind to the homodimer interface of NAPE-PLD for therapeutic application, starting from crystal structure coordinates.

Validated is also the innovative relationship between targeting the homodimer interface of NAPE-PLD with chemical entities and the therapeutic efficacy of these chemical entities.

Results reported herein show that NAPE-PLD is a previously unknown renal and extrarenal target of compounds having antihypertensive and vascular protective activity, a sought-after missing piece in the treatment of many human disorders (Hughes 2005 JRASS 5: 155-160).

The three-dimensional structure coordinates of NAPE-PLD in complex with different compounds (PDB IDs: 8PC4, 8P90, 8P96) and the reported images, representations and methodologies are useful to accelerate and facilitate the drug discovery process, and almost essential for drug approval. The approach and structure complex coordinates reported herein will be integral for developing and approval of novel and more efficacious therapeutic agents for the management of vascular risk factors.

Protein binding pockets, also referred to as binding sites in the present invention, are of significant utility in fields such as drug discovery. The association of natural ligands or substrates with the binding pockets of their corresponding receptors or enzymes is the basis of many biological mechanisms of action. Similarly, many drugs exert their biological effects through association with the binding pockets of receptors and enzymes. Such associations may occur with all or part of the binding pocket. An understanding of such associations helps to design novel or modified compounds having more favourable interactions with their protein targets receptor, and thus, having improved biological effects. Therefore, this information is valuable in designing novel potential ligands of the binding pockets of biologically important targets. The homodimer interface of NAPE-PLD (or “thiazide-binding site”) of this invention may be important for the entire drug discovery and development process, as well as to progress promising candidates to clinical studies. Those of skill in the art understand that a set of structure coordinates for a molecule or a molecular complex or a portion thereof, is a relative set of points that define a shape in three dimensions. Thus, it is possible that an entirely different set of coordinates could define a similar or identical shape. Moreover, slight variations in the individual coordinates will have little effect on overall shape. In terms of binding pockets, these variations would not be expected to significantly alter the nature of ligands that could associate with those pockets.

The variations in coordinates discussed above may be generated because of mathematical manipulations of the NAPE-PLD structure coordinates.

Alternatively, modifications in the crystal structure due to mutations, additions, substitutions, and/or deletions of amino acids, or other changes in any of the components that make up the crystal could also account for variations in structure coordinates. If such variations are within a certain root mean square deviation as compared to the original coordinates, the resulting three-dimensional shape is considered encompassed by this invention. Thus, for example, a ligand that binds to the homodimer interface of NAPE-PLD would also be expected to bind to another binding pocket whose structure coordinates define a shape that falls within the acceptable root mean square deviation.

The reported coordinates (PDB IDs: 8PC4, 8P90, 8P96) and process are useful to identify and develop novel chemical entities that bind to the homodimer interface of NAPE-PLD for therapeutic application (e.g., naphthalene trisu Ifonic acid).

This finding in vivo confirms (i) the usefulness of the reported approach to discover chemical entities for therapeutic intervention, and (ii) the direct and innovative relationship between targeting the thiazide-binding site of NAPE- PLD and therapeutic application.

Examples

Protein cloning, expression, and purification.

The region encoding a truncated protein form of human NAPE-PLD (from amino acid D47 to F393) was amplified using polymerase-chain reaction (PCR) methodologies. The resulting vector harbouring the full-length gene was transformed into E. coli (e.g., using Rosetta gamiB (DE3) pLysS cells, Novagen) to produce the encoded recombinant protein with fusion partners and/or tags (e.g., N-terminus maltose binding protein (MBP) tag and/or a N- terminus or C-terminus hexahistidine tag). The expressed protein samples were purified by affinity chromatography and, after tag-cleavage, by one or multiple size exclusion chromatography elutions (e.g., using a buffer 20-200 mM TRIS or HEPES (pH 7.0-9.0), 50-250 mM NaCI, in the presence (0.01 - 0.25%) or absence of deoxycholic acid (DCA).

X-ray structure determination

The protein samples purified according to the preceding paragraph were used for x-ray structure determination of the protein in complex with ligands (ID codes 8PC4, 8P90, 8P96), for direct crystallization screenings, and for binding assays (e.g., surface plasmon resonance or thermal shift analysis). Chemical entities

All compounds used for x-ray structure determination and in vitro and in vivo analysis were from Merck (Sigma). The compound naphthalene trisulfonic acid was an isomeric mixture of (1 ,3,6) and (1 ,3,7)-naphthalene trisulfonic acid (~ 80:20, respectively).

Crystallization and structure determination

For crystallization, the purified protein was concentrated in the elution buffer to centrifugal filter devices (e.g., up to 20-60 mg/mL). To obtain the protein in complex with the selected ligand (e.g., hydrochlorothiazide, or pyridoxal phosphate, or naphthalene trisulfonic acid), the chemical entity was added to the concentrated NAPE-PLD protein solution or directly to the soaking solution, up to a final concentration of 0.1 -10 mM. The selected ligand could also be dissolved previously in solution containing DMSO.

Crystals were obtained at room temperature (e.g., 20-25°C) by vapor diffusion, using a buffer of 0.8-1 .2 M lithium sulphate (LiSO ) and 20-200 mM TRIS or HEPES [pH 7.0-9.0], in the presence or absence of 1 -5% ethanol. Before x-ray data collection at cryogenic temperature (100 K), crystals were cryoprotected in mother liquor containing glycerol (e.g., 25-30% w/v) and the selected ligand (0.1 -10 mM), and subsequently flash-cooled in liquid nitrogen.

Data collections and analysis

Data collections were acquired as images (from 0.1 ° to 1.0°) on the DECTRIS PILATUS 6M detector at the beamline XRD2 of the synchrotron light source ELETTRA (Trieste, Italy). Diffraction images were indexed, integrated, and scaled using the CCP4i programs package and the XDS programs package. The phase problem was solved by the method of molecular replacement using the PHENIX programs package, starting from the atomic coordinates of the native protein structure having PDB ID accession number: 4QN9 (https://www.rcsb.org). The structures of the ligand complexes were refined by cycles of automatic and manual building and analysed using COOT (Emsley et al., 2010) and the PHENIX programs package. The figures of the complex’s coordinates were prepared with the software PyMol (www.pymol.org). Binding kinetics by Surface Plasmon Resonance

Kinetics parameters for the interaction between the protein NAPE-PLD (or single point protein mutants) and the selected ligands were measured by the label-free methodology of surface plasmon resonance (SPR) at room temperature (20 °C), using the Biosensor SensiQ Pioneer (ForteBio). For the immobilization of the target on the SPR chip, the protein sample was minimally biotinylated on ice for 3h using an equimolar concentration of sulfo-NHS-LC- biotin and purified by gel filtration chromatography (buffer 20 mM TRIS pH 8.0, 200 mM NaCI) to remove the excess of free biotin. Streptavidin was immobilized onto a COOH5 sensor chip using a standard amine-coupling method and Hepes buffered saline (HBS) as the running buffer. This coupling method resulted in a density ~10,000 RU (resonance units) of neutravidin on all flow cells. Biotinylated NAPE-PLD was then captured to densities of ~2000 RU.

SPR assays were performed using the conventional method of multiple standard fixed concentration injections, or the method of one-step injections. In the standard method, the protein was titrated using concentrations spanning from 500 pM to 250 nM before injection into the SPR system, at a flow rate spanning from 20 to 30 pL/min. The spr profiles were used to calculate the KD of each analyte, from kinetic fitting of multiple injections at different concentration (Figure 2A). In the one-step injection method, the spr profile of a single injection in automatic concentration gradient (from 250 pM to 0 nM) was used for the kinetic fitting to calculate the KD of each analyte (Figure 2B, 2C). Experiments and analyses were performed normally from 3 to 5 replicates, and at least in duplicate. The response data were processed with the analytic program (Qdat) of the SPR system, using a reference surface to correct for any bulk refractive index changes, and multiple blank injections for double referencing. The binding profiles were fit globally to 1 :1 or 1 :2 interaction model, to determine the kinetic parameters of the interaction - association rate constant K_a = K_on (M^-1 s^-1 ), dissociation rate constant K_d = K_off (s^-1), equilibrium dissociation constant KD (M).

The interaction of the selected compounds that can bind to the homodimer interface of NAPE-PLD protein was validated experimentally using approaches of single point mutagenesis (e.g., using spr assays with the protein mutant Seri 52lle), and confirmed by x-ray diffraction.

Thermal shift assay

Fluorescence thermal shift (TS) assays were carried out using a ViiA™ 7 Real-Time PCR System. 18pL of the protein solution of NAPE-PLD (0.25 mg/mL), in the absence or presence of the ligand (0.1 mM), were mixed with 2pL of Protein Thermal Shift Dye (Life technologies) diluted 1 :100 in buffer 20 mM TRIS pH 8.0, 200 mM NaCI, 0.1 % DCA. Samples were denatured by heat (from 50 to 99°C) at a rate of 0.2°C per minute. Protein thermal unfolding curves were monitored by detecting changes in dye fluorescence (Figure 2D). The inflection point of the fluorescence-versus-temperature curves was identified by plotting the first derivative over the temperature, and the minima were referred to as the melting temperature (Tm). Buffer fluorescence was used as control.

Structure-based screening and design in silico

To identify potential chemical entities able to bind to the homodimer interface of NAPE-PLD protein, docking calculations were performed using conventional methodologies with AutoDock Vina, with all parameters kept at default values. The compound libraries used for docking were selected compounds of different chemical libraries, including that of the Istituto Italiano di Tecnologia, of FDA-approved and EMA-approved drugs, and of natural compounds. Two-dimensional compound coordinates were downloaded (or generated) and converted to three-dimensional coordinates using Open Babel. A region at the homodimer interface of NAPE-PLD protein structure was defined as the docking site using a box extended of 35 A (corresponding to the distance of the protein channel from Lys97 to Glu201 ) x 26 A (corresponding to the distance from Gln158 to Lys108) x 15 A (corresponding to the distance from Pro169 of one subunit to Pro169 of the other subunit). Compounds were scored and ranked based on their steric and electrostatic interactions with the homodimer interface of NAPE-PLD. The results in silico provided a binding score or a binding energy (expressed in kJ/mol or kcal/mol with smaller, more negative energies being more favourable). Most promising compounds were tested with bioassays. Validated compounds were used to search, design and optimize compounds to increase potency, thus affinity for the NAPE-PLD subunits interface.

The interaction of the selected in silico compounds that can bind to the homodimer interface of NAPE-PLD protein was validated experimentally using approaches of single point mutagenesis coupled with binding assays (SPR and TS) (e.g., using spr assays with the use of the protein mutant Seri 52lle), and possibly proved directly by crystallography.

Example of the process

The complex structure between NAPE-PLD and the homodimer interface- binding drug hydrochlorothiazide was discovered by screening directly selected approved drugs using x-ray crystallography. The complex structure coordinates showed the key role of NAPE-PLD residues Ser151 , Ser52, Lys163, Ile172 and Asp 192 for the interaction of the ligand (Figure 1 A). The coordinates of the complex structure of NAPE-PLD with hydrochlorothiazide were used to discover other chemical entities that can bind the target protein, similarly, based on the interactions of the molecule with the target dimeric interface (Figure 1A). This step was performed iteratively using in silico and in vitro assays (above).

Among the compounds selected with this methodology, the natural ligand pyridoxal phosphate demonstrated experimentally to bind the homodimer interface of NAPE-PLD in the low pM range (Figure 1 B). This compound is the active form of vitamin B6, and it is known to attenuate hypertension in vivo, as the compound hydrochlorothiazide.

The compound (3,7) naphthalene disulfonic acid, was identified by screening in silico. The obtained computational complex was used to design chemical analogues from information derived from the NAPE-PLD complex structure coordinates. The designed analogues are listed in Figure 4. Binding energies obtained by virtual screening are reported for each one of said compounds in the graph in Figure 5. This process allowed the identification of the compounds (3), (4), (17), (34), (40) which showed a binding energy higher than -6 kcal/mol.

Compound (3) was then tested in vitro, showing a potency in the nM range (Figure 2B) and proved to bind the homodimer interface of NAPE-PLD, as verified by X-ray crystallography (Figure 1 C).

Finally, the compound (1 ,3,7)-naphthalene trisulfonic acid was used in vivo on an animal model of hypertension (Spontaneous Hypertensive Rats, SHR), demonstrating its previously unknown potent anti-hypertensive action (Figure 3), and the innovative correlation between targeting the homodimer interface of NAPE-PLD and therapeutic application (below).

Synergistic combination

The relative positions of the thiazide-binding site and BA-binding site in the crystal structure coordinates indicate a cooperative binding to NAPE-PLD of compounds that stabilized allosterically the target. We measured by SPR the binding affinity of the drug hydrochlorothiazide (1 ) in the presence of different concentrations of deoxycholic acid (gradient of concentrations spanning from 0 pM to 250 pM) using “onestep” injections (SPR instrument Pioneer) (Figure 3B). This analysis shows that the equilibrium dissociation constant of the drug, from a value of KD = 8 (±2) pM in the absence of the bile acid, increased up to KD = 0.9 (±0.4) pM, thus with an increase in potency of about one order of magnitude. The analysis using the “classic” SPR method, with different concentrations of the analyte - e.g., from 0.01 pM to 100 pM - at fixed concentrations of the bile acid - e.g., 10 pM -, or with fixed concentrations of the analyte - e.g., 10 pM - at different concentrations of the bile acid - e.g., from 0.1 pM to 100 pM - provides similar results (Figure 3C).

Analogously, the binding kinetics of the compound pyridoxal phosphate (2), in the presence of different concentrations of deoxycholic acid (from 0 to 250 pM) gives an equilibrium dissociation constant that from a value of KD = 32.7 (±0.1 ) pM gets up to KD = 3.6 (±0.5) pM, about one order of gain in magnitude. Furthermore, the potency of naphthalene trisulfonic acid (3) from a value of KD = 730 (±20 nM) increases up to KD = 195 (±63) nM in presence of the bile acid (Fig. 3B and 3C).

Results in vivo using a test composition of the compound naphthalene trisulfonic acid and deoxycholic acid demonstrate a positive change of the anti- hypertensive profiles in SHR rats (Figure 3B), in line with the results measured in vitro by SPR.

Therapeutic potential on animal model

Fifteen male spontaneous hypertensive rats (SHR) from Charles River (250-350 g) were used at different ages, of 8 weeks (Figure 3A) and 14 weeks (Figure 3B) and studied in groups (n=3-4). Body weight and blood pressure were measured daily before any treatment. All rats were housed in polyethylene cages, under a 12/12 h light/dark cycle (light 8:00-20:00 h) at constant temperature (24 ± 1 °C) and humidity (60 ± 5%), with free access to food and water. The compounds used, naphthalene trisulfonic acid (1 and 2 mg/kg/d) or deoxycholic acid (2.5 mg/kg/d), were dissolved in phosphate buffer (pH 7.4) and administered via syringe-feeding method. The pressure of each rat was determined at 1 to 3-day intervals by means of the microphonic method of Friedman and Freed, without anaesthesia. Blood pressure data were summarized as mean ± SD and plotted as mean ± standard error of the mean (SEM).

This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institute of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Pisa and Ministry of Health (Permit Number: 157/2017-PR).

Claims

1. Compounds of Formula (I) or pharmacologically acceptable derivatives thereof, wherein

A is C, N or S;

R³ is H, S, CH₃;

R⁴ is H,

R⁵ is SO₃H, PO₃H₂, SO₂NH₂, SO₂CH₂OH, or R⁴ and R⁵ close to form a cycle which is for use in the treatment of NAPE-PLD related diseases selected from the group comprising: hypertension, including cardiovascular disease, hypervolemia, electrolyte disorders, kidney disease, diabetes, stone prevention, hypercalciuria, metabolic disorder, neuromuscular disease, neurodegeneration, multiple sclerosis, myelination disorders and white matter disease progression, epilepsy, and autism spectrum disorders.

2. A composition comprising a bile acid and a compound that bind to homodimer interface of NAPE-PLD for use in the treatment of NAPE- PLD related diseases selected from the group comprising: hypertension, including cardiovascular disease, hypervolemia, electrolyte disorders, kidney disease, diabetes, stone prevention, hypercalciuria, metabolic disorder, neuromuscular disease, neurodegeneration, multiple sclerosis, myelination disorders and white matter disease progression, epilepsy, and autism spectrum disorders.

3. The compounds for use according to claim 1 , wherein said compounds are compounds of Formula (I), or pharmacologically acceptable derivatives thereof, wherein

A is C, N or S;

R² is SO₃H, SO₂NH₂, PO₃H₂, halogen, COOH, CONH₂, C1 -C4 alkyl, C1 -C4 alkyl halogen substituted, or optionally substituted NHAr;

R³ is H, S;

R⁴ is H,

R⁵ is SO₃H, PO₃H₂, SO₃H, PO₃H₂, SO₂NH₂, SO₂CH₂OH, or R⁴ and R⁵ close to form a cycle which is

4. The compounds for use according to claim 1 , wherein said compounds are compounds of Formula (I), or pharmacologically acceptable derivatives thereof, wherein A is C, N or S;

R¹ is SO₃H, SO₂NH₂, or PO₃H₂;

R² is SO₃H, SO₂NH₂, PO₃H₂, CF3, or Cl;

R³ is H, S;

R⁴ is H;

R⁵ is SO₃H, SO₂NH₂, PO₃H₂.

5. The compounds for use according to claim 1 , wherein the compounds are selected in the group comprising or consisting of:

6. The compounds for use according to claim 1 , wherein the compound is (1 ,3,7)-naphthalene trisulfonic acid, N3SA, compound (3).

7. The compounds for use according to claim 1 or claim 2, wherein said disease is hypertension.

8. A method to identify compounds capable to stabilise NAPE-PLD, wherein said method comprises:

- Making available the subunit interface on NAPE-PLD, wherein said subunit interface is defined as the volume of NAPE-PLD structure delimited by residues PHE150, SER151 , SER152, ARG153, ALA154, SER155, PRO156, SER157, GLN158, TYR159, MET160, GLY161 , PRO162, LYS163, ARG164, PHE165, ARG166, ARG167, SER168, PRO169, CYS170, THR171 , ILE172, SER173, GLU174, ASP192, TYR193, ASN194, SER195, VAL196, ILE197, ALA198, and GLU201 , wherein said numbering is referred to the crystal structure PDB ID: 4QN9;

- Selecting, within said subunit interface of NAPE-PLD, at least one residue in the group comprising SER151 , SER152, LYS163, ARG167, ILE172, whose interactions with selected atoms and compounds are useful to stabilize, promote, enhance, protract, the association of its protein subunits;

- Identifying compounds that bind to said subunit interface of NAPE-PLD.

9. The method according to claim 8, wherein said selected residues on the subunit interface of NAPE-PLD are PHE150, SER151 , SER152, ASN158, LYS163, ARG167, THR171 , ILE172, ASN194.

10. The complex coordinates of the stabilised NAPE-PLD, being defined by PDB ID codes 8PC4 (NAPE-PLD in complex with HCTZ), 8P90 (NAPE- PLD in complex with PLP), 8P96 (NAPE-PLD in complex with N3SA), including images and graphical or virtual representations of these coordinates and of NAPE-PLD homologues, for use in the identification, design and development of compounds that bind to the dimer interface of the protein NAPE-PLD.