Patent Application
20220370482
The invention relates to a compound for use in the treatment of nephrotic syndrome, particularly a compound which is an inhibitor of binding between a target protein and keratin 8. The invention also relates to a kit comprising the compound, a method of treatment using the compound, and use of the compound in a method of manufacturing a medicament.
Nephrotic syndrome (NS) is described as a triad of heavy proteinuria (>40 mg/m2/hr), oedema and hypoalbuminaemia (<3.0 g/dL), and often results in end-stage renal disease (ESRD), accounting for 15% of the cases in the European population. Even though NS is linked to various types of renal disease, the most common form (90%) identified in children is idiopathic NS, which progresses in the absence of any clinical features of primary extrarenal disorder (T.-S. Ha, Korean J. Pediatr., vol. 60, no. 3, p. 55, 2017).
Clinically, NS can be classified into two groups by the response to steroid therapy: steroid-sensitive NS (SSNS) and steroid-resistant NS (SRNS). Most patients with idiopathic NS initially respond well to steroids and enter the remission stage with a good renal prognosis; however, around 90% of these patients will relapse, with another half of those becoming steroid-dependent. The remaining 10% are recognized to have SRNS. However, this fails to explain why some patients who originally respond to steroid treatment later become resistant. NS is considered to be the most prevalent glomerular disease of childhood, with an incidence of around 2 in 100,000 children. Approximately 20% of children will be steroid resistant, with a further 60% of these cases presenting with focal segmental glomerulosclerosis (FSGS), indicated by biopsy (M. A. Saleem, Pediatr. Nephrol., vol. 28, no. 5, pp. 699-709, May 2013.). FSGS is described as scarring of the glomerulus that includes several distinct changes, where only a segment of the glomerulus and some, but not all, glomeruli are affected. Progression to ESRD is closely correlated with the development of FSGS. Therefore, because most cases of genetic NS are clinically steroid-resistant with pathologically prevalent FSGS, genetic NS is hard to treat, has poor renal prognosis and often results in ESRD.
The majority of the SRNS genetic forms show structural alterations in the glomerulus or, more precisely, the podocyte. Molecular research work on the genetics of hereditary NS has revealed the podocyte as a key player in regulating glomerular filtration, whose structure and function are essential in the maintenance of the slit diaphragm membrane. To date, around 75 genes including those encoding nephrin, transient receptor potential canonical channel-6 (TRPC6), CD2AP, α-actinin-4 and podocin, have been reported to cause NS, and yet more remain to be identified. The significance of several of them, including nephrin, CD2AP and podocin, which participate in the slit diaphragm assembly, has been shown by the presence of heavy proteinuria, when they are mutated (M.-C. Gubler, J. Am. Soc. Nephrol., vol. 14, no. 90001, p. 22S-26, Jun. 2003).
One such protein, podocin, is the protein product of the NPHS2 gene that is mutated in a subset of patients with autosomal recessive SRNS, which manifests as early childhood onset of proteinuria, fast progression to ESRN and FSGS. Podocin is a novel 42 kDa podocyte specific integral membrane protein, and is a member of the stomatin family of proteins. In podocytes, podocin is exclusively localized to the slit diaphragm, where it is involved in mechanotransduction events (T. B. Huber et al., J. Biol. Chem., vol. 276, no. 45, pp. 41543— 6, Nov. 2001). This crucial localization of podocin to the slit diaphragm is underscored by the fact that mice lacking podocin present with massive proteinuria, foot process effacement and absence of slit diaphragms. Multiple experimental studies show that podocin in its oligomeric form localizes to lipid raft microdomains in the slit diaphragm, where it recruits and colocalizes with nephrin. As mentioned previously, the slit diaphragm of podocytes is essential for the correct function of the GFB and is assembled in lipid rafts.
Paediatric patients carrying truncating mutations of podocin (nonsense or frameshift), or homozygous p.R138Q, present with a severe, early-onset from of SRNS, which is found in 98.2% of cases studied (B. Hinkes et al., J. Am. Soc. Nephrol., vol. 19, no. 2, pp. 365-71, Feb. 2008). The replacement of the arginine residue at position 138, which is highly conserved amongst the stomatin-like family of proteins and is crucial for podocin function, with glutamine results in podocin with the R138Q mutation (N. Boute et al., Nat. Genet., vol. 24, no. 4, pp. 349-54, Apr. 2000). Functional data have shown that this protein is retained in the endoplasmic reticulum (ER) and loses its ability to target nephrin to lipid raft microdomains, therefore augmenting its signaling (T. B. Huber et al., Hum. Mol. Genet., vol. 12, no. 24, pp. 3397-405, December 2003.
As set out above, genetic NS is hard to treat, has poor renal prognosis and often results in ESRD. There is therefore a significant unmet need in provision of a therapeutic for treatment of NS.
The present invention provides a compound for use in the treatment of nephrotic syndrome in a subject in need thereof, wherein the compound is an inhibitor of binding between a target protein and keratin 8.
The present invention also provides a compound for use in the treatment of nephrotic syndrome in a subject in need thereof, wherein the compound is a compound of formula I, or a pharmaceutically acceptable salt thereof:
wherein x is 0 or an integer from 1 to 6;
Y is selected from the group consisting of: direct bond, C(O), C(O)O, 0, C(R1)(OH), C(O)NR1, S(O), S(O)(O) and P(O)(OH),
Z is selected from the group consisting of: C(0), C(O)O, 0, C(R′)(OH), C(O)NRI, S(0), S(0)(0) and P(0)(OR1);
wherein R1 is selected from the group consisting of: hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl and optionally substituted heterocyclyl;
R2 is selected from the group consisting of: hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl and optionally substituted heterocyclyl; and
R3 is selected from the group consisting of: hydrogen, OH, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl, optionally substituted alkoxy, COOH, optionally substituted carboxylate ester, optionally substituted amide, optionally substituted amine, optionally substituted ether, phosphinic acid and phosphinate ester.
The present invention also provides a compound for use in the treatment of nephrotic syndrome in a subject in need thereof, wherein the compound is a compound of formula II, or a pharmaceutically acceptable salt thereof:
wherein R1 is selected from the group consisting of: hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl and optionally substituted heterocyclyl;
R2 is selected from the group consisting of: hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl and optionally substituted heterocyclyl;
x is 0 or an integer from 1 to 6;
Y is selected from the group consisting of: direct bond, C(0), S(0), C(O)O, C(O)NH, and P(O)(OH); and
R3 is selected from the group consisting of: hydrogen, OH, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl, optionally substituted alkoxy, COOH, optionally substituted carboxylate ester, optionally substituted amide, optionally substituted amine, optionally substituted ether, phosphinic acid and phosphinate ester.
The present invention also provides a kit comprising a compound which is an inhibitor of binding between a target protein and keratin 8 together with instructions for treating nephrotic syndrome.
The present invention also provides a method of treating nephrotic syndrome in a subject in need thereof comprising administering to said subject an effective amount of a compound which is an inhibitor of binding between a target protein and keratin 8.
The present invention also provides the use of a compound which is an inhibitor of binding between a target protein and keratin 8 in the manufacture of a medicament for the treatment of nephrotic syndrome in a subject in need thereof.
One or more embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:
The term “pharmaceutically acceptable salt” used herein refers to a salt of the compounds described herein which is generally safe, non-toxic and neither biologically nor otherwise undesirable and includes that which is suitable for veterinary use as well as human pharmaceutical use. Examples of pharmaceutically acceptable salts are discussed in Berge et al (J. Pharm. Sci., 1977, 66, 1-19). Pharmaceutically acceptable salt forms include pharmaceutically acceptable acidic/anionic or basic/cationic salts.
Such salts include acid addition salts formed with inorganic acids, or with organic acids. Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Generally, such salts are, for example, prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of the two. Generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. Examples of the acid addition salts include acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glyceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, malonate, mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate, pamoate, pantothenate, phosphate/diphospate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, hydrogensulfate, tannate, tartrate, teoclate, tosylate, methanesulfonate and p-toluenesulfonate and triethiodide salts. Examples of the alkali addition salts include inorganic salts such as, for example, sodium, potassium, calcium and magnesium, and organic alkali salts such as, for example, ethylenediamine, ethanolamine, N,N-dialkylenethanolamine, triethanolamine and basic amino acids salts. Examples of suitable organic cations include ammonium ion (i.e., NH4+) and substituted ammonium ions (e.g. NH3R+, NH2R2+, NHR3+, NR4+, where R is an alkyl group).
If the compound has both a cationic functional group, or a functional group that can become cationic, and an anionic functional group, or a functional group that can become anionic, then the compound may be present as a zwitterion.
The term “hydrogen” or “hydrogen atom” as used herein refers to a —H moiety.
The term “halo”, “halogen” or “halogen atom” as used herein refers to a —F,-C1, —Br or —I moiety.
The term “hydroxy” as used herein refers to an —OH moiety.
The term “alkyl” as used herein refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 12 carbon atoms (unless otherwise specified), which may be aliphatic or alicyclic, which may be saturated or unsaturated (e.g. partially unsaturated or fully unsaturated), and which may be linear or branched. Thus, the term “alkyl” includes the sub-classes alkenyl, alkynyl, cycloalkyl, cycloalkenyl and cylcoalkynyl below.
In the context of alkyl groups, the prefix C1-12 denotes the number of carbon atoms, or range of number of carbon atoms present in that group. Thus, the term “C1-12 alkyl” refers to an alkyl group having from 1 to 12 carbon atoms. The first prefix may vary according to the nature of the alkyl group. Thus, if the alkyl group is an alkenyl or alkynyl group, then the first prefix must be at least 2 (e.g. C2-12). For cyclic (e.g. cycloalkyl, cycloalkenyl, cylcoalkynyl) or branched alkyl groups, the first prefix must be at least 3 (e.g. C3-12).
Examples of saturated alkyl groups include methyl (C1), ethyl (C2), propyl (C3), butyl (C4), pentyl (C5), hexyl (C6), heptyl (C7), octyl (C8), nonyl (C9) and decyl (C10). Examples of saturated linear alkyl groups include, but are not limited to, methyl (CO, ethyl (C2), n-propyl (C3), n-butyl (C4), n-pentyl (amyl) (C5), n-hexyl (C6), and n-heptyl (C7). Examples of saturated branched alkyl groups include iso-propyl (C3), iso-butyl (C4), sec-butyl (C4), tert-butyl (C4), iso-pentyl (Cs), and neo-pentyl (C5).
The term “alkenyl” refers to an alkyl group having one or more carbon-carbon double bonds. Examples of unsaturated alkenyl groups include ethenyl (vinyl, —CH═CH2), 1-propenyl (—CH═CH—CH3) and 2-propenyl (allyl,-CH-CH═CH2).
The term “alkynyl” refers to an alkyl group having one or more carbon-carbon triple bonds. Examples of unsaturated alkynyl groups include, but are not limited to, ethynyl (ethinyl,-CECH) and 2-propynyl (propargyl, —CH2—C≡CH).
The term “cycloalkyl” refers an alkyl group which is also a cyclyl group; that is, a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a carbocyclic compound (i.e. a compound where all of the ring atoms are carbon atoms). The ring may be saturated or unsaturated (e.g. partially unsaturated or fully unsaturated), which moiety has from 3 to 12 carbon atoms (unless otherwise specified). Thus, the term “cycloalkyl” includes the sub-classes cycloalkenyl and cycloalkynyl. In an embodiment, each ring has from 3 to 7 ring carbon atoms. Examples of cycloalkyl groups include those derived from (i) saturated monocyclic hydrocarbon compounds: cyclopropane (C3), cyclobutane (C4), cyclopentane (C5), cyclohexane (C6), cycloheptane (C7) and methylcyclopropane (C4); (ii) unsaturated monocyclic hydrocarbon compounds: cyclopropene (C3), cyclobutene (C4), cyclopentene (C5), cyclohexene (C6), methylcyclopropene (C4) and dimethylcyclopropene (C5); (iii) saturated polycyclic hydrocarbon compounds: thujane (C10), carane (C10), pinane (C10), bornane (C10), norcarane (C7), norpinane (C7), norbornane (C7), adamantane (C10), decalin (C10); (iv) unsaturated polycyclic hydrocarbon compounds: camphene (C10), limonene (C10), pinene (C10); and (v) polycyclic hydrocarbon compounds having an aromatic ring: indene (C9), indane (C9) and tetraline (C10).
In an embodiment, a reference to an alkyl group described herein is a C1-12 alkyl group, such as a C1-8 alkyl group, for example a C1-6 alkyl group, or a C1-4 alkyl group. The alkyl groups in the invention can be saturated alkyl groups or saturated cycloalkyl groups, for example saturated, unbranched alkyl groups.
The phrase “optionally substituted” as used herein refers to a parent group which may be unsubstituted or which may be substituted with one or more, for example one or two, substituents. The substituents on an “optionally substituted” group may for example be selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclyl groups; carboxylic acids and carboxylate ions; carboxylate esters; carbamates; alkoxyl groups; ketone and aldehyde groups; amine and amide groups; —OH; —CN; —NO2; and halogens.
The term “substituents” is used herein in the conventional sense and refers to a chemical moiety, which is covalently attached to, or if appropriate, fused to, a parent group.
In some embodiments, substituents can themselves be substituted. For example, a C1-12alkyl group may be substituted with, for example, hydroxy (referred to as a hydroxy-C1-12alkyl group) or a halogen atom (referred to as a halo-C1-12 alkyl group), and a C1-12alkoxy group may be substituted with, for example, a halogen atom (referred to as a halo-C1-12alkoxy group).
The term “aryl” as used herein refers to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 6 to 10 ring carbon atoms (unless otherwise specified). In an embodiment, the aryl group is a phenyl group.
The term “heteroaryl” as used herein refers to a monovalent moiety obtained by removing a hydrogen atom from a heteroaromatic compound, which moiety may for example be a monocyclic or bicyclic group. The heteroaryl moiety may contain from 1 to 12 carbon atoms (unless otherwise specified) and one or more N, O or S atoms. The heteroaryl moiety may be a 5 or 6-membered ring containing one or more N atoms.
The term “heterocyclyl” as used herein refers to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which moiety may for example be a monocyclic or bicyclic group. The heterocyclyl group may contain from 1 to 12 carbon atoms (unless otherwise specified) and one or more N, O or S atoms.
The term “alkoxy” used herein refers to an alkyl-oxy group, where the alkyl group is as defined above and has from 1 to 12 carbon atoms (unless otherwise specified). In an embodiment, the alkyl moiety in an alkoxy group is a saturated alkyl group or a saturated cycloalkyl group. In an embodiment, the alkyl moiety is a saturated, unbranched alkyl group. Examples of C1-12 alkoxy groups include —OMe (methoxy), —OEt (ethoxy), —O(nPr) (n-propoxy), —O(iPr) (isopropoxy), —O(nBu) (n-butoxy), —O(sBu) (sec-butoxy), —O(iBu) (isobutoxy), and —O(tBu) (tert-butoxy).
The term “phosphinic acid moiety” used herein refers to a functional group containing a P(O)OH group.
The term “phosphinate ester moiety” used herein refers to an ester of a phosphinic acid moiety, i.e. a phosphinic acid moiety wherein the hydrogen of the acid (P(0)OH) group has been replaced by an organic substituent, for example an alkyl, cycloalkyl, aryl, heteroaryl, alkenyl or alkynyl group (e.g. an alkyl group).
Where present, the functional groups C(O)O and C(O)NR can be found in either orientation. In other words, C(O)O represents —C(O)O— and —OC(O)—; and C(O)NR represents —C(O)NR— and —NRC(0)—.
Certain compounds may exist in one or more particular geometric, enantiomeric, diasteriomeric, tautomeric, or conformational forms. Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof. Methods for the preparation and separation of such isomeric forms are either known in the art.
The term “subject” used herein includes humans, non-human animals (e.g. dogs, cats, rabbits, cattle, horses, sheep, goats, swine, deer and the like) and non-mammals (e.g. birds and the like).
Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, mean “including but not limited to”, and do not exclude other components, integers or steps. Moreover the singular encompasses the plural unless the context otherwise requires: in particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
Preferred features of each aspect of the invention may be as described in connection with any of the other aspects. Within the scope of this application it is expressly intended that the various aspects, embodiments, examples and alternatives set out in the preceding paragraphs, in the claims and/or in the following description and drawings, and in particular the individual features thereof, may be taken independently or in any combination. That is, all embodiments and/or features of any embodiment can be combined in any way and/or combination, unless such features are incompatible.
The present invention provides a compound for use in the treatment of nephrotic syndrome in a subject in need thereof. Preferably, the present invention provides a compound for use in the treatment of nephrotic syndrome in a subject in need thereof, wherein the compound is an inhibitor of binding between a target protein and keratin 8.
Without wishing to be bound by theory, the inventors have identified the mechanisms in podocyte cells whereby certain proteins involved in nephrotic syndrome are prevented from reaching their target site. These mechanisms involve binding by keratin 8, which leads to retention of the protein-keratin 8 complex in the podocyte endoplasmic reticulum and ultimately proteasomal degradation chaperoned by keratin 8. The inventors have further identified that the binding of these proteins by keratin 8 can be disrupted by inhibitors, leading to restoration of these proteins at their podocyte target sites and ultimately the restoration of kidney function. These compounds represent a first in class opportunity for the treatment of nephrotic syndrome, and in particular offer a real opportunity for the treatment of steroid resistant nephrotic syndrome.
Preferably, the target protein is podocin. Preferably, the target protein is mutated podocin. The mutated podocin may have a truncating or a missense mutation. The truncating mutation may be nonsense or frameshift. Preferably the mutation is one which causes the podocin to bind keratin 8 (e.g. by causing a hydrophobic patch within the podocin which binds keratin 8). An example of such a mutation is the R138Q mutation. The target protein is, therefore, preferably podocin with the R138Q mutation (also described herein as “R138Q podocin”). The podocin may preferably be homozygous p.R138Q podocin.
The compounds described herein may be used in the treatment of nephrotic syndrome (NS). The nephrotic syndrome is preferably steroid-resistant nephrotic syndrome (SRNS). The nephrotic syndrome is preferably genetic nephrotic syndrome (e.g. SRNS with pathologically prevalent focal segmental glomerulosclerosis (FSGS)). The genetic nephrotic syndrome may preferably be autosomal recessive SRNS.
Preferably, the nephrotic syndrome (NS) is NS associated with (e.g. caused by) a podocin mutation. In particular, the NS is NS associated with (e.g. caused by) a mutation that causes binding with keratin 8. More preferably, the NS is NS associated with (e.g. caused by) an R138Q podocin mutation.
Preferably, the compound comprises at least one moiety selected from phosphinic acid moieties, phosphinate ester moieties and pharmaceutically acceptable salts thereof. It has surprisingly been found that compounds comprising at least one phosphinic acid moiety and/or phosphinate ester moiety are effective in the treatment of nephrotic syndrome. Without wishing to be bound by theory, it is believed that compounds comprising such moieties disrupt the binding of keratin 8 to proteins such as podocin leading to restoration of these proteins at their target sites.
Thus, the present invention provides a compound for use in the treatment of nephrotic syndrome in a subject in need thereof, wherein the compound comprises at least one moiety selected from phosphinic acid moieties, phosphinate ester moieties and pharmaceutically acceptable salts thereof.
Pharmaceutically acceptable salts of the compound for use according to the present invention may be of any suitable type. Preferred pharmaceutically acceptable salts are alkali metal salts, such as sodium and potassium salts.
A preferred compound for use in the treatment of nephrotic syndrome is a compound of formula I, or a pharmaceutically acceptable salt thereof:
wherein x is 0 or an integer from 1 to 6; Y is selected from the group consisting of: direct bond, C(0), C(O)O, 0, C(R′)(OH), C(O)NRI, S(0), S(0)(0) and P(0)(OR′);
Z is selected from the group consisting of: C(0), C(O)O, 0, C(R′)(OH), C(O)NRI, S(0), S(0)(0) and P(0)(OR′);
wherein R1 is selected from the group consisting of: hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl and optionally substituted heterocyclyl;
R2 is selected from the group consisting of: hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl and optionally substituted heterocyclyl; and
R3 is selected from the group consisting of: hydrogen, OH, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl, optionally substituted alkoxy, COOH, optionally substituted carboxylate ester, optionally substituted amide, optionally substituted amine, optionally substituted ether, phosphinic acid and phosphinate ester.
A particularly preferred compound for use in the treatment of nephrotic syndrome is a compound of formula II, or a pharmaceutically acceptable salt thereof:
wherein R1 is selected from the group consisting of: hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl and optionally substituted heterocyclyl;
R2 is selected from the group consisting of: hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl and optionally substituted heterocyclyl;
Y is selected from the group consisting of: direct bond, C(0), S(0), C(O)O, C(O)NH, and P(O)(OH); and
R3 is selected from the group consisting of: hydrogen, OH, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl, optionally substituted alkoxy, COOH, optionally substituted carboxylate ester, optionally substituted amide, optionally substituted amine, optionally substituted ether, phosphinic acid and phosphinate ester.
In the compound of formula I and/or the compound of formula II, R1 is selected from the group consisting of: hydrogen, optionally substituted alkyl (e.g. preferably unsubstituted C1-12 alkyl or preferably unsubstituted C1-6 alkyl), optionally substituted cycloalkyl (e.g. preferably unsubstituted C3-12 cycloalkyl or preferably unsubstituted C3-6 cycloalkyl), optionally substituted alkenyl (e.g. preferably unsubstituted C1-12 alkenyl or preferably unsubstituted C1-6 alkenyl), optionally substituted alkynyl (e.g. preferably unsubstituted C1-12 alkynyl or preferably unsubstituted C1-6 alkynyl), optionally substituted aryl (e.g. preferably unsubstituted C6-12 aryl or preferably unsubstituted C6 aryl), optionally substituted heteroaryl (e.g. preferably unsubstituted C5-12 heteroaryl or preferably unsubstituted C5-6 heteroaryl), and optionally substituted heterocyclyl (e.g. preferably unsubstituted C3-12 heterocyclyl or preferably unsubstituted C3-6 heterocyclyl). Preferably R1 is selected from the group consisting of: hydrogen, optionally substituted alkyl and optionally substituted cycloalkyl, more preferably, hydrogen and optionally substituted alkyl. Still more preferably R1 is selected from the group consisting of: hydrogen and optionally substituted alkyl (e.g. unsubstituted C1-12 alkyl). Preferred alkyl groups include methyl, ethyl, propyl (e.g. n-propyl, i-propyl), butyl (e.g. n-butyl, i-butyl, s-butyl, t-butyl), pentyl, hexyl and heptyl, more preferably methyl, ethyl, propyl and butyl.
In the compound of formula I and/or the compound of formula II, R2 is selected from the group consisting of: hydrogen, optionally substituted alkyl (e.g. preferably unsubstituted C1-12 alkyl or preferably unsubstituted C1-6 alkyl), optionally substituted cycloalkyl (e.g. preferably unsubstituted C3-12 cycloalkyl or preferably unsubstituted C3-6 cycloalkyl), optionally substituted alkenyl (e.g. preferably unsubstituted C1-12 alkenyl or preferably unsubstituted C1-6 alkenyl), optionally substituted alkynyl (e.g. preferably unsubstituted C1-12 alkynyl or preferably unsubstituted C1-6 alkynyl), optionally substituted aryl (e.g. preferably unsubstituted C6-12 aryl or preferably unsubstituted C6 aryl), optionally substituted heteroaryl (e.g. preferably unsubstituted C5-12 heteroaryl or preferably unsubstituted C5-6 heteroaryl), and optionally substituted heterocyclyl (e.g. preferably unsubstituted C3-12 heterocyclyl or preferably unsubstituted C3-6 heterocyclyl). Preferably R2is selected from the group consisting of: optionally substituted aryl (e.g. C6-12 aryl) and optionally substituted heteroaryl (e.g. C5-12 heteroaryl). Preferred aryl groups include optionally substituted phenyl and optionally substituted naphthyl groups, more preferably optionally substituted phenyl. Preferred heteroaryl groups include pyrryl, furyl, thiophenyl, pyridinyl, indolyl and imidazolyl. When substituted, preferred substituents include halo (e.g. fluoro, chloro, bromo, iodo), hydroxy, C1-12 alkyl, C1-12 alkoxy (e.g. methoxy, ethoxy, propoxy, butoxy), COOH, carboxylate ester, nitrile, nitro and amine, more preferably halo (e.g. fluoro, chloro, bromo, iodo), hydroxy, C1-12 alkyl, C1-12 alkoxy (e.g. methoxy, ethoxy, propoxy, butoxy) and COOH, still more preferably halo. R2 is especially preferably unsubstituted or substituted phenyl (e.g. halophenyl such as fluorophenyl, chlorophenyl, bromophenyl, iodophenyl). R2 may preferably be unsubstituted phenyl.
In the compound of formula I and/or the compound of formula II, x is 0 or an integer from 1 to 6 (e.g. 1, 2, 3, 4, 5 or 6). Preferably, x is 0 or an integer from 1 to 3. More, preferably,
In the compound of formula I, Y is selected from the group consisting of: direct bond, C(0),
C(O)O, 0, C(R′)(OH), C(O)NRI, S(0), S(0)(0) and P(0)(OR′). In the compound of formula II, Y is selected from the group consisting of: direct bond, C(0), S(0), and P(O)(OH). Preferably, Y is selected from the group consisting of: direct bond, C(0), and P(O)(OH). Y may preferably be P(O)(OH).
In the compound of formula I and/or the compound of formula II, R3 is selected from the group consisting of: hydrogen, OH, optionally substituted alkyl (e.g. preferably unsubstituted C1-12 alkyl or preferably unsubstituted C1-6 alkyl), optionally substituted cycloalkyl (e.g. preferably unsubstituted C3-12 cycloalkyl or preferably unsubstituted C3-6 cycloalkyl), optionally substituted alkenyl (e.g. preferably unsubstituted C1._1.2 alkenyl or preferably unsubstituted C1-6 alkenyl), optionally substituted alkynyl (e.g. preferably unsubstituted C1-12 alkynyl or preferably unsubstituted C1-6 alkynyl), optionally substituted aryl (e.g. preferably unsubstituted C6-12 aryl or preferably unsubstituted C6 aryl), optionally substituted heteroaryl (e.g. preferably unsubstituted C5-12 heteroaryl or preferably unsubstituted C5-6 heteroaryl), optionally substituted heterocyclyl (e.g. preferably unsubstituted C3-12 heterocyclyl or preferably unsubstituted C3-6 heterocyclyl)., optionally substituted alkoxy (e.g. preferably unsubstituted C1-6 alkoxy), COOH, optionally substituted carboxylate ester (e.g. preferably unsubstituted C1-6 carboxylate ester), optionally substituted amide (e.g. preferably unsubstituted C1-6 carboxylate amide), optionally substituted amine, optionally substituted ether (e.g. preferably unsubstituted C1-6 ether), phosphinic acid (e.g. P(O)(OH)R) and phosphinate ester (e.g. P(0)(OR′)R). Preferably, R3 is selected from the group consisting of: OH, optionally substituted alkyl (e.g. preferably unsubstituted C1-12 alkyl or preferably unsubstituted C1-6 alkyl), optionally substituted cycloalkyl (e.g. preferably unsubstituted C3-12 cycloalkyl or preferably unsubstituted C3-6 cycloalkyl), optionally substituted aryl (e.g. preferably unsubstituted C6-12 aryl or preferably unsubstituted C6 aryl), optionally substituted heteroaryl (e.g. preferably unsubstituted C5-12 heteroaryl or preferably unsubstituted C5-6 heteroaryl), and optionally substituted alkoxy (e.g. preferably unsubstituted C1-6 alkoxy). More preferably, R3 is selected from the group consisting of: OH, optionally substituted (e.g. unsubstituted) C1-6 alkyl and optionally substituted C6-12 aryl (e.g. substituted or unsubstituted phenyl). Preferred alkyl groups include methyl, ethyl, propyl (e.g. n-propyl, i-propyl), butyl (e.g. n-butyl, i-butyl, s-butyl, t-butyl), pentyl, hexyl and heptyl, more preferably methyl, ethyl, propyl and butyl. When substituted, preferred substituents include halo (e.g. fluoro, chloro, bromo, iodo), hydroxy,
C1-12 alkyl, C1-12 alkoxy (e.g. methoxy, ethoxy, propoxy, butoxy), COOH, carboxylate ester, nitrile, nitro and amine. R3 is especially preferably hydroxy, unsubstituted C1-6 alkyl or optionally substituted phenyl (e.g. unsubstituted phenyl, halophenyl or hydroxyphenyl, preferably unsubstituted phenyl or hydroxyphenyl).
In a particularly preferred compound of formula II for use according to the invention:
R1 is selected from hydrogen, optionally substituted C1-12 alkyl and optionally substituted C3-12 cycloalkyl;
R2 is selected from optionally substituted C6-12 aryl and optionally substituted C5-12 heteroaryl;
Y is selected from the group consisting of: direct bond, C(0), S(0), C(O)O, C(O)NH, and
P(O)(OH); and
R3 is selected from the group consisting of: hydrogen, optionally substituted C1-12 alkyl, optionally substituted C3-12 cycloalkyl, optionally substituted C6-12 aryl, optionally substituted C5-12 heteroaryl, COOH, optionally substituted carboxylate ester, phosphinic acid and phosphinate ester;
wherein the optional substituents are selected from halo, hydroxy, C1-12 alkyl, C1-12 alkoxy, COOH, carboxylate ester, nitrile, nitro and amine.
An especially preferred compound of formula II for use in the treatment of nephrotic syndrome is one wherein:
R1 is selected from hydrogen and C1-6 alkyl (e.g. methyl, ethyl, propyl, butyl, pentyl; hexyl);
R2 is selected from optionally substituted C6-12 aryl and optionally substituted C5-12 heteroaryl;
x is an integer from 1 to 3;
Y is selected from the group consisting of: P(O)(OH); and
R3 is selected from the group consisting of: hydrogen, C1-6 alkyl, optionally substituted phenyl, COOH, and phosphinic acid;
wherein the optional substituents are selected from halo, hydroxy, C1-12 alkyl, C1-12 alkoxy, COOH, carboxylate ester, nitrile, nitro and amine (e.g. halo, hydroxy and alkoxy).
Especially preferred compounds for use in the treatment of nephrotic syndrome in a subject in need thereof are selected from compounds of formulae IIa-IIh:
A particularly preferred compound is the compound of formula IIa.
In one embodiment, the compound is other than a bisphosphonate drug. For example, the compound is other than risedronic acid, etidronic acid, alendronic acid, minodronic acid, zoledronic acid, pamidronic acid, tildronic acid, monidronic acid, neridronic acid, olpadronic acid, clodronic acid and ibandronic acid. In one embodiment, the compound does not have one or two or more terminal phosphonate (P(O)(OH)2) functional groups.
The present invention provides a compound as hereinbefore described for use in the treatment of nephrotic syndrome in a subject in need thereof. Preferably the subject is a mammal, more preferably a human.
The present invention also provides a kit comprising a compound which is an inhibitor of binding between a target protein and keratin 8 together with instructions for treating nephrotic syndrome. Preferred features of the kit, including preferred features of the compound, target protein, nephrotic syndrome and subject, are as defined above with respect to the compound for use in the treatment of nephrotic syndrome.
The present invention also provides a method of treating nephrotic syndrome in a subject in need thereof comprising administering to said subject an effective amount of a compound which is an inhibitor of binding between a target protein and keratin 8. Preferred features of the method, including preferred features of the compound, target protein, nephrotic syndrome and subject, are as defined above with respect to the compound for use in the treatment of nephrotic syndrome.
The therapeutically effective amount of the compound administered to the patient is an amount which confers a therapeutic effect in accordance with the present invention on the treated subject, at a reasonable benefit/risk ratio applicable to any medical treatment. The therapeutic effect may be objective (i.e. measurable by some test or marker) or subjective (i.e. subject gives an indication of or feels an effect).
The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or contemporaneously with the specific compound employed; and like factors well known in the medical arts.
The compounds may be administered in any effective manner. Suitable examples of the administration form include without limitation oral, topical, parenteral, sublingual, rectal, vaginal, ocular, and intranasal. Parenteral administration includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.
The present invention also provides the use of a compound which is an inhibitor of binding between a target protein and keratin 8 in the manufacture of a medicament for the treatment of nephrotic syndrome in a subject in need thereof. Preferred features of the method, including preferred features of the compound, target protein, nephrotic syndrome and subject, are as defined above with respect to the compound for use in the treatment of nephrotic syndrome.
The present work describes previously unknown machinery that plays role in the R138Q podocin intracellular retention and firmly suggests that keratin 8-R138Q podocin association facilitates the ER retention of the mutant protein. Keratins have been previously reported to be up-regulated in different disease models, such as animal models of pancreas and liver injury, while keratin 8 and 18 have been recently described to be novel markers of renal epithelial cell injury (S. Djudjaj et al., Kidney Int., vol. 89, pp. 792-808, 2016.). The current study reports an increased expression of keratin 8 in R138Q mutant human podocytes for the first time. There is also an increased interaction of keratin 8 and podocin in close vicinity of the ER in R138Q mutant cells, which is reversed by treating cells with compound Ia, resulting in the rescue of the mutant protein localization.
A full rescue of adhesion function of mutant cells was achieved upon treatment with compound Ia. Furthermore, computer modelling of protein folding revealed that R138Q mutation creates a large hydrophobic region, which becomes exposed for interaction with other proteins or small molecules, such as compound Ia. Therefore, it is tempting to hypothesize that K8-R138Q interaction occurs as a result of the appearance of this hydrophobic pocket, and further structural modelling shows that inhibitor compounds are able to bind to this pocket blocking the interaction with K8 and thus allowing for the correction of the R138Q podocin processing defect with small molecule intervention.
Here, we demonstrate that keratin 8 shRNA results in the correct localization of the R138Q mutant protein and the recovery of its function. The fact that silencing of keratin 8 recovers the function of the mutant protein further suggests that keratin 8 network can act as a potential therapeutic target.
In summary, our study provides direct evidence that keratin 8 is involved in the podocyte dysfunction associated with the most common R138Q podocin mutation in NS. This work further demonstrates that pharmacological targeting of keratin 8 with inhibitor compounds, such as compound Ia, in vivo leads to prevention and correction of proteinuria in NPHS2flox/R140Q mice.
In this work, the consequence of the R138Q mutation of podocin was studied by expressing the GFP-tagged wild-type and R138Q mutant podocins in human podocytes. As shown in
In the next set of experiments, the intracellular distribution of keratin 8/18 was analysed in human podocytes expressing either the GFP-tagged wild-type or R138Q mutant podocins using confocal microscopy. Localization of keratin 8 and WT podocin was shown by immunofluorescence at the cytosol level, while localization of keratin 8 was seen in the perinuclear area in close vicinity of R138Q podocin (
Co-immunoprecipitation (Co-IP) assay of endogenous proteins was then conducted to see whether keratin 8 and podocin interact directly in human podocytes. Co-immunoprecipitation experiments demonstrated that endogenous keratin 8 preferably interacts with R138Q podocin over its wild-type counterpart (
It was observed that inhibiting the keratin 8-R138Q podocin interaction by keratin 8 silencing rescued the lipid raft association of the mutant protein and restore its functional defect. All K8-shRNA sequences considerably decreased K8 expression in both human podocyte cell lines (
Immunofluorescence studies were performed to see whether treatment with compound Ia for 24 hours rescued mutant podocin localization back to the plasma membrane.
To test whether compound Ia interrupts the protein-protein interaction, a series of proximity ligation experiments were performed on human podocytes derived either from a healthy patient or patient with the R138Q mutation.
To understand whether compound Ia could prevent the development of NS in NPHS2flox/R140Q mice, doxycycline was administrated for 3 weeks to induce proteinuria, at the same time as one group was treated with compound Ia and another group was treated with vehicle (0.9% NaCl) for 4 weeks via osmotic mini pumps. A third group with vehicle pumps served as no disease controls and was administrated drinking water for the duration of the study.
Doxycycline induced animals treated with vehicle, developed proteinuria within the first two weeks of R140Q hemizygosity induction, which peaked at Week 4 as anticipated (
Decreased podocin protein expression was observed at week 4 in in NPHS2flox/R140Q mice, which were given doxycycline for 3 weeks compared to that of healthy controls. In vivo compound Ia treatment restored mutant podocin localization back to the plasma membrane as seen by immunofluorescence on mice kidney sections (
A number of compound Ia analogues have been tested in a robust cell adhesion assay. This is a rapid in vitro assay that provides information on any given compound's ability to restore the WT-phenotype. As can be seen in
It was demonstrated that high throughput screening of inhibitor candidates can be conducted using an InCell analyser with GlomSpheres. GlomSpheres can be used to mimic the glomerulus. They are formed by coating podocytes and endothelial cells with inert nanoparticles, and co-culturing them under magnetic levitation, so that they self organise into a glomerulus-like structure. The use of human podocytes with a podocin mutation can be used to represent the disease condition, and for high-throughput testing of inhibitor candidates. The results of the screening assay conducted using compound 407 are shown in
Further evidence of Keratin 8 involvement in removal of R138Q mutated podocin was established through immunohistochemistry performed on human biopsy samples.
All the laboratory reagents were of the highest quality and purity and purchased from Sigma Aldrich unless otherwise specifies in the text or Appendix 1. Appendix 1 also contains list of solutions used, cell culture and cell extraction reagents.
The wild-type and R138Q podocin mutant conditionally immortalized human podocyte cell lines were developed at Bristol Renal Unit by transduction with the temperature-sensitive SV40-T transgene as previously described (M. A. Saleem et al., J. Am. Soc. Nephroi., vol.
13, no. 3, pp. 630-8, Mar. 2002). These cells can proliferate at the permissive temperature of 33° C., and thereafter they can be transferred to the nonpermissive temperature of 37° C., where the cells enter growth arrest and show key characteristics of podocyte differentiation and function. The wild-type podocyte cell line is referred to as WT, while the podocin mutant podocyte cell line is called PM. The wild-type and R138Q GFP-tagged immortalized human podocyte cell lines were created using PCR-based molecular cloning approach and used in experiments, where stated. All cells were grown and maintained in CO2 incubators with a temperature of 33° C./37° C., 5% CO2 concentration and 95% relative humidity. All cell work was performed in aseptic conditions in a class two biological safety hood. Cell culture media was changed every 34 days.
All human conditionally immortalised podocyte cell lines were grown in RPMI 1640 media with supplements as detailed in Appendix. Cells were cultured at 33° C. until 70% confluent, and then switched to 37° C. for 10-14 days differentiation. All podocyte cell lines were cultured under sterile conditions in tissue culture vessels including T175 cm2, T75 cm2 and
T25 cm2 flasks, and 6 well plates. For immunofluorescence cells were grown as described above either on glass coverslips in 6 well plates or in 6 cm2 glass bottom dishes.
Stock flasks of proliferating cells were maintained under an atmosphere of 5% (v/v) CO2 and 95% air at the permissive temperature of 33° C. for podocytes. Cells were grown to the desired confluency (70%-90%) and sub-cultured to maintain them at a logarithmic growth rate. For sub-culturing, growth medium was removed, and the adherent cells were washed with 1ml sterile trypsin. The cells were detached from the flask by mild tryptic digestion using 0.5 ml 0.25% Trypsin-EDTA and incubation at 37° C. for 3-5 mins. The cells were then resuspended in the appropriate growth medium and aliquoted into fresh tissue culture flasks or dishes. Each time the cells were split, a consecutive passage number was assigned. Passages 13 to 22 were used for experiments.
The inhibitor compounds were commercially available or synthesized using routine methods known in the art. Compound Ia was a kind gift from Prof. Aleksander Edelman, although it is noted this compound can be synthesized, for example, according to methods disclosed in Acta Crystallogr Sect E Struct Rep Online. 2012 Aug 1; 68(Pt 8): o2456.
Compounds Ib-f were obtained from the vendor ChemBridge, with catalogue numbers as follows: compound Ib=ChemBridge 5116054; compound Ic=ChemBridge 5116157; compound Id=Chembridge 5231281; compound Ie=ChemBridge 5243784; compound If =ChemBridge 5304459; compound Ig=ChemBridge 5304787; compound Ih=ChemBridge 5307860.
Cell adhesion assays are widely used to assess the adhesion properties of many cell types, for example epithelial cells to the extracellular matrix, other cells, or specially coated surfaces. In addition, this type of assay can be used to determine the effects of various treatments, such as pharmacological compounds and small molecules, on the ability of cells to adhere. An adapted cell adhesion assay protocol is detailed here for studying the adhesion characteristics of human podocytes in vitro. Podocytes were grown in a T75 cm2 flask and differentiated at 37° C. for 10-14 days. When fully differentiated, cells were trypsinised with 0.025% trypsin/EDTA for 5 min at 37° C. Podocytes were then resuspended in cell culture media to stop enzyme activity and collected in a 15 ml falcon. Cells were centrifuged for 5 min at 1000 g, and then gently resuspended in 1 ml of cell media. 10-15p1 of cells with trypan blue were pipetted onto the disposable slide and counted with the Luna-FLTM automated cell counter. Podocytes were again resuspended to a concentration of 5×105/ml in cell media. Cells were allowed to recover from trypsinisation in an upright falcon tube with the lid off at 37° C. for 10 min. 50p1 of PBS and 50p1 of cells were added to each well of a 96 well plate. Three experimental wells were assigned as the 100% attachment control, to which 20%, 50% and 100% of the total volume of cells were added. Cells were left to adhere for 45 min at 37° C. Control wells for 100% attachment were fixed with 100 μl 4% PFA for 20 min at room temperature. Thereafter, the plate was tapped to remove lose and non-adherent podocytes, and washed twice with 100 μl PBS, and the experimental wells were then fixed with 100 μl 4% PFA for 20 min (note that the 100% attachment wells were not washed). The PFA was then washed off three times with 100p1 of distilled water, and cells were stained 100p1 0.1% crystal violet in 2% ethanol for 60 minutes at room temperature. Crystal violet was also added to three empty wells in order to measure binding of the dye to the plastic as a control. Crystal violet was removed, and wells were washed 3 times with 400p1 of distilled water. 100p1 of 10% acetic acid was added to each well to solubilise the dye. A 96 well plate was incubated on an orbital shaker at 150 rpm for 5 minutes at room temperature. Absorbance of the plate was measured at 570 nM in a plate reader. Results were expressed as a percentage of 100% attachment and normalised against the adhesion of the human wild type podocytes cell line.
Podocyte adhesion in vitro is widely accepted as a surrogate of podocyte dysregulation in nephrotic syndrome in vivo (ref Welsh GI, Saleem MA. Nat Rev Nephrol. 2011 Oct 25;8(1):14-21). It reflects disruption of cytoskeletal dynamics, and the subsequent podocyte foot process effacement which is common to all forms of human NS.
Spheroids were formed using a modified version of N3D bioscience standard protocol for magnetic spheroid bioprinting (Nano3D Biosciences Inc). A T75 flask of cells (i.e. endothelial cells or podocytes) was incubated overnight with 100p1 nanoshuttle-PL (Nano3D Biosciences Inc), added to 10 ml of fresh cell culture medium. Cells were then washed with 5 ml sterile phosphate-buffered saline (PBS) and with trypsin-EDTA (Lonza). Cells were then pelleted via 1500 rpm centrifugation (5 mins) and counted with a Luna cell counter (Logos Biosystems). For monoculture experiments, 10,000 cells were pipetted into each well of an ultra-low attachment plate (Greiner) and a 96-magnet MagDrive (Nano3D Biosciences) was placed underneath. Spheroids were left to form overnight at 33° C.
For co-culture/GlomSphere spheroids, a spheroid of 5000 glomerular endothelial cells (GEnCs) was generated as above and allowed to aggregate for 1 hr at 33° C. The MagDrive was then removed and 5000 podocytes were pipetted into each well before the MagDrive was replaced. This forces the newly added podocytes to form a peripheral coating around the GEnC core. The spheroids were then thermoswitched to 37° C. and the podocyte layer migrates (
GlomSpheres were magnetically transferred to Eppendorf tubes and fixed in 4% Paraformaldehyde (Sigma) containing 1% Triton-x100 (Sigma) (20 mins, 20° C.). To block, spheroids were incubated with 5% bovine-serum-albumin (BSA) (Sigma) containing 0.1% Triton-x100 (overnight, 4° C.). Spheroids were then incubated with primary antibody, diluted in 5% BSA containing 0.1% Triton-x100 (48 hours, 4° C.). Spheroids were then washed in PBS containing 1% Triton-x100 (3x30 minutes, room temp). Spheroids were then incubated with secondary fluorescent antibodies diluted 1:400 in 5% donkey serum (Sigma) containing 0.1% Triton-x100 (24 hours, 4° C.). Spheroids were then washed in PBS containing 1% Triton-x100 (3x30 minutes, 20° C.).
Sections were cut as previously described (Tuffin J, Burke M, Richardson T, Johnson T, Saleem MA, Satchel) S, et al. A Composite Hydrogel Scaffold Permits Self-Organization and Matrix Deposition by Cocultured Human Glomerular Cells. Adv Healthc Mater. 2019;8(17):e1900698). Sections or fixed 2D-cultured cells on glass coverslips were washed in PBS (2x 5 mins, 20° C.). To block, samples were incubated in 5% BSA containing 0.1% Triton-x100 (45 minutes, 20° C.), then incubated with primary antibody, diluted in 5% BSA containing 0.1% Triton-x100 (1 hour, 20° C.). Primary antibodies were then washed with PBS (2x 5 mins, 20° C.) before being incubated with fluorescent secondary antibodies diluted in 1:400 in 5% donkey serum (1 hour, 20° C.). Samples were then washed with PBS (2x 5 mins, 20° C.) and mounted to microscope slides with 5p1 Mowiol (Sigma).
Spheroids were formed using either GFP CI podocytes (WT) or GFP CI podocin mutant podocytes (PM). Compounds were added 24 hrs after culture and re-dosed daily. Images were taken daily and those shown and quantified are from Day 5. Imaging was performed using an IN cell analyser (GE lifesciences).
To test whether inhibitor compounds can rescue mutant podocin localization in vivo, a transgenic mouse model was developed and characterized. This mouse was designed to carry the R140Q mutation, the mouse analogue of human R138Q, on one allele and floxed WT NPHS2 on the other, which can be excised upon induction with doxycycline.
A mouse model carrying R140Q podocin mutation, which is analogous to human p.R138Q variant, was generated using a 6.6 kb targeting construct (A. Philippe et al., Kidney Int., vol. 73, no. 9, pp. 1038-47, May 2008). Using site-directed mutagenesis, the c.505G>A, c.506A>G mutations were introduced into exon 3 of the NPHS2 gene of the targeting vector, while a phosphoglycerate kinase— hygromycin cassette flanked by flox sites was inserted into intron 3 to select positive embryonic stem (ES) cell clones. Following successful homologous recombination, two ES cell clones were selected and injected into the murine blastocyst of C57BL/6 mice. Several rounds of breeding were undertaken to generate a mouse with germline incorporation of the mutant allele, which was further mated with mice constitutively expressing Cre recombinase that aids the removal of the floxed hygromycin insert. Using this model, the group of C. Antignac have demonstrated that homozygous NPHS2 R140Q/R140Q mice die within the first week of life with some mice dying during embryogenesis, while heterozygous NPHS2R140Q/+mice did not develop albuminuria or any renal anomalies. The above models are characterized either by severe renal phenotype resulting in early death or by insufficient expression of the mutated protein. Therefore, the conditional inactivation of wild-type protein is essential to elucidate the role of podocin in health and disease in mice at postnatal age.
In more detail, homozygous NPHS2flox/flox and heterozygous NPHS2R140Q/+transgenic mice on a 129Sv/PasCrl genetic background were a kind gift from Prof. Corinne Antignac (INSERM, Paris). Homozygous NPHS2flox/flox mice were generated to carry a floxed NPHS2 exon 2 alleles, which can be excised, when Cre recombinase is expressed leading to a conditional podocin inactivation in mature kidneys (
Postnatal induction of R140Q hemizygosity in NPHS2flox/R14Q mice was achieved by administration of doxycycline at 2 mg/ml in 5% sucrose dissolved in drinking water for three weeks. Doxycycline, a tetracycline derivative, binds with high affinity to rtTA, thus inducing a Tet-off system in transgenic mice. Doxycycline was shown to have an excellent medical safety record and deep tissue penetration with relatively low toxicity in eukaryotic cells (I. M. Redelsperger et al., J. Am. Assoc. Lab. Anim. Sci., vol. 55, no. 4, pp. 467-74, 2016.). Doxycycline was made fresh every 4 days and administered in black bottles to protect from light. Control mice with the same genotype (NPHS2flox/R140Q) were given the same volume of drinking water without doxycycline. Other control groups of mice, which lacked either Pod-rtTA or TetO-Cre or both, received the equivalent volume of drinking water with doxycycline at the same concentration as used for the experimental mice. The rationale for having mice without the Pod-rtTA gene, for example, is that the induction efficiency of the floxed wild-type podocin can be monitored. Mice were maintained and managed by skilled animal technicians in the pathogen-free conditions of ASU unit, University of Bristol.
Beginning from the first week after doxycycline induction, weekly urine collections were performed to look for the onset and control the duration of proteinuria until the point of sacrificing. Animals were sacrificed at weeks 10-12 after doxycycline administration. In case of weight loss (over 10%), the affected animal was culled immediately. At designated experimental time points mice were maintained under general anaesthesia with isoflurane, and 1 ml of blood was withdrawn by cardiac puncture. Animals were subsequently sacrificed by Schedule 1, and both kidneys were removed (flash frozen, put in PFA or EM buffer).
Samples were collected and used for RNA and protein extraction, immunofluorescence studies, immunohistochemistry and electron microscopy. Urine albumin concentration was analysed using human albumin ELISA kit (Bethyl Laboratories). Whole blood samples were sent to Diagnostic Laboratories (Langford Vets, University of Bristol) to measure serum creatinine, albumin and urea. Urine samples were also sent to Diagnostic Laboratories (Langford Vets) to analyse creatinine in the urine.
Doxycycline induction of a hemizygous state in the NPHS2flox/R140Q mice led to severe proteinuria detectable within few days, which peaked to a maximum at 2-4 weeks after induction of NPHS2nU!!/R140Q and was maintained at week 12 (
Pod-rtTA+/−TetO-Cre+/−NPHS2flox/R140QRG+/−or Pod-rtTA
Directly after surgery, subcutaneous injection of rimadyl (0.1 mg/kg) was administrated as analgesia. Mice were closely monitored for 24 h and expected to make a rapid recovery from implantation of osmotic pumps. Wound clips were removed at day 11 post-surgery. ALZET Osmotic pumps are designed to have a fixed volume-delivery rate, therefore accurate calculations based on the volume of impermeable pump reservoir were performed to fill each pump with appropriate volume of inhibitor to achieve a dose of 22.3 mg/month/mouse. ALZET Osmotic Pumps 2004 operate based on the difference in an osmotic pressure between the osmotic layer of the pump and the subcutaneous tissue layer of the living animal. The high osmolarity of the osmotic layer results in water being diffused into the pump through a semipermeable membrane creating an increase in pressure within the pump, which in turn displaces the contents from the pump at a controlled rate. This further results in the impermeable reservoir of the pump being compressed; thus, the pump is designed for a single use only.
Urine samples were obtained from all mice prior to the start of any experimental procedures, such as doxycycline induction or implantation of the osmotic pumps. Beginning from the first week after doxycycline induction, weekly urine collections were performed to look for the onset and control the duration of proteinuria until the point of sacrificing (10-12 weeks)in NPHS2flox/R140Q mice. In the compound Ia/saline osmotic pump model, urine was collected twice a week for a 28-day period. To collect samples, mice were placed individually in a clean plastic container and watched constantly. Once the mouse urinated, 50-100 μl of urine was pipetted off and stored at −20° C. until analysis. Siemens Multistix Urinalysis strips were used as a basic diagnostic tool to give an estimation of proteinuria as indicated by a colour change on the protein pad.
The kidneys harvested from the NPHS2flOx/R140Q mice were processed in several ways to provide a phenotypic profile for each animal after the transgene induction and to examine the effects of compound Ia on the renal function.
Mice were culled in accordance with Schedule 1 humane killing methods by confirmation of permanent cessation of the circulation. Both kidneys were collected and cut in half longitudinally. Half of one kidney was fixed in 10% (v/v) formalin in PBS for four days, before being placed in 70% ethanol. Two other halves were placed in cryovials and snap frozen immediately in liquid nitrogen. The cryovials were then stored at −80° C. until required for IF, RNA and protein extraction. The last half of the kidney was cut into 1 mm pieces and placed in the EM buffer for fixation and storage at 4° C. Spleen was also harvested and snap frozen immediately from each animal to be used as a control tissue for RNA and protein.
Kidney tissues were fixed in 10% (v/v) formalin, further processed and paraffin embedded after ethanol dehydration. Tissues were then cut by University of Bristol histopathology staff within the Bristol Medical School histopathology laboratory to give sections 3 μm thick.
PAS staining was performed on paraffin-embedded tissue sections to evaluate the degree of sclerosis within the glomerular tuft using PAS staining system (SIGMA-ALDRICH) as per manufacturer's protocol. Slides were immersed twice in Histo-Clear II (National Diagnostics HS-202) for 5 min to deparaffinize sections. Sections were then rehydrated by passing the slides through alcohol series (100% ethanol, 90% ethanol, 70% ethanol, 50% ethanol) to deionized water. Slides were oxidized in periodic acid solution for 5 min at room temperature, before being rinsed in several changes of distilled water. Slides were then placed in Schiff's reagent for 15 min and washed in running tap water for 5 min. The tissue sections were counterstained in hematoxylin solution for 90 seconds and immediately washed again in running tap water. The sections were then dehydrated by being passed through alcohol series (50% ethanol, 70% ethanol, 90% ethanol, 100% ethanol), dried and mounted in DPX (Sigma #44581). The sections were then imaged using a Leica light microscope that allows to assess morphology of the kidney.
Masson's Trichrome staining was performed on paraffin-embedded tissue sections to evaluate the degree of glomerular fibrosis (collagen deposits) using Trichrome Stain
(Masson) Kit ((SIGMA-ALDRICH) as per manufacturer's protocol. Slides were immersed twice in xylene for 5 min to deparaffinize sections. Sections were then rehydrated by passing the slides through alcohol series (100% ethanol, 90% ethanol, 70% ethanol, 50% ethanol) to deionized water. Slides were stained in Weigert's iron hematoxylin working solution for 5-10 mins and then rinsed in running warm tap water for another 10 mins, followed by the distilled water wash. The tissue sections were then stained in Biebrich scarlet-acid fuchsin solution for 10-15 mins, washed in distilled water and differentiated in phosphomolybdic-phosphotungstic acid solution for another 5-10 mins (or until collagen is not red). Sections were then transferred directly to aniline blue solution and stained for 5-10 mins, followed by a brief rinse in distilled water and differentiation in 1% acetic acid solution for 2-5 mins, followed by a brief wash in distilled water. The sections were then dehydrated by being passed through alcohol series and cleared in xylene, dried and mounted in DPX (Sigma #44581). The sections were then imaged using a Leica light microscope that allows to assess morphology of the kidney.
IHC was performed with Wilm's tumour-1 protein (WT1) antibody (Santa Cruz) to look for the glomerular podocyte number in mice kidney sections. Slides were immersed twice in histoclear for 5 min to deparaffinize sections. Sections were then rehydrated by passing the slides through alcohol series (100% ethanol, 90% ethanol, 70% ethanol, 50% ethanol) to deionized water. Slides were next boiled in the antigen retrieval buffer (10 mM Sodium citrate tribasic, pH 6) for 10 min in microwave and allowed to cool on bench top for 30 min.
Sections were then washed in deionized water 3 times for 5 min each. PAP pen was used to draw around sections, which were then incubated in 3% hydrogen peroxide for 20 min in a humidified box, followed by wash in deionized water twice for 5 min each. Sections were blocked in blocking solution (5% goat serum in TBST 0.1%) for 30 min at room temperature. Blocking solution was then removed, and sections were incubated with primary antibody (20u1/sample) overnight at 4° C. Next morning, primary antibody was removed, and sections were washed with wash buffer (TBST 0.1%) 3 times for 5 min each. Sections were then covered with 1-3 drops of SignalStain® Boost Detection Reagent and incubated in a humidified chamber for 30 min at room temperature. Meanwhile, 1 drop (30 p1) SignalStain® DAB Chromogen Concentrate was added to 1 ml SignalStain® DAB Diluent and mixed well before use (20u1/sample). Following 3 washes with wash buffer, SignalStain® DAB was applied to each section for 1 to 10 mins, or until the brown staining became apparent. Slides were then immersed in deionized water and counterstained with hematoxylin for 10 seconds. Next, slides were washed in tap water, until water became clear. The sections were then dehydrated by being passed through alcohol series and cleared in histoclear, dried and mounted in DPX (Sigma #44581). The sections were then imaged using a Leica light microscope.
Kidney tissue preparation for immersion fixation involved rapid excision of animal tissues and immersion of small tissue pieces (1 mm cubes) in fixative (2.5% glutaraldehyde in 0.1 M cacodylate at pH 7.3) in order to get the best ultrastructure. Tissues were then washed in 0.1 M cacodylate buffer 3 times for 10 minutes, followed by post fixation in 1% osmium tetroxide in 0.1M cacodylate buffer pH 7.3 at 4-8° C. for 1 hour. Kidney tissues were washed in 0.1 M cacodylate buffer for 15 minutes, followed by 3 washes in deionised water. Tissues were then put into 1-3% Uranyl acetate en bloc staining overnight, followed by 15-minute deionised water wash next morning. Kidney tissues were washed twice in propylene oxide for 15 minutes and infiltrated with Epon resin mix: propylene oxide (1:1) for 2 hours. Infiltrated tissues were then embedded in fresh Epon resin mix in a silicon rubber mould and left to polymerize for >24 hours at 60° C. Note, Epon resin mix consists of resin, hardener, accelerator and plasticiser. Finally, tissues were cut into sections (70 nm thick) on an Ultracut S ultramicrotome, which were then stained with 3% uranyl acetate (5 mins) and Reynolds' lead citrate (2 mins) with deionised water wash in between and after the lead stain. Sections of glomerulus were imaged at various magnifications in a Tecnai T12 microscope (FEI Ltd).
| Number | Date | Country | Kind |
|---|---|---|---|
| 1916071.2 | Nov 2019 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/081114 | 11/5/2020 | WO |
