Suppressors of Premature Termination Codons as Therapeutics and Methods for Their Use

Information

  • Patent Application
  • 20180258123
  • Publication Number
    20180258123
  • Date Filed
    September 23, 2016
    7 years ago
  • Date Published
    September 13, 2018
    5 years ago
Abstract
This invention discloses the use of aminoglycoside antibiotics such as gentamicin B1 to suppress premature termination codons during translation and promote the full length read-through of transcripts such as p53 that incorporate nonsense mutations and to treat disease conditions such as cancer caused by such genetic mutations.
Description
TECHNICAL FIELD

This invention relates to therapeutic compounds and compositions, and methods for their use in the treatment or amelioration of various indications, including medical conditions associated with premature termination codons (PTCs) in RNA, including various cancers. In particular the invention relates to therapies and methods of treatment that would at least partially restore translation of full-length protein products.


CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Ser. No. 62/232,789 filed 25 Sep. 2015.


BACKGROUND

Genomics advances will soon make it routine to identify the precise molecular lesions responsible for many of the rare genetic diseases that afflict our population. Unfortunately, most of these diseases have no treatments, in Canada about 30% of patients die in childhood, and it is exceedingly difficult to develop disease-specific treatments because of the small number of patients for each disease and the high cost of developing new drugs. About 10% of disease-causing mutations are nonsense mutations that introduce a PTC.


The European Organization for Rare Diseases estimates that there are at least 5,000 rare genetic diseases, defined as affecting less than 1 in 2,000 people and the genes for about 4,000 have been identified (Online Mendelian Inheritance in Man database). Rare genetic diseases are believed to affect 5-6% of the population, or about 25 million people in the EU, 16 million in the USA and 1.8 million in Canada. It is estimated that 95% of rare genetic diseases have no specific treatment. Furthermore, genetic diseases that would not have the rare classification, also have nonsense mutations. It is estimated that 20.3% of the ˜43,000 disease-associated single-base pair substitutions affecting gene coding regions that are cataloged in the Human Gene Mutation Database (HGMD 2007-Mort M. et al. 2008) are PTCs.


For nearly all these diseases, about 10-11% of patients have nonsense point mutations. These mutations change an amino acid codon to a PTC (i.e. UAA, UAG and UGA). PTCs may result in decreased mRNA stability via nonsense-mediated mRNA decay (NMD), as well as production of some truncated non-functional protein, if any protein is produced. Compounds that allow insertion of an amino acid at a PTC, without affecting normal termination codons, can enable production of functional full-length protein. This approach, termed both nonsense mutation suppression and PTC read-through, offers the possibility of developing a single treatment for large numbers of patients across multiple diseases. In reality, a proportion of these patients would likely not benefit from such a therapy, for example those children born with irreversible neurological damage. Nevertheless, for 50% of rare genetic diseases, the onset of disease occurs in childhood and progressively worsens, and these patients are the ones who stand to benefit most from nonsense suppression therapy.


The therapeutic potential of nonsense suppression is not limited to inherited disorders. Nonsense mutations also occur in tumour suppressor genes in about 10% of cases of sporadic cancer, which affects 40% of the population and is far from rare. To illustrate, the R213X mutation in protein p53 is present in 1% of all human cancers. This corresponds to about 220,000 cases worldwide (Hoe, K. K. Verma, C. S. and Lane, D. P. 2014) that could theoretically benefit from nonsense suppression therapy. A further 70 cancer-driver tumour suppressor genes have been identified (Vogelstein B, et al. 2013). Tumour sequencing and mutation analysis is not yet routine for cancer diagnosis. However, the concept of personalized medicine has taken huge steps in the cancer field and it is anticipated that identifying nonsense mutations in cancer will become routine in the next decade.


Accordingly, the targeting of nonsense mutations could eliminate the “rare” element of rare genetic diseases in some cases where the genetic disease is caused at least in part by a nonsense mutation and nonsense suppression may also be of use in the treatment of some cancers.


Compounds that enable PTC read-through, offer the possibility of using the same treatment for large numbers of patients across multiple diseases based on the mechanism of the PTC and not the particular gene having the PTC.


High concentrations of aminoglycoside antibiotics were shown 30 years ago to induce PTC read-through in some yeast genes (Singh A et al. 1979) and in a reporter gene in mammalian cells (Burke J F and Mogg A E. 1985). The potential for using gentamicin to treat cystic fibrosis patients with a PTC in the CFTR gene was shown when gentamicin was used to induce CFTR protein expression from the endogenous gene in a patient-derived bronchial epithelial cell line (Bedwell D M et al. 1997), recovery of function in mice bearing the human CFTR G542X transgene (Du M et al. 2002) and increases in CFTR chloride conductance in patients (Clancy J P et al. 2001; and Wilschanski M et al. 2003). Similarly, paromomycin, geneticin (G418) and PTC124 (3-[5-(2-fluorophenyl)-[1,2,4]oxadiazole-3-yl]-benzoic acid) are all reported to have nonsense suppressive properties (Karijolich J, and Yu, Y-T 2014). In all cases the improvement was small and patient response was variable (Linde L et al. 2007). The lack of potency, the recognized renal and otic toxicities of high dose gentamicin and the need for intravenous or intramuscular administration likely limited its further development.


Read-through by gentamicin was demonstrated in mdx mice (Barton-Davis E R et al. 1999) with a PTC introduced into the mouse dystrophin gene to model human Duchenne Muscular Dystrophy (DMD). The first small trial in DMD patients showed no effect of gentamicin. Two others showed dystrophin expression in some patients (Malik V et al. 2010) but the level of expression was insufficient for patient improvement. Again, dose-limiting toxicities prevented further development.


Major efforts have been put into developing aminoglycoside derivatives with reduced toxicity e.g. (Shulman E et al. 2014; and Xue X et al. 2014) and discovering non-aminoglycoside RT compounds such as RTC13, RTC14, GJ71, GJ72 and PTC124 (Gatti R A. 2012; and Welch E M et al. 2007). These compounds increased protein production in several cell culture and animal disease models, but often at the limit of detection by western blotting for endogenous gene expression and with variable responses between genes, cell lines, and PTC mutations.


Furthermore, there are numerous approaches to read-through therapy. For example, read-through drugs, suppressor tRNAs, PTC pseudouridylation, and inhibition of nonsense-mediated mRNA decay (Keeling, K. M. et al. 2104).


PTC124 (Translarna™) is the sole new compound to have entered clinical trials. It is orally bioavailable and has a good safety profile compared with aminoglycosides. PTC124's PTC RT activity has been challenged based on artifactual activity in luciferase reporter assays of the type used for its discovery and lack of demonstrable RT activity in other reporter assays (McElroy S P et al. 2013). Nevertheless, it has shown activity in higher model systems, including increased dystrophin expression and muscle function in the mdx mouse (Welch E M et al. 2007) and CFTR protein expression and improved chloride conductance in the intestine of the G542X-hCFTR mouse (Du M et al. 2008). Recently, a phase 3 clinical trial in CFTR (Kerem E. 2014) and a phase 2b trial in DMD patients (Bushby K et al. 2014) both failed to reach statistical significance. However, retrospective analyses hinted at signs of efficacy in subgroups of authorization for DMD treatment in the European Union, conditional upon completion of a phase 3 trial (mid-2015) and submission of additional safety and efficacy data (Ryan N J. 2014).


Overall, currently available RT compounds suffer two major limitations: they display low activity, typically inducing less than 5% of wild-type (wt) protein levels; and they show unpredictable activity in only a small subset of genetic disease systems tested.


SUMMARY

This invention is based in part on the discovery that compounds described herein suppress premature termination codons. Specifically, compounds identified herein, show the ability to read through premature stop codons.


In accordance with one embodiment, there is provided a pharmaceutical composition including 1) a compound, or a pharmaceutically acceptable salt thereof, in an amount effective for treating or ameliorating a medical condition associated with premature termination codons (PTCs) in RNA, wherein the compound has the structure of Formula II:




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wherein M may be




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and 2) a pharmaceutically acceptable excipient or pharmaceutically acceptable carrier.


In accordance with a further embodiment, there is provided a pharmaceutical composition including i) a compound, or a pharmaceutically acceptable salt thereof, in an amount effective for treating or ameliorating a medical condition associated with premature termination codons (PTCs) in RNA, wherein the compound has the structure of Formula I:




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wherein R may be OH or NH2;


M may be




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when R is OH and M may be




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when R is NH2; and 2) a pharmaceutically acceptable excipient or pharmaceutically acceptable carrier.


In accordance with a further embodiment, there is provided a method of treating or ameliorating a medical condition associated with premature termination codons (PTCs) in RNA, the method including administering a compound, or a pharmaceutically acceptable salt thereof, in an amount effective for treating or ameliorating a medical condition associated with a PTC in RNA, wherein the compound has the structure of Formula II:




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wherein M may be




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to a subject in need thereof. Alternatively, the method may have a compound of Formula I.


In accordance with a further embodiment, there is provided a method of promoting read-through of a premature termination codon (PTC) in a RNA sequence, the method including administering a compound, or a pharmaceutically acceptable salt thereof, in an amount effective for treating or ameliorating a medical condition associated with a PTC in RNA, wherein the compound has the structure of Formula II:




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wherein M may be




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to a subject in need thereof. Alternatively, the compound may be of Formula I.


In accordance with a further embodiment, there is provided a method of promoting production of a functional protein in a cell, the protein encoded by a nucleotide sequence comprising a premature termination codon (PTC), the method comprising contacting the cell with an effective amount of a compound having the structure of Formula II:




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wherein M may be




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to a subject in need thereof. Alternatively, the compound may be of Formula I.


In accordance with a further embodiment, there is provided a compound, wherein the compound has the structure:




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In accordance with a further embodiment, there is provided a pharmaceutical composition, the pharmaceutical composition comprising: a compound having the structure




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and a steroid.


In accordance with a further embodiment, there is provided a method of treating or ameliorating a medical condition associated with premature termination codons (PTCs) in RNA, the method including administering a compound, or a pharmaceutically acceptable salt thereof, in an amount effective to treat or ameliorate a medical condition associated with a PTC in RNA, wherein the compound has the structure of




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in combination with a steroid, to a subject in need thereof.


In accordance with a further embodiment, there is provided a use of a compound, or a pharmaceutically acceptable salt thereof, in an amount effective for treating or ameliorating a medical condition associated with premature termination codons (PTCs) in RNA, wherein the compound has the structure of Formula II:




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wherein M may be




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Alternatively, the compound may be of Formula I.


In accordance with a further embodiment, there is provided a use of a compound in the manufacture of a medicament for treatment or amelioration of a medical condition associated with premature termination codons (PTCs) in RNA, wherein the compound has the structure of Formula II:




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and wherein M may be




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Alternatively, the compound may be of Formula I.


In accordance with a further embodiment, there is provided a commercial package comprising: (a) a compound having the structure of Formula II:




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wherein M may be




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and (b) instructions for treating or ameliorating a medical condition associated with premature termination codons (PTCs) in RNA. Alternatively, the compound ma be of Formula I.


The compound may be selected from one or more of the following:




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The compound may be selected from one or more of the following:




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The compound may be selected from one or more of the following:




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The medical condition may be selected from one or more of the conditions listed in TABLE 1 or TABLE 2. The medical condition may be selected from TABLE 1 or TABLE 2. The medical condition may be selected from TABLE 1. The medical condition may be selected from TABLE 2.


The medical condition may be selected from the group consisting of: central nervous system disease; peripheral nervous system disease; neurodegenerative disease; autoimmune disease; DNA repair disease; inflammatory disease; collagen disease; kidney disease; pulmonary disease; eye disease; cardiovascular disease; blood disease; metabolic disease; neuromuscular diseases; neoplastic disease; and any genetic disorder caused by nonsense mutation(s).


The medical condition may be selected from the group consisting of: ataxia-telangiectasia; muscular dystrophy; Duchenne muscular dystrophy; Dravet syndrome; myotonic dystrophy; multiple sclerosis; infantile neuronal ceroid lipofuscinosis; Alzheimer's disease; Tay-Sachs disease; neural tissue degeneration; Parkinson's disease; chronic rheumatoid arthritis; lupus erythematosus; graft-versus-host disease; primary immunodeficiencies; severe combined immunodeficiency; DNA Ligase IV deficiency; Nijmegen breakage disorders; xeroderma pigmentosum (XP); rheumatoid arthritis; hemophilia; von Willebrand disease; thalassemia (for example; β-thalassemia); familial erythrocytosis; nephrolithiasis; osteogenesis imperfecta; cirrhosis; neurofibroma; bullous disease; lysosomal storage diseases; Hurler's disease; familial cholesterolemia; cerebellar ataxia; tuberous sclerosis; immune deficiency; cystic fibrosis; familial hypercholesterolemia; pigmentary retinopathy; retinitis pigmentosa; amyloidosis; atherosclerosis; giantism; dwarfism; hypothyroidism; hyperthyroidism; aging; obesity; diabetes mellitus; familial polycythemia; Niemann-Pick disease; epidermolysis bullosa; Marfan syndrome; Becker muscular dystrophy (BMD); spinal muscular atrophy; cancer; and any genetic disorder caused by nonsense mutation(s).


The medical condition may be cancer. The cancer may be of the head and neck, eye, skin, mouth, throat, esophagus, chest, bone, blood, lung, colon, sigmoid, rectum, stomach, prostate, breast, ovaries, kidney, liver, pancreas, brain, intestine, heart or adrenals. The cancer may be sarcoma, carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, Kaposi's sarcoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, retinoblastoma, a blood-born tumor or multiple myeloma. The cancer may be acute lymphoblastic leukemia, acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute monoblastic leukemia, acute erythroleukemic leukemia, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acute nonlymphocyctic leukemia, acute undifferentiated leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, or multiple myeloma.


The premature termination codon may be UGA or UAG. The premature termination codon may be UGA. The premature termination codon may be UAG. The premature termination codon may be UAA.


The method may further include the administration of a steroid to the subject. The steroid may be selected from one or more of the following: Medroxyprogesterone; Betamethasone; Dexamethasone; Beclomethasone; Budesonide; Clobetasol propionate; Cortisone acetate; Flumethasone Pivalate; Fluticasone Propionate; Hydrocortisone; Methylprednisolone; Paramethasone; Prednisolone; Prednisone; Triamcinolone; Danazol; Fludrocortisone; Mifepristone; Megestrol acetate; and Progesterone.


The compound may be




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BRIEF DESCRIPTION OF THE DRAWINGS


FIG. 1 shows the structures of Gentamicins C1, C1a, C2, C2a, C2b, B, B1, A, G418, X2, Sisomicin, Garamine and Ring C, as well as the structure of some of the steroids tested in combination with G418.



FIG. 2 shows the induction of PTC read-through by gentamicin B1 and X2 using the 96-well plate immunofluorescence assay, wherein those not shown on the graph had no read-through activity.



FIG. 3A shows the induction of full-length p53 by gentamicin B1, gentamicin X2, G418 and gentamicin measured by western analysis, where the intensity of the full-length (FL) and truncated p53 (TR) bands is shown relative to the intensity of the truncated p53 band seen in untreated cells and is displayed under the lanes.



FIG. 3B shows the induction of PTC read-through by G418, gentamicin, gentamicin B1 and gentamicin X2 using western analysis, wherein the amount of full-length p53 observed in FIG. 3A was plotted versus the concentration of the different compounds on a log scale.



FIG. 4 shows the induction of full-length p53 by gentamicin G418 in combination with a steroid (A) Dexamethasone; and (B) Betamethasone and Medroxyprogesterone Acetate (Medroxy Pro).



FIG. 5 shows the induction of premature termination codon (PTC) readthrough by gentamicin B1 and gentamicin X2.



FIG. 6 shows induction of PTC readthrough at TGA, TAG and TAA termination codons by gentamicin B1.



FIG. 7 shows induction of PTC readthrough in variety of cancer cell lines—SW900; NCI-H1688; ESS-1; SK-MES-1; HCC1937; H1299; and HCT116.



FIG. 8 shows induction of PTC readthrough in a mouse in vivo assay.



FIG. 9 shows induction of PTC readthrough by Gentamicin B1 in cells derived from patients with rare genetic diseases, wherein Panels A and B show Neuronal Ceroid Lipofuscinosis; Panel C shows Duchenne Muscular Dystrophy; Panel D shows Schimke Immuno-Osseous Dysplasia; and Panel E shows Recessive Dystrophic Epidermolysis Bullosa.





DETAILED DESCRIPTION

In some embodiments, the compounds described herein may be used to treat or ameliorate various indications, including medical conditions associated with premature termination codons (PTCs) in RNA, including various cancers. The various conditions may be found in TABLE 1.









TABLE 1







Medical Conditions Associated with PTC









Gene


Medical Condition Associated with PTC
symbol





Pk synthase deficiency (p phenotype)
A4GALT


Triple-A syndrome
AAAS


Ichthyosis, harlequin
ABCA12


Ichthyosiform erythroderma, congenital, nonbullous
ABCA12


Fatal surfactant deficiency
ABCA3


Fundus flavimaculatus, late onset
ABCA4


Stargardt disease
ABCA4


Intrahepatic cholestasis, familial progressive 2
ABCB11


Intrahepatic cholestasis of pregnancy
ABCB4


Intrahepatic cholestasis, familial progressive
ABCB4


Dubin-Johnson syndrome
ABCC2


Pseudoxanthoma elasticum
ABCC6


Pseudoxanthoma elasticum, autosomal recessive
ABCC6


Pseudoxanthoma elasticum, autosomal dominant
ABCC6


Hyperinsulinism
ABCC8


Hypoglycaemia, persistent hyperinsulinaemic
ABCC8


Adrenoleukodystrophy
ABCD1


Sitosterolaemia
ABCG5


Sitosterolaemia
ABCG8


Chanarin-Dorfman syndrome
ABHD5


Medium chain acyl CoA dehydrogenase deficiency
ACADM


Very long chain acyl-CoA dehydrogenase deficiency
ACADVL


Alpha actin 3 deficiency
ACTN3


Haemorrhagic telangiectasia 2
ACVRL1


Adenosine deaminase deficiency
ADA


Weill-Marchesani syndrome
ADAMTS10


Thrombotic thrombocytopaenic purpura
ADAMTS13


Upshaw-Schulman syndrome
ADAMTS13


Geleophysic dysplasia
ADAMTSL2


Ectopia lentis, isolated form
ADAMTSL4


Dyschromatosis symmetrica hereditaria
ADAR


Parkinson disease, association with
ADH1C


Glycogen storage disease 3
AGL


Glycogen storage disease 3a
AGL


Renal tubular dysgenesis
AGT


Hyperoxaluria
AGXT


Molar tooth sign & superior vermian dysplasia
AHI1


Joubert syndrome
AHI1


Pituitary adenoma
AIP


Leber congenital amaurosis IV
AIPL1


APECED
AIRE


Adenylate kinase deficiency
AK1


Analbuminaemia
ALB


Sjoegren-Larsson syndrome
ALDH3A2


Succinic semialdehyde dehydrogenase deficiency
ALDH5A1


Epilepsy, pyridoxine-dependent
ALDH7A1


Aldolase A deficiency
ALDOA


Fructose intolerance
ALDOB


Alstrom syndrome
ALMS1


Ichthyosis, congenital, autosomal recessive
ALOX12B


Ichthyosis, congenital, autosomal recessive
ALOXE3


Hypophosphatasia
ALPL


Spastic paralysis, infantile-onset
ALS2


Frontorhiny
ALX3


Amelogenesis imperfecta
AMELX


Adenosine monophosphate deaminase deficiency
AMPD1


Spherocytosis
ANK1


Mental retardation
AP1S2


Hermansky-Pudlak syndrome
AP3B1


Adenomatous polyposis coli
APC


Apolipoprotein A1 deficiency
APOA1


HDL deficiency with periorbital xanthelasmas
APOA1


HDL deficiency
APOA1


Apolipoprotein A1 deficiency
APOA1


Hypertriglyceridaemia
APOA5


Hypobetalipoproteinaemia
APOB


Apolipoprotein B deficiency
APOB


Apolipoprotein C2 deficiency
APOC2


Adenine phosphoribosyltransferase deficiency
APRT


Diabetes insipidus, nephrogenic
AQP2


Androgen insensitivity syndrome
AR


Arginase deficiency
ARG1


X-linked with epilepsy
ARHGEF9


Mental retardation
ARHGEF9


Bardet-Biedl syndrome
ARL6


Cancer, association with
ARL11


Metachromatic leukodystrophy
ARSA


Mucopolysaccharidosis VI
ARSB


Chondrodysplasia punctata
ARSE


Dombrock blood group variation
ART4


Lissencephaly, X-linked, with abnormal genitalia
ARX


Argininosuccinate lyase deficiency
ASL


Canavan disease
ASPA


Primary microcephaly
ASPM


Polycystic kidney disease 1
ASS1


Citrullinaemia
ASS1


Ataxia telangiectasia
ATM


Mantle cell lymphoma
ATM


Hemiplegic migraine
ATP1A2


Darier disease
ATP2A2


Hailey-Hailey disease
ATP2C1


Cutis laxa, autosomal recessive, type 2
ATP6V0A2


Distal renal tubular acidosis, autosomal recessive
ATP6V0A4


Menkes syndrome
ATP7A


Wilson disease
ATP7B


Intrahepatic cholestasis, familial progressive
ATP8B1


Intrahepatic cholestasis, benign recurrent
ATP8B1


ATRX syndrome
ATRX


3-methylglutaconic aciduria type 1
AUH


Diabetes insipidus, neurohypophyseal
AVP


Diabetes insipidus, central
AVP


Diabetes insipidus, nephrogenic
AVPR2


Tooth agenesis and colorectal cancer
AXIN2


B3GALNT1 deficiency (P2K phenotype)
B3GALNT1


Cholinesterasaemia
BCHE


Butyrylcholinesterase variant
BCHE


Maple syrup urine disease
BCKDHA


Maple syrup urine disease
BCKDHB


Oculofaciocardiodental syndrome
BCOR


Bestrophinopathy
BEST1


Cleft lip and palate
BMP4


Juvenile polyposis syndrome
BMPR1A


Polyposis, juvenile intestinal
BMPR1A


Pulmonary hypertension, primary
BMPR2


Pulmonary arterial hypertension
BMPR2


Pulmonary hypertension, primary
BMPR2


Breast cancer
BRCA1


Breast and/or ovarian cancer
BRCA1


Breast cancer
BRCA2


Breast and/or ovarian cancer
BRCA2


Berardinelli-Seip lipodystrophy
BSCL2


Bartter syndrome with sensorineural deafness
BSND


Biotinidase deficiency
BTD


Agammaglobulinaemia
BTK


Premature chromatid separation syndrome
BUB1B


Complement C1S deficiency
C1S


Complement C3 deficiency
C3


Complement C5 deficiency
C5


Complement C7 deficiency
C7


Complement C8 alpha-gamma deficiency
C8A


Carbonic anhydrase deficiency
CA2


Cone-rod synaptic disorder
CABP4


Episodic ataxia 2
CACNA1A


Night blindness, congenital stationary, incomplete
CACNA1F


Muscular dystrophy, limb girdle
CAPN3


Ventricular tachycardia, polymorphic
CASQ2


Hypercalcaemia, hypocalciuric
CASR


Berardinelli-Seip lipodystrophy
CAV1


Joubert syndrome
CC2D2A


Joubert syndrome
CC2D2A


Cerebral cavernous malformations
CCM2


CD36 deficiency
CD36


Hyper-IgM syndrome
CD40LG


Cromer blood group
CD55


Agammaglobulinaemia
CD79B


Hyperparathyroidism, primary
CDC73


Gastric cancer
CDH1


Usher syndrome 1d
CDH23


Hypotrichosis with juvenile macular dystrophy
CDH3


Rett syndrome, atypical
CDKL5


Pituitary and parathyroid tumours
CDKN1B


Melanoma
CDKN2A


Hypotrichosis simplex of the scalp
CDSN


Bardet-Biedl syndrome
CEP290


Leber congenital amaurosis
CEP290


Cholesterol ester transfer protein deficiency
CETP


Drusen, basal laminar
CFH


Cystic fibrosis
CFTR


Congenital absence of vas deferens
CFTR


Elevated sweat chloride concentration
CFTR


CHARGE syndrome
CHD7


Choroideraemia
CHM


Frontotemporal dementia
CHMP2B


Fetal akinesia deformation sequence disorder
CHRND


Congenital myasthenic syndrome
CHRNE


Slow channel myasthenic syndrome
CHRNE


Macular corneal dystrophy, type 1
CHST6


Immunodeficiency
CIITA


Myotonia congenita
CLCN1


Myotonia, Becker
CLCN1


Myotonia
CLCN1


Low molecular weight proteinuria
CLCN5


Dent disease
CLCN5


Dent (Japan) disease
CLCN5


Bartter syndrome 4, digenic
CLCNKA


Bartter syndrome 3
CLCNKB


Neuronal ceroid lipofuscinosis, juvenile
CLN3


Neuronal ceroid lipofuscinosis, late infantile
CLN5


Neuronal ceroid lipofuscinosis, late infantile
CLN6


Retinitis pigmentosa
CNGA1


Achromatopsia
CNGB3


Congenital disorder of glycosylation IIh
COG8


Metaphyseal chondrodysplasia, Schmid
COL10A1


Stickler syndrome, without eye involvement
COL11A2


Epidermolysis bullosa
COL17A1


Epidermolysis bullosa, junctional
COL17A1


Epidermolysis bullosa, atrophic benign
COL17A1


Osteogenesis imperfecta I
COL1A1


Osteogenesis imperfecta
COL1A1


Ehlers-Danlos syndrome VII
COL1A2


Stickler syndrome
COL2A1


Spondyloperipheral dysplasia
COL2A1


Ehlers-Danlos syndrome IV
COL3A1


Alport syndrome
COL4A3


Alport syndrome
COL4A5


Ullrich congenital muscular dystrophy
COL6A1


Myosclerosis myopathy
COL6A2


Ullrich congenital muscular dystrophy
COL6A3


Epidermolysis bullosa
COL7A1


Epidermolysis bullosa dystrophica
COL7A1


Endplate acetylcholinesterase deficiency
COLQ


Aceruloplasminaemia with diabetes
CP


Aceruloplasminaemia
CP


Coproporphyria
CPOX


Harderoporphyria
CPOX


Coproporphyria
CPOX


Carbamoyl phosphate synthetase I deficiency
CPS1


Carnitine palmitoyltransferase 1 deficiency
CPT1A


Leber congenital amaurosis
CRB1


Mental retardation, non-syndromic, autosomal recessive
CRBN


Rubinstein-Taybi syndrome
CREBBP


Crisponi syndrome
CRLF1


Congenital cataract
CRYAA


Cataract, autosomal dominant
CRYBB1


Cataract
CRYGC


Cataract, pediatric
CRYGD


Cataract
CRYGD


Cystinosis
CTNS


Pancreatitis, chronic
CTRC


Papillon-Lefevre syndrome
CTSC


Pycnodysostosis
CTSK


3-M syndrome
CUL7


Methaemoglobinaemia 2
CYB5R3


Methaemoglobinaemia
CYB5R3


Chronic granulomatous disease
CYBA


Chronic granulomatous disease
CYBB


Trichoepithelioma, multiple familial
CYLD


Adrenal hyperplasia
CYP11B1


Steroid-11 beta-hydroxylase deficiency
CYP11B1


Adrenal hyperplasia
CYP11B1


Steroid-11 beta-hydroxylase deficiency
CYP11B1


17-alpha-hydroxylase/17,20-lyase deficiency
CYP17A1


Glaucoma, primary congenital
CYP1B1


Adrenal hyperplasia
CYP21A2


Non-classic 21-hydroxylase deficiency
CYP21A2


Adrenal hyperplasia
CYP21A2


Pseudovitamin D-deficiency rickets
CYP27B1


Null allele
CYP2A13


Cytochrome P450 deficiency
CYP2D6


CYP2G deficiency, association with
CYP2G2P


Null allele
CYP4A22


Bietti crystalline corneoretinal dystrophy
CYP4V2


Spastic paraplegia
CYP7B1


Maple syrup urine disease
DBT


Immunodeficiency, severe combined
DCLRE1C


Subcortical band heterotopia
DCX


Double cortex syndrome
DCX


Usher syndrome 2
DFNB31


Progressive hearing loss, autosomal recessive
DFNB59


Mitochondrial DNA depletion syndrome
DGUOK


Smith-Lemli-Opitz syndrome
DHCR7


Spondylocostal dysostosis
DLL3


Muscular dystrophy, Duchenne
DMD


Dystrophinopathy
DMD


Muscular dystrophy, Becker
DMD


Primary ciliary dyskinesia and situs inversus
DNAH11


Primary ciliary dyskinesia
DNAH5


Primary ciliary dyskinesia
DNAI1


Primary ciliary dyskinesia
DNAI2


Systemic lupus erythematosus
DNASE1


Immunodeficiency, centromeric instability and facial anomalies
DNMT3B


syndrome


Dihydropyrimidine dehydrogenase deficiency
DPYD


Receptor deficiency
DRD5


Striate palmoplantar keratoderma
DSG1


Cardiomyopathy, arrhythmogenic right ventricular
DSG2


Dilated cardiomyopathy, woolly hair, keratoderma
DSP


Dentinogenesis imperfecta Shields type II
DSPP


Hypothyroidism
DUOX2


Hypothyroidism, transient
DUOX2


Hypothyroidism, transient
DUOX2


Hypothyroidism
DUOX2


Hypothyroidism
DUOXA2


Smith-McCort dysplasia
DYM


Dyggve-Melchior-Clausen syndrome
DYM


Muscular dystrophy, limb girdle
DYSF


Miyoshi myopathy
DYSF


Chondrodysplasia punctata, X-linked
EBP


CHILD syndrome
EBP


Lipoid proteinosis
ECM1


Ectodermal dysplasia
EDA


Ectodermal dysplasia, hypohidrotic
EDAR


Waardenburg-Hirschsprung disease
EDNRB


ABCD syndrome
EDNRB


Craniofrontonasal syndrome
EFNB1


Erythrocytosis
EGLN1


Mental retardation
EHMT1


Wolcott-Rallison syndrome
EIF2AK3


Leukoencephalopathy with vanishing white matter
EIF2B4


Prostate cancer
ELAC2


Supravalvular aortic stenosis
ELN


Amelogenesis imperfecta, hypoplastic
ENAM


Haemorrhagic telangiectasia 1
ENG


Idiopathic infantile arterial calcification
ENPP1


Prostate cancer, increased risk, in African Americans, association with
EPHB2


Erythrocytosis
EPOR


Xeroderma pigmentosum (B)
ERCC3


Xeroderma pigmentosum/Cockayne syndrome
ERCC3


Cockayne syndrome
ERCC8


SC Phocomelia
ESCO2


Glutaricacidaemia 2a
ETFA


Electron transfer flavoprotein deficiency
ETFA


Multiple exostoses
EXT1


Multiple exostoses
EXT2


Branchio-oto-renal/branchiootic syndrome
EYA1


Branchio-oto-renal syndrome
EYA1


Factor XI deficiency
F11


Factor XIII deficiency
F13A1


Factor V deficiency
F5


Factor VII deficiency
F7


Haemophilia A
F8


Haemophilia B
F9


Tyrosinaemia 1
FAH


Amelogenesis imperfecta, hypoplastic local
FAM83H


Amelogenesis imperfecta, hypocalcified
FAM83H


Fanconi anaemia
FANCA


Fanconi anaemia
FANCC


Fanconi anaemia
FANCG


Cytochrome c oxidase deficiency
FASTKD2


Marfan syndrome
FBN1


Ectopia lentis
FBN1


Fibrillinopathy
FBN1


Kindler syndrome
FERMT1


Afibrinogenaemia
FGA


Dysfibrinogenaemia
FGA


Hypofibrinogenaemia
FGB


Afibrinogenaemia
FGB


Charcot-Marie-Tooth disease 4H
FGD4


Lacrimo-auriculo-dento-digital syndrome
FGF10


Kallmann syndrome
FGFR1


Afibrinogenaemia
FGG


Leiomyomatosis and renal cell cancer
FH


Fumarase deficiency
FH


Cutaneous leiomyomatosis
FH


Muscular dystrophy, Fukuyama
FKTN


Pneumothorax, primary spontaneous
FLCN


Birt-Hogg-Dub syndrome
FLCN


Ichthyosis vulgaris
FLG


Ichthyosis vulgaris
flg10.2


Heterotopia, periventricular
FLNA


Myopathy, myofibrillar
FLNC


FMO1 variant
FMO1


FMO2 variant
FMO2


Trimethylaminuria
FMO3


fmo6 variant
FMO6P


Axenfeld-Rieger & Peters' anomaly
FOXC1


Axenfeld-Rieger anomaly
FOXC1


Lymphoedema-distichiasis
FOXC2


Aphakia, congenital, primary
FOXE3


ACD/MPV with cardiovascular malformations
FOXF1


Blepharophimosis/ptosis/epicanthus inversus syndrome
FOXL2


Developmental verbal dyspraxia
FOXP2


Follicle-stimulating hormone deficiency
FSHB


Mental retardation
FTSJ1


Fucosidosis
FUCA1


H antigen, Bombay phenotype
FUT1


H antigen, para-Bombay phenotype
FUT1


Non-secretor phenotype
FUT2


Fucosyltransferase deficiency
FUT2


Fucosyltransferase deficiency
FUT6


Friedreich ataxia
FXN


Exudative vitreoretinopathy
FZD4


Glycogen storage disease 1a
G6PC


Glucose-6-phosphate dehydrogenase deficiency
G6PD


Glycogen storage disease 2
GAA


Krabbe disease
GALC


Galactosaemia epimerase deficiency
GALE


Mucopolysaccharidosis IVa
GALNS


Tumoural calcinosis
GALNT3


Galactosaemia
GALT


Giant axonal neuropathy
GAN


Hypoparathyroidism, deafness and renal dysplasia
GATA3


Gaucher disease 2
GBA


Glycogen storage disease 4
GBE1


Dystonia, dopa-responsive
GCH1


Diabetes, NIDDM
GCK


Diabetes, MODY2
GCK


Diabetes, MODY
GCK


Congenital cataract
GCNT2


Demyelinating peripheral neuropathy
GDAP1


Charcot-Marie-Tooth disease 4A
GDAP1


Charcot-Marie-Tooth disease, autosomal recessive
GDAP1


Brachydactyly, type C
GDF5


Laron dwarfism
GHR


Growth hormone insensitivity
GHR


Growth hormone deficiency
GHRHR


Growth hormone deficiency, isolated
GHSR


Oculodentodigital dysplasia
GJA1


Charcot-Marie-Tooth disease
GJB1


Deafness, autosomal recessive 1
GJB2


Deafness
GJB2


Deafness, non-syndromic, autosomal dominant
GJB3


Pelizaeus-Merzbacher-like disease
GJC2


Glycerol kinase deficiency
GK


Fabry disease
GLA


Gangliosidosis GM1
GLB1


Hyperglycinaemia, non-ketotic
GLDC


Hyperglycinaemia, non-ketotic
GLDC


Hyperglycinaemia, non-ketotic
GLDC


Hyperglycinaemia, non-ketotic
GLDC


Pallister-Hall syndrome
GLI3


Greig cephalopolysyndactyly syndrome
GLI3


Postaxial polydactyly A/B
GLI3


Hyperekplexia
GLRA1


Gangliosidosis GM2
GM2A


Albright hereditary osteodystrophy
GNAS


Progressive osseous heteroplasia
GNAS


Mucolipidosis II
GNPTAB


Mucopolysaccharidosis IIId
GNS


Bernard-Soulier syndrome
GP1BA


Giant platelet disorder
GP1BB


Bernard-Soulier syndrome
GP9


Simpson-Golabi-Behmel syndrome
GPC3


Glucosephosphate isomerase deficiency
GPI


Albinism, ocular
GPR143


Febrile and afebrile seizures
GPR98


Hyperoxaluria II
GRHPR


Frontotemporal dementia
GRN


Alzheimer disease
GRN


Glutathione synthetase deficiency
GSS


Leber congenital amaurosis
GUCY2D


Mucopolysaccharidosis VII
GUSB


Hypoglycaemia, hyperinsulinaemic
HADH


Thalassaemia alpha
HBA2


Thalassaemia beta
HBB


Microphthalmia, syndromic 7
HCCS


Tay-Sachs disease
HEXA


Sandhoff disease
HEXB


Haemochromatosis
HFE


Haemochromatosis
HFE2


Alkaptonuria
HGD


Mucopolysaccharidosis IIIC
HGSNAT


HLA-A null allele
HLA-A


HLA-B null allele
HLA-B


Holocarboxylase synthetase deficiency
HLCS


Porphyria, acute intermittent
HMBS


HMG-CoA lyase deficiency
HMGCL


3-hydroxy-3-methylglutaric aciduria
HMGCL


HMG-CoA lyase deficiency
HMGCL


Diabetes, MODY3
HNF1A


Diabetes, MODY
HNF1B


GCKD with early-onset diabetes
HNF1B


Diabetes, MODY1
HNF4A


Hand-foot-genital syndrome
HOXA13


Tyrosinaemia 3
HPD


Lesch-Nyhan syndrome
HPRT1


Hypoxanthine guanine phosphoribosyltransferase deficiency
HPRT1


Hermansky-Pudlak syndrome
HPS1


Hermansky-Pudlak syndrome
HPS4


Atrichia with papular lesions
HR


Congenital atrichia
HR


Adrenal hyperplasia
HSD3B2


Cataract, autosomal recessive
hsf4b


Schwartz-Jampel syndrome type 1
HSPG2


CARASIL
HTRA1


Mucopolysaccharidosis II
IDS


Scheie syndrome
IDUA


Hurler syndrome
IDUA


Reduced activity
IFIH1


Growth retardation
IGF1R


Acid-labile subunit deficiency
IGFALS


Spinal muscular atrophy with respiratory distress 1
IGHMBP2


Spinal muscular atrophy with respiratory distress 1
IGHMBP2


Spinal muscular atrophy with respiratory distress 1
IGHMBP2


Incontinentia pigmenti
IKBKG


Incontinentia pigmenti, familial
IKBKG


Mental retardation, X-linked
IL1RAPL1


Immunodeficiency, severe combined
IL2RG


Immunodeficiency, severe combined
IL7R


Leprechaunism
INSR


Insulin resistance
INSR


Insulin resistance A
INSR


Senior-Loken syndrome 5
IQCB1


Van der Woude syndrome
IRF6


Popliteal pterygium syndrome
IRF6


Diabetes, type 2
ISL1


Glanzmann thrombasthenia
ITGA2B


Leukocyte adhesion deficiency
ITGB2


Glanzmann thrombasthenia
ITGB3


Epidermolysis bullosa with pyloric atresia
ITGB4


Alagille syndrome
JAG1


Immunodeficiency, severe combined
JAK3


Kallmann syndrome
KAL1


Atrial fibrillation
KCNA5


Miscarriage and intrauterine foetal loss
KCNH2


Long QT syndrome
KCNH2


Hyperinsulinism
KCNJ11


Long QT syndrome
KCNQ1


Cone dystrophy with supernormal rod ERG
KCNV2


Mental retardation, X-linked
KDM5C


Kell blood group variation
KEL


K(null) phenotype
KEL


Cornea plana 2
KERA


Goldberg-Shprintzen syndrome
KIAA1279


Piebaldism
KIT


Prostate cancer
KLF6


Cerebral cavernous malformations
KRIT1


Dowling-Degos disease
KRT5


Epidermolysis bullosa, Dowling-Meara
KRT5


Epidermolysis bullosa simplex
KRT5


Epidermolytic hyperkeratosis
KRT10


Epidermolysis bullosa, Koebner
KRT14


Naegeli syndrome
KRT14


Dermatopathia pigmentosa reticularis
KRT14


Hydrocephalus, X-linked
L1CAM


L-2-Hydroxyglutaric aciduria
L2HGDH


Muscular dystrophy, merosin deficient
LAMA2


Laminin alpha 2 chain deficiency, partial
LAMA2


Epidermolysis bullosa, Herlitz
LAMA3


Laryngo-onycho-cutaneous syndrome
LAMA3


Cardiomyopathy, dilated
LAMA4


Epidermolysis bullosa, Herlitz
LAMB3


Epidermolysis bullosa, junctional
LAMB3


Epidermolysis bullosa, Herlitz
LAMC2


Epidermolysis bullosa, junctional
LAMC2


Danon disease
LAMP2


Pelger-Huet anomaly
LBR


Leber congenital amaurosis
LCA5


Lecithin:cholesterol acyltransferase deficiency
LCAT


Lactase deficiency, congenital
LCT


Lactate dehydrogenase deficiency
LDHB


Hypercholesterolaemia
LDLR


Left-right axis malformation
LEFTY2


Osteopoikilosis
LEMD3


Leydig cell hypoplasia
LHCGR


Pseudohermaphroditism
LHCGR


Wolman syndrome
LIPA


Factor V and factor VIII deficiency, combined
LMAN1


Factor V and factor VIII deficiency, combined
LMAN1


Muscular dystrophy, limb girdle
LMNA


Muscular dystrophy, Emery-Dreifuss
LMNA


Cardiomyopathy, dilated
LMNA


Nail patella syndrome
LMX1B


Lipoprotein lipase deficiency
LPL


Hypertriglyceridaemia
LPL


Lipoprotein lipase deficiency, association with
LPL


Deafness, non-syndromic
lrtomt2


Oligodontia
LTBP3


Chediak-Higashi syndrome
LYST


Hypospadias
MAMLD1


Mannosidosis, alpha
MAN2B1


Mannosidosis, beta, lysosomal
MANBA


Obesity, autosomal dominant
MC4R


3-methylcrotonyl-CoA carboxylase deficiency
MCCC1


3-methylcrotonyl-CoA carboxylase deficiency
MCCC2


Methylmalonic aciduria
MCEE


Factor V and Factor VIII deficiency, combined
MCFD2


Mucolipidosis IV
MCOLN1


Rett syndrome
MECP2


Myocardial infarction
MEF2A


Mediterranean fever, familial
MEFV


Multiple endocrine neoplasia 1
MEN1


Spondylocostal dysostosis
MESP2


Neuronal ceroid lipofuscinoses, late infantile
MFSD8


Opitz G/BBB syndrome
MID1


Bardet-Biedl syndrome
MKKS


Colorectal cancer, non-polyposis
MLH1


Colorectal cancer, young-onset
MLH1


Colorectal cancer
MLH1


Gastrointestinal cancer
MLH1


Lynch syndrome-associated breast cancer
MLH1


Colorectal cancer, early onset
MLH1


Methylmalonic aciduria
MMAB


Methylmalonic aciduria, cblB type
MMAB


Fetomaternal alloimmunisation
MME


Osteolysis, idiopathic, Saudi type
MMP2


Currarino syndrome
MNX1


Xanthinuria, type 2
MOCOS


Amegakaryocytic thrombocytopaenia, congenital
MPL


Mercaptopyruvate sulphurtransferase deficiency, association with
MPST


Mitochondrial DNA depletion syndrome, hepatocerebral
MPV17


Charcot-Marie-Tooth disease 1b
MPZ


Charcot-Marie-Tooth disease 1
MPZ


Ataxia telangiectasia-like disease
MRE11A


Mitochondrial respiratory chain disorder
MRPS16


Atopy
MS4A2


Colorectal cancer, non-polyposis
MSH2


Colorectal cancer, non-polyposis
MSH6


Prostate cancer
MSR1


Witkop syndrome
MSX1


Homocystinuria
MTHFR


Methylenetetrahydrofolate reductase deficiency
MTHFR


Homocystinuria
MTHFR


Myotubular myopathy
MTM1


Methionine synthase deficiency
MTR


Methionine synthase reductase deficiency
MTRR


Abetalipoproteinaemia
MTTP


Methylmalonic aciduria
MUT


Mevalonic kinase deficiency
MVK


Hyperimmunoglobulin D and periodic fever syndrome
MVK


Hyperimmunoglobulin D and periodic fever syndrome
MVK


Cardiomyopathy, hypertrophic
MYBPC3


Cardiomyopathy, hypertrophic
MYBPC3


Feingold syndrome
MYCN


Hearing impairment
MYH14


Cardiomyopathy, hypertrophic
MYH7


May-Hegglin anomaly
MYH9


Deafness, non-syndromic, autosomal recessive
MYO15A


Sensorineural deafness, nonsyndromic
MYO1A


Microvillus inclusion disease
MYO5B


Deafness, autosomal dominant 22
MYO6


Deafness, autosomal recessive
MYO6


Usher syndrome 1b
MYO7A


Sanfilippo syndrome B
NAGLU


Fertility defects
NBN


Chronic granulomatous disease
NCF1


Chronic granulomatous disease
NCF2


Norrie disease
NDP


Mitochondrial complex I deficiency
NDUFAF2


Complex 1 deficiency
NDUFS4


Nemaline myopathy
NEB


Charcot-Marie-Tooth disease
NEFL


Sialidosis
NEU1


Sialidosis 2
NEU1


Neurofibromatosis 1
NF1


Neurofibromatosis 2
NF2


Ectodermal dysplasia, anhidrotic with immune deficiency
NFKBIA


Myoclonic epilepsy of Lafora
NHLRC1


Ichthyosis, autosomal recessive
NIPAL4


Cornelia de Lange syndrome
NIPBL


Benign hereditary chorea
NKX2-1


Hypothyroidism
NKX2-1


Periodic fever syndrome
NLRP12


Familial cold autoinflammatory syndrome
NLRP3


Primary ciliary dyskinesia
NME8


Stapes ankylosis with broad thumb and toes
NOG


Niemann-Pick disease C
NPC1


Niemann-Pick type C2 disease
NPC2


Nephronophthisis 1
NPHP1


Nephronophthisis 3
NPHP3


Nephronophthisis 4
NPHP4


Congenital nephrotic syndrome, Finnish type
NPHS1


Nephrotic syndrome
NPHS1


Nephrotic syndrome, steroid resistant
NPHS2


Nephrotic syndrome
NPHS2


Acromesomelic dysplasia, Maroteaux type
NPR2


Adrenal hypoplasia
NR0B1


Enhanced S cone syndrome
NR2E3


Pseudohypoaldosteronism 1
NR3C2


XY sex reversal, without adrenal failure
NR5A1


Sotos syndrome
NSD1


CHILD syndrome
NSDHL


Pain insensitivity, congenital
NTRK1


Gyrate atrophy
OAT


Albinism, oculocutaneous II
OCA2


Lowe oculocerebrorenal syndrome
OCRL


Oral-facial-digital syndrome 1
OFD1


Optic atrophy 1
OPA1


Mental retardation syndrome, X-linked
OPHN1


X-linked cone dystrophy
orf15


Atrophic macular degeneration
orf15


Retinitis pigmentosa, X-linked
orf15


Osteopetrosis, autosomal recessive
OSTM1


Ornithine transcarbamylase deficiency
OTC


Ornithine transcarbamylase deficiency
OTC


Ornithine transcarbamylase deficiency
OTC


Deafness, autosomal recessive 9
OTOF


Deafness, non-syndromic
OTOF


Lissencephaly, isolated
PAFAH1B1


Subcortical band heterotopia
PAFAH1B1


Phenylketonuria
PAH


HARP syndrome
PANK2


Pantothenate kinase-associated neurodegeneration
PANK2


Spondyloepiphyseal dysplasia
PAPSS2


Parkinsonism, juvenile, autosomal recessive
PARK2


Renal hypoplasia
PAX2


Waardenburg syndrome
PAX3


Aniridia
PAX6


Oligodontia
PAX9


Hyperphenylalaninaemia
PCBD1


Propionic acidaemia
PCCA


Propionic acidaemia
PCCB


Usher syndrome 1f
PCDH15


Epilepsy and mental retardation limited to females
PCDH19


Schizophrenia
PCM1


Obesity and impaired prohormone processing
PCSK1


Low LDL cholesterol
PCSK9


Cerebral cavernous malformation
PDCD10


Retinitis pigmentosa
PDE6B


Pyruvate dehydrogenase deficiency
PDHA1


Pyruvate dehydrogenase complex deficiency
PDHX


Pyruvate dehydrogenase phosphatase deficiency
PDP1


Prolidase deficiency
PEPD


Zellweger syndrome
PEX1


Peroxisome biogenesis disorder
PEX1


Neonatal adrenoleukodystrophy
PEX10


Zellweger syndrome H
PEX13


Zellweger syndrome
PEX14


Zellweger syndrome, complementation group D
PEX16


Rhizomelic chondrodysplasia punctata
PEX7


Glycogen storage disease 7
PFKM


Rickets, hypophosphataemic
PHEX


X-linked mental retardation & cleft lip/palate
PHF8


Phosphorylase kinase deficiency
PHKA1


Liver glycogenosis 1
PHKA2


Liver glycogenosis
PHKB


Fibrosis of extraocular muscles type 2
PHOX2A


Central hypoventilation syndrome
PHOX2B


Parkinson disease, early-onset
PINK1


Axenfeld-Rieger syndrome
PITX2


Polycystic kidney disease 1
PKD1


Polycystic kidney disease 2
PKD2


Polycystic kidney disease
PKHD1


Pyruvate kinase deficiency
PKLR


Haemolytic anaemia
PKLR


Pyruvate kinase deficiency
PKLR


Ectodermal dysplasia/skin fragility syndrome
PKP1


Infantile neuroaxonal dystrophy 1
PLA2G6


Epidermolysis bullosa with pyloric atresia
PLEC


Muscular dystrophy with epidermolysis bullosa
PLEC


Epidermolysis bullosa simplex
PLEC


Plasminogen deficiency
PLG


Ehlers-Danlos syndrome VI
PLOD1


Pelizaeus-Merzbacher disease
PLP1


Spastic paraplegia
PLP1


Congenital disorder of glycosylation 1a
PMM2


Turcot syndrome
PMS2


PNPO deficiency
PNPO


Alpers syndrome
POLG


Xeroderma pigmentosum, variant
POLH


Xeroderma pigmentosum, variant
POLH


Obesity
POMC


Walker-Warburg syndrome
POMT1


Focal dermal hypoplasia
PORCN


Pituitary hormone deficiency
POU1F1


Partial lipodystrophy
PPARG


Porphyria, variegate
PPOX


Neuronal ceroid lipofuscinosis, juvenile
PPT1


Neuronal ceroid lipofuscinosis, infantile
PPT1


Neuronal ceroid lipofuscinosis, late infantile
PPT1


Haemophagocytic lymphohistiocytosis, familial
PRF1


Perforin deficiency
PRF1


Camptodactyly-arthropathy-coxa vara-pericarditis
PRG4


Carney complex
PRKAR1A


Azoospermia
PRM2


Protein C deficiency
PROC


Hypogonadotropic hypogonadism
PROKR2


Hypogonadotropic hypogonadism
PROP1


Protein S deficiency
PROS1


High myopia
PRPH


Pattern dystrophy
PRPH2


Pancreatitis, protection against
PRSS1


Dejerine-Sottas syndrome
PRX


Charcot-Marie-Tooth disease 4
PRX


Gaucher disease, atypical
PSAP


Nevoid basal cell carcinoma syndrome
PTCH1


Cowden disease
PTEN


Hypertension
PTGIS


Osteochondrodysplasia, Blomstrand, type 1
PTH1R


Mitochondrial myopathy and sideroblastic anaemia
PUS1


McArdle disease
PYGM


Dihydropteridine reductase deficiency
QDPR


Acrocephalopolysyndactyly, type II
RAB23


Immunodeficiency, severe combined
RAG2


Immunodeficiency, severe combined, B cell −ve
RAG2


Omenn syndrome
RAG2


Smith-Magenis syndrome
RAI1


Anophthalmia
RAX


Retinoblastoma
RB1


RAPADILINO syndrome
RECQL4


Spastic paraplegia 31
REEP1


Hirschsprung disease
RET


MHC class II deficiency
RFXANK


Retinitis pigmentosa
RHO


Ribonuclease L deficiency
RNASEL


Brachydactyly, type B
ROR2


Robinow syndrome, autosomal recessive
ROR2


Brachydactyly, type B
ROR2


Retinitis pigmentosa
RP1


Retinitis pigmentosa, X-linked
RP2


Leber congenital amaurosis
RPE65


Retinitis pigmentosa, X-linked
RPGR


Diamond-Blackfan anaemia
RPS24


Coffin-Lowry syndrome
RPS6KA3


Mitochondrial DNA depletion syndrome
RRM2B


Retinoschisis, X linked juvenile
RS1


Platelet disorder, familial
RUNX1


Cleidocranial dysplasia
RUNX2


Townes-Brocks syndrome
SALL1


Goldenhar syndrome
SALL1


Townes-Brocks syndrome
SALL1


Okihiro syndrome
SALL4


Tumoural calcinosis, normophosphataemic
SAMD9


Chylomicron retention disease
SAR1B


Cleft palate, osteoporosis and cognitive defects
SATB2


Charcot-Marie-Tooth disease 4b2
SBF2


Action myoclonus-renal failure syndrome
SCARB2


Myoclonic epilepsy of infancy
SCN1A


Dravet syndrome or Dravet syndrome C or Dravet syndrome B
SCN1A


Generalized epilepsy with febrile seizures plus
SCN1A


Intractable epilepsy
SCN1A


Intractable epilepsy and mental decline
SCN2A


Brugada syndrome
SCN5A


Cardiac conduction disease
SCN5A


Channelopathy-associated insensitivity to pain
SCN9A


Cardioencephalomyopathy, fatal infantile
SCO2


Cytochrome c oxidase deficiency
SCO2


Leigh syndrome
SDHA


Phaeochromocytoma
SDHB


Paraganglioma, autosomal dominant 3
SDHC


Paraganglioma
SDHD


SEPN-related myopathy
SEPN1


Antitrypsin alpha 1 deficiency
SERPINA1


Venous thromboembolic disease
SERPINA10


Thyroxine-binding globulin deficiency
SERPINA7


Antithrombin deficiency
SERPINC1


Deep vein thrombosis
SERPINC1


Angioneurotic oedema
SERPING1


Surfactant protein B deficiency
SFTPB


Muscular dystrophy, limb girdle
SGCD


Myoclonus dystonia
SGCE


Muscular dystrophy, limb girdle
SGCG


Sanfilippo syndrome A
SGSH


Lymphoproliferative syndrome, X-linked
SH2D1A


Holoprosencephaly
SHH


Leri-Weill dyschondrosteosis
SHOX


JK-null variant
SLC14A1


Cataract, juvenile with microcornea and renal glucosuria
SLC16A12


Monocarboxylate transporter 8 deficiency
SLC16A2


Salla disease
SLC17A5


Sialic acid storage disease, infantile
SLC17A5


Megaloblastic anaemia, thiamine responsive
SLC19A2


Organic cation transporter deficiency
SLC22A4


Intrahepatic cholestasis, neonatal
SLC25A13


HHH syndrome
SLC25A15


Diarrhoea, congenital chloride
SLC26A3


Glucose transporter 1 deficiency syndrome
SLC2A1


Fanconi-Bickel syndrome
SLC2A2


Hereditary hypophosphataemic rickets with hypercalciuria
SLC34A3


Acrodermatitis enteropathica
SLC39A4


Cystinuria
SLC3A1


Spherocytosis
SLC4A1


Corneal endothelial dystrophy 2
SLC4A11


Proximal renal tubular acidosis
SLC4A4


Glucose/galactose malabsorption
SLC5A1


Renal glucosuria
SLC5A2


Iodide transport defect
SLC5A5


Hyperekplexia
SLC6A5


Creatine deficiency
SLC6A8


Lysinuric protein intolerance
SLC7A7


Cystinuria, type I/III
SLC7A9


Mal de Meleda
SLURP1


Juvenile polyposis syndrome
SMAD4


Pulmonary arterial hypertension
SMAD9


Schimke immuno-osseous dysplasia
SMARCAL1


Schimke immuno-osseous dysplasia
SMARCAL1


Spinal muscular atrophy
SMN1


Niemann-Pick disease
SMPD1


Amyotrophic lateral sclerosis
SOD1


Sclerosteosis
SOST


PCWH
SOX10


Shah-Waardenburg syndrome and neuropathy
SOX10


Hypotrichosis-Lymphoedema-Telangiectasia
SOX18


Anophthalmia, hearing loss and brain abnormalities
SOX2


Anophthalmia-oesophageal-genital syndrome
SOX2


Campomelic dysplasia
SOX9


Spastic paraplegia
SPAST


Spastic paraplegia, autosomal dominant
SPAST


Retiniitis pigmentosa, juvenile
SPATA7


Leber congenital amaurosis IV
SPATA7


Spastic paraplegia, autosomal recessive
SPG11


Spastic paraplegia with thin corpus callosum
SPG11


Netherton syndrome
SPINK5


Neurofibromatosis 1-like syndrome
SPRED1


Legius syndrome
SPRED1


Cafe-au-lait macules
SPRED1


Pyropoikilocytosis
SPTA1


Spherocytosis
SPTB


Steroid-5 alpha-reductase deficiency
SRD5A2


XY sex reversal
SRY


Gonadal dysgenesis
SRY


Amish infantile epilepsy syndrome
ST3GAL5


Congenital lipoid adrenal hyperplasia
STAR


Growth hormone insensitivity
STAT5B


Gonadotrophin-independent precocious puberty
STK11


Peutz-Jeghers syndrome
STK11


Microphthalmia
STRA6


Haemophagocytic lymphohistiocytosis, familial
STX11


Glutaric aciduria 3
SUGCT


Sulphite oxidase deficiency
SUOX


Leigh syndrome
SURF1


Epilepsy
SYN1


Schizophrenia
syngr1c


Corneal dystrophy, gelatinous drop-like
TACSTD2


Tyrosinaemia 2
TAT


Barth syndrome
TAZ


Cardiomyopathy, X-linked infantile
TAZ


Amyotrophic lateral sclerosis
TBK1


ACTH deficiency, isolated
TBX19


Congenital heart disease
TBX20


Cleft palate and ankyloglossia
TBX22


Ulnar-mammary syndrome
TBX3


Holt-Oram syndrome
TBX5


Osteopetrosis, autosomal recessive
TCIRG1


Transcobalamin II deficiency
TCN2


Treacher-Collins syndrome
TCOF1


Haemochromatosis
TFR2


Goitre with hypothyroidism
TG


Goitre, simple
TG


Holoprosencephaly
TGIF1


Ichthyosis, congenital, autosomal recessive
TGIF1


Ichthyosis, lamellar
TGM1


Dystonia
THAP1


Thyroid hormone resistance
THRB


Epidermodysplasia verruciformis
TMC6


Epidermodysplasia verruciformis
TMC8


Enteropeptidase deficiency
TMPRSS15


Microcytic anaemia & iron deficiency
TMPRSS6


Microcytic anaemia & iron deficiency
TMPRSS6


Li-Fraumeni syndrome
TP53


Multiple cancers
TP53


Osteosarcoma
TP53


Adrenocortical carcinoma
TP53


Split-hand/split-foot malformation
TP63


Nemaline myopathy
TPM3


Goitrous hypothyroidism
TPO


Neuronal ceroid lipofuscinosis, late infantile
TPP1


Spondyloepiphyseal dysplasia tarda
TRAPPC2


Deafness, non-syndromic
TRIOBP


Hypomagnesaemia with secondary hypocalcaemia
TRPM6


Tricho-rhino-phalangeal syndrome I
TRPS1


Tuberous sclerosis
TSC1


Tuberous sclerosis
TSC2


Hypothyroidism
TSHB


Hyperthyroidism
TSHR


Tibial muscular dystrophy
TTN


Cardiomyopathy, dilated
ttntvn2b


Saethre-Chotzen syndrome
TWIST1


Baller-Gerold syndrome
TWIST1


Albinism, oculocutaneous 1
TYR


Albinism, oculocutaneous 1A
TYR


Albinism, oculocutaneous 3
TYRP1


Hypotrichosis, Marie Unna type
u2hr


Angelman syndrome
UBE3A


Crigler-Najjar syndrome 1
UGT1A1


Crigler-Najjar syndrome 2
UGT1A1


Haemophagocytic lymphohistiocytosis, familial
UNC13D


Mental retardation
UPF3B


Porphyria, hepatoerythropoietic
UROD


Porphyria, cutanea tarda
UROD


Porphyria, erythropoietic
UROS


Usher syndrome 1c
USH1C


Usher syndrome 1g
USH1G


Usher syndrome 2a
USH2A


Retinitis pigmentosa, recessive, no hearing loss
USH2A


Usher syndrome 2
USH2A


Rickets, vitamin D resistant
VDR


Von Hippel-Lindau syndrome
VHL


Cerebellar hypoplasia and quadrupedal locomotion
VLDLR


Dysequilibrium syndrome
VLDLR


Chorea-acanthocytosis
VPS13A


Cohen syndrome
VPS13B


Von Willebrand disease 3
VWF


Von Willebrand disease
VWF


Von Willebrand disease 2n
VWF


Wiskott-Aldrich syndrome
WAS


Wolfram syndrome
WFS1


Neuropathy, hereditary sensory, type II
wnk1tv3


Odonto-onycho-dermal dysplasia
WNT10A


Tetra-amelia
WNT3


Werner syndrome
WRN


Wilms tumour
WT1


Renal dysfunction & renal blastema
WT1


Xanthinuria, type 1
XDH


Xeroderma pigmentosum (A)
XPA


Xeroderma pigmentosum (C)
XPC


Posterior polymorphous corneal dystrophy
ZEB1


Mowat-Wilson syndrome
ZEB2


Cardiac malformation
ZIC3


Situs abnormality
ZIC3


Mental retardation, X-linked
ZNF674
















TABLE 2







Short List of Medical Conditions Associated with PTC








Medical Condition Associated with PTC
Gene symbol





Muscular Dystrophy (Duchenne or Becker)
DMD


Chronic granulomatous disease
CYBB or NCF1 or NCF2


Late infantile neuronal ceroid lipofuscinosis
TPP1


Neuronal ceroid lipofuscinosis (juvenile, infantile or late
PPT1


infantile)


Neuronal ceroid lipofuscinosis (juvenile or late or late
CLN3, CLN5, CLN6, or


infantile)
MFSD8


Frontotemporal dementia
GRN or CHMP2B


Epidermolysis bullosa (dystrophic/dystrophica,
COL7A1 or COL17A1


junctional, atrophic benign)


Rett syndrome
MECP2


Congenital disorder of deglycosylation (IIh or 1a)
COG8 or PMM2 or NGLY1


Cystic fibrosis
CFTR


Schimke immuno-osseous dysplasia
SMARCAL1


Adenomatous polyposis coli
APC


Li-Fraumeni syndrome
TP53


Sporadic cancer
various tumour suppressor



genes including TP53









The codon changes resulting in all of the above medical conditions are well known in the art and new codon changes that result in PTC are still being discovered. Nevertheless, there is an expectation that the compounds described herein will have some degree of readthrough activity for all such PTCs.


Compounds as described herein may be in the free form or in the form of a salt thereof. In some embodiment, compounds as described herein may be in the form of a pharmaceutically acceptable salt, which are known in the art (Berge S. M. et al., J. Pharm. Sci. (1977) 66(1):1-19). Pharmaceutically acceptable salt as used herein includes, for example, salts that have the desired pharmacological activity of the parent compound (salts which retain the biological effectiveness and/or properties of the parent compound and which are not biologically and/or otherwise undesirable). Compounds as described herein having one or more functional groups capable of forming a salt may be, for example, formed as a pharmaceutically acceptable salt. Compounds containing one or more basic functional groups may be capable of forming a pharmaceutically acceptable salt with, for example, a pharmaceutically acceptable organic or inorganic acid. Pharmaceutically acceptable salts may be derived from, for example, and without limitation, acetic acid, adipic acid, alginic acid, aspartic acid, ascorbic acid, benzoic acid, benzenesulfonic acid, butyric acid, cinnamic acid, citric acid, camphoric acid, camphorsulfonic acid, cyclopentanepropionic acid, diethylacetic acid, digluconic acid, dodecylsulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, glucoheptanoic acid, gluconic acid, glycerophosphoric acid, glycolic acid, hemisulfonic acid, heptanoic acid, hexanoic acid, hydrochloric acid, hydrobromic acid, hydriodic acid, 2-hydroxyethanesulfonic acid, isonicotinic acid, lactic acid, malic acid, maleic acid, malonic acid, mandelic acid, methanesulfonic acid, 2-napthalenesulfonic acid, naphthalenedisulphonic acid, p-toluenesulfonic acid, nicotinic acid, nitric acid, oxalic acid, pamoic acid, pectinic acid, 3-phenylpropionic acid, phosphoric acid, picric acid, pimelic acid, pivalic acid, propionic acid, pyruvic acid, salicylic acid, succinic acid, sulfuric acid, sulfamic acid, tartaric acid, thiocyanic acid or undecanoic acid. Compounds containing one or more acidic functional groups may be capable of forming pharmaceutically acceptable salts with a pharmaceutically acceptable base, for example, and without limitation, inorganic bases based on alkaline metals or alkaline earth metals or organic bases such as primary amine compounds, secondary amine compounds, tertiary amine compounds, quaternary amine compounds, substituted amines, naturally occurring substituted amines, cyclic amines or basic ion-exchange resins. Pharmaceutically acceptable salts may be derived from, for example, and without limitation, a hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation such as ammonium, sodium, potassium, lithium, calcium, magnesium, iron, zinc, copper, manganese or aluminum, ammonia, benzathine, meglumine, methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, isopropylamine, tripropylamine, tributylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, glucamine, methylglucamine, theobromine, purines, piperazine, piperidine, procaine, N-ethylpiperidine, theobromine, tetramethylammonium compounds, tetraethylammonium compounds, pyridine, N,N-dimethylaniline, N-methylpiperidine, morpholine, N-methylmorpholine, N-ethylmorpholine, dicyclohexylamine, dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine, N,N′-dibenzylethylenediamine or polyamine resins. In some embodiments, compounds as described herein may contain both acidic and basic groups and may be in the form of inner salts or zwitterions, for example, and without limitation, betaines. Salts as described herein may be prepared by conventional processes known to a person skilled in the art, for example, and without limitation, by reacting the free form with an organic acid or inorganic acid or base, or by anion exchange or cation exchange from other salts. Those skilled in the art will appreciate that preparation of salts may occur in situ during isolation and purification of the compounds or preparation of salts may occur by separately reacting an isolated and purified compound.


In some embodiments, compounds and all different forms thereof (e.g. free forms, salts, polymorphs, isomeric forms) as described herein may be in the solvent addition form, for example, solvates. Solvates contain either stoichiometric or non-stoichiometric amounts of a solvent in physical association the compound or salt thereof. The solvent may be, for example, and without limitation, a pharmaceutically acceptable solvent. For example, hydrates are formed when the solvent is water or alcoholates are formed when the solvent is an alcohol.


In some embodiments, compounds and all different forms thereof (e.g. free forms, salts, solvates, isomeric forms) as described herein may include crystalline and amorphous forms, for example, polymorphs, pseudopolymorphs, conformational polymorphs, amorphous forms, or a combination thereof. Polymorphs include different crystal packing arrangements of the same elemental composition of a compound. Polymorphs usually have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability and/or solubility. Those skilled in the art will appreciate that various factors including recrystallization solvent, rate of crystallization and storage temperature may cause a single crystal form to dominate.


A PTC read-through compound may provide a therapeutic benefit if the compound permits read-through of a PTC in a protein coding sequence to produce the full length protein. Wherein the full length protein may have sequence variations and may not be the same as the native protein. Generally, the full length protein produced by the read-through is functional and can stand in for the wild-type protein. In some cases, as little as 5% of the normal total amount of the full length protein, wherein the total amount of protein, is what a subject not having the medical condition associated with the PTC would normally produce. However, depending on the medical condition associated with the PTC, as little as 1% of the normal total amount of the full length protein may be sufficient to have a therapeutic benefit. Provided that the PTC read-through compound provides read-through of a PTC in a protein coding sequence to produce at least about 1% of the normal total amount of the full length protein a therapeutic benefit may be achieved. Provided that the PTC read-through compound provides read-through of a PTC in a protein coding sequence to produce at least about 2% of the normal total amount of the full length protein a therapeutic benefit may be achieved. Provided that the PTC read-through compound provides read-through of a PTC in a protein coding sequence to produce at least about 3% of the normal total amount of the full length protein a therapeutic benefit may be achieved. Provided that the PTC read-through compound provides read-through of a PTC in a protein coding sequence to produce at least about 4% of the normal total amount of the full length protein a therapeutic benefit may be achieved. Provided that the PTC read-through compound provides read-through of a PTC in a protein coding sequence to produce at least about 5% of the normal total amount of the full length protein a therapeutic benefit may be achieved. Provided that the PTC read-through compound provides read-through of a PTC in a protein coding sequence to produce at least about 6% of the normal total amount of the full length protein a therapeutic benefit may be achieved. Provided that the PTC read-through compound provides read-through of a PTC in a protein coding sequence to produce at least about 7% of the normal total amount of the full length protein a therapeutic benefit may be achieved. Provided that the PTC read-through compound provides read-through of a PTC in a protein coding sequence to produce at least about 8% of the normal total amount of the full length protein a therapeutic benefit may be achieved. Provided that the PTC read-through compound provides read-through of a PTC in a protein coding sequence to produce at least about 9% of the normal total amount of the full length protein a therapeutic benefit may be achieved. Provided that the PTC read-through compound provides read-through of a PTC in a protein coding sequence to produce at least about 10% of the normal total amount of the full length protein a therapeutic benefit may be achieved.


Alternatively, a PTC read-through compound may provide a therapeutic benefit if the compound permits sufficient read-through of a PTC in a protein coding sequence to provide some therapeutic benefit to the subject or achieve some therapeutic result. The therapeutic benefit may be determined functionally by measuring some therapeutic result. A therapeutic result may result from a therapeutically effective amount or a prophylactically effective amount of the compound, and may include, for example, reduced tumor size, increased life span, a delay of symptom onset or disease onset, increase metabolic efficiency or increased life expectancy. A therapeutically effective amount of a compound or a prophylactically effective amount of a compound may vary according to the disease state, age, sex, other health factors unrelated to or related to the disease and weight of the subject, and the ability of the compound to elicit a desired response in the subject.


Furthermore, the read-through efficiently may be greater at TGA than TAG, and in some circumstances there may be no read-through at TAA. Accordingly, treatments may be tailored to particular stop codons.


In some embodiments, compounds and all different forms thereof (e.g. free forms, salts, solvates, polymorphs, protonated forms) as described herein include isomers such as geometrical isomers, optical isomers based on asymmetric carbon, stereoisomers, tautomers, individual enantiomers, individual diastereomers, racemates, diastereomeric mixtures and combinations thereof, and are not limited by the description of the formula illustrated for the sake of convenience.


For example, Gentamicin B1 may be represented as follows:




embedded image


In some embodiments, compounds may include analogs, isomers, stereoisomers, or related derivatives. In some embodiments the compounds may be used in conjunction with another compound to form a pharmaceutical composition.


In some embodiments, pharmaceutical compositions as described herein may comprise a salt of such a compound, preferably a pharmaceutically or physiologically acceptable salt. Pharmaceutical preparations will typically comprise one or more carriers, excipients or diluents acceptable for the mode of administration of the preparation, be it by injection, inhalation, topical administration, lavage, or other modes suitable for the selected treatment. Suitable carriers, excipients or diluents (used interchangeably herein) are those known in the art for use in such modes of administration.


Suitable pharmaceutical compositions may be formulated by means known in the art and their mode of administration and dose determined by the skilled practitioner. For parenteral administration, a compound may be dissolved in sterile water or saline or a pharmaceutically acceptable vehicle used for administration of non-water soluble compounds such as those used for vitamin K. For enteral administration, the compound may be administered in a tablet, capsule or dissolved in liquid form. The tablet or capsule may be enteric coated, or in a formulation for sustained release. Many suitable formulations are known, including, polymeric or protein microparticles encapsulating a compound to be released, ointments, pastes, gels, hydrogels, or solutions which can be used topically or locally to administer a compound. A sustained release patch or implant may be employed to provide release over a prolonged period of time. Many techniques known to one of skill in the art are described in Remington: the Science & Practice of Pharmacy by Alfonso Gennaro, 20th ed., Lippencott Williams & Wilkins, (2000). Formulations for parenteral administration may, for example, contain excipients, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for modulatory compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.


Compounds or pharmaceutical compositions as described herein or for use as described herein may be administered by means of a medical device or appliance such as an implant, graft, prosthesis, stent, etc. Also, implants may be devised which are intended to contain and release such compounds or compositions. An example would be an implant made of a polymeric material adapted to release the compound over a period of time.


An “effective amount” of a pharmaceutical composition as described herein includes a therapeutically effective amount or a prophylactically effective amount. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as reduced tumor size, increased life span or increased life expectancy. A therapeutically effective amount of a compound may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the compound to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result (for example, smaller tumors, increased life span, increased life expectancy or prevention of the progression of the medical condition associated with premature termination codons). Typically, a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a therapeutically effective amount.


It is to be noted that dosage values may vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners. The amount of active compound(s) in the composition may vary according to factors such as the disease state, age, sex, and weight of the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.


In some embodiments, compounds and all different forms thereof as described herein may be used, for example, and without limitation, in combination with other treatment methods for at least one indication selected from the group set out in TABLE 1 or TABLE 2.


In general, compounds as described herein should be used without causing substantial toxicity. Toxicity of the compounds as described herein can be determined using standard techniques, for example, by testing in cell cultures or experimental animals and determining the therapeutic index, i.e., the ratio between the LD50 (the dose lethal to 50% of the population) and the LD100 (the dose lethal to 100% of the population). In some circumstances however, such as in severe disease conditions, it may be appropriate to administer substantial excesses of the compositions. Some compounds as described herein may be toxic at some concentrations. Titration studies may be used to determine toxic and non-toxic concentrations. Toxicity may be evaluated by examining a particular compound's or composition's specificity across cell lines or in an animal model.


Compounds as described herein may be administered to a subject. As used herein, a “subject” may be a human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc. The subject may be suspected of having or at risk of having a medical condition associated with premature termination codons (PTCs).


As used herein, a “medical condition associated with premature termination codons” may be defined as any medical condition caused in whole or in part by a nonsense codon, which may result in decreased mRNA stability as well as protein truncation resulting in a non-functional protein, which in turn may directly or indirectly result in the medical condition. For example, the medical condition associated with premature termination codons may be selected from TABLE 1 or TABLE 2.


There are about 5000 or so such genetic diseases which may be grouped into broad categories, as follows: an autoimmune disease; a blood disease; a collagen disease; diabetes; a neurodegenerative disease; a cardiovascular disease; a pulmonary disease; or an inflammatory disease; a neoplastic disease or central nervous system disease. One third of the cases of genetic inherited diseases involve a premature termination codon (PTC) (Frischmeyer P A and Dietz H C 1999). In most cases, the primary mechanism whereby a nonsense mutation has an effect is through the degradation of that mRNA by a surveillance mechanism called nonsense-mediated mRNA decay (NMD) (see: Chang Y F et al. 2007; Isken O and Maquat L E 2007; Rebbapragada I and Lykke-Andersen J 2009; Rehwinkel J et al. 2006; and Muhlemann 0 et al. 2008).


Diagnostic methods for various medical conditions associated with premature termination codons are known in the art. Depending on the condition genetic diagnostics may be readily available or may be determined with directed sequencing. For example, the medical condition may be selected from the group consisting of central nervous system diseases, ataxia-telangiectasia, muscular dystrophy, Duchenne muscular dystrophy, Dravet syndrome, myotonic dystrophy, multiple sclerosis, infantile neuronal ceroid lipofuscinosis, Alzheimer's disease, Tay-Sachs disease, neural tissue degeneration, Parkinson's disease, autoimmune diseases, chronic rheumatoid arthritis, lupus erythematosus, graft-versus-host disease, primary immunodeficiencies, severe combined immunodeficiency, DNA Ligase IV deficiency, DNA repair disorders, Nijmegen breakage disorders, xeroderma pigmentosum (XP), inflammatory diseases, rheumatoid arthritis, blood diseases, hemophilia, von Willebrand disease, thalassemia (for example, β-thalassemia), familial erythrocytosis, nephrolithiasis, collagen diseases, osteogenesis imperfecta, cirrhosis, neurofibroma, bullous disease, lysosomal storage disease, Hurler's disease, familial cholesterolemia, cerebellar ataxia, tuberous sclerosis, immune deficiency, kidney disease, lung disease, cystic fibrosis, familial hypercholesterolemia, pigmentary retinopathy, retinitis pigmentosa, amyloidosis, atherosclerosis, giantism, dwarfism, hypothyroidism, hyperthyroidism, aging, obesity, diabetes mellitus, familial polycythemia, Niemann-Pick disease, epidermolysis bullosa, Marfan syndrome, neuromuscular diseases, Becker muscular dystrophy (BMD), spinal muscular atrophy, cancer, and any genetic disorder caused by nonsense mutation(s). Furthermore, where the medical condition associated with a premature termination codon is a cancer, the cancer may be selected from one or more of cancer is of the head and neck, eye, skin, mouth, throat, esophagus, chest, bone, blood, lung, colon, sigmoid, rectum, stomach, prostate, breast, ovaries, kidney, liver, pancreas, brain, intestine, heart or adrenals. Alternatively, the cancer may be selected from sarcoma, carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, Kaposi's sarcoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, retinoblastoma, a blood-born tumor or multiple myeloma. Tests for determining whether a PTC is involved in the condition are known to those of ordinary skill in the art.


Compounds tested and to be tested are set out below in TABLES A and B respectively.











TABLE A





Compound

PTC Read-


Identifier
Structure
through







Gentamicin B1 or B1


embedded image


Y





G-418 or G418


embedded image


Y





Gentamicin X2 or X2


embedded image


Y


















TABLE B





Compound

PTC Read-


Identifier
Structure
through







JI-20B CAS 51846-98-1


embedded image


Not yet tested





Analogue A


embedded image


Not yet tested





CAS# 52945-42-3


embedded image


Not yet tested









The Gentamicin complex or Gentamicin C complex as used herein includes gentamicin C1, gentamicin C1a, and gentamicin C2 (˜80% of complex) and are reported to have the most significant antibacterial activity. The remaining ˜20% of the complex is made up of Gentamicins A, B, X, et al. The exact compositions may vary between different production runs and based on the producer.


Various alternative embodiments and examples of the invention are described herein. These embodiments and examples are illustrative and should not be construed as limiting the scope of the invention.


Materials and Methods
p53 PTC Read-Through in a Human Cell Line.

Compounds were tested for PTC read-through in human cells, wherein mammary carcinoma HDQ-P1 cells homozygous for TGA (R213X) in exon 6 of the TP53 gene (Wang et al., 2000) were selected on the basis of convincing evidence of read-through by the aminoglycoside G418 (Floquet, C. et al. 2011). Western analysis using a quantitative automated capillary electrophoresis system showed that HDQ-P1 cells express very low levels of truncated p53 and no full-length p53 and that 50 μM G418 induces the formation of full-length p53 while also increasing truncated p53 levels as reported (Floquet, C. et al. 2011).


Nuclear localization sequences and a tetramerization domain located in the p53 C-terminus contribute to retaining p53 in the nucleus (Shaulsky, G et al. 1990; Liang, S. H and Clark, M. F. 2001) and p53 truncated at R213 lacks these sequences. To enable analysis of p53 R213X read-through at high throughput an automated 96-well fluorescence microscopy assay was established to detect and quantitate nuclear p53 signal. G418 induced a concentration-dependent increase in nuclear p53 consistent with read-through induction. During 72 h exposure, 50 μM G418 induced nuclear 53 expression in 9% of cells while 250 μM G418 induced nuclear p53 expression in nearly all cells.


Automated p53 Immunofluorescence 96-Well Plate Assay

HDQ-P1 cells cultured in DMEM containing 10% FBS and 1× Gibco™ antibioticantimicotic were seeded at 4000 per well of PerkinElmer View™ 96-well plates. The next day, the medium was replaced with fresh culture medium containing the compounds to be tested. After 72 h, the culture medium was removed by aspiration, the cells were fixed with 3% paraformaldehyde, 0.3% Triton X-100 and 1.5 μg/ml Hoechst 33323 in phosphate-buffered saline pH 7.2 (PBS) for 20 min at room temp. The cells were rinsed once with PBS and incubated for 2 h at room temp with a blocking solution of 3% BSA in PBS. The blocking solution was removed by aspiration and cells were incubated with 0.1 μg/ml DO-1 p53 mouse monoclonal p53 antibody (Santa Cruz™) in blocking solution for 90 min at room temp. The wells were washed once with PBS for 5 min and the cells were incubated with Alexa 488-conjugated goat anti-mouse antibody (Invitrogen Life Technologies A11029™) in blocking buffer for 90 min at room temp. The wells were washed once with PBS for 5 min, 75 μl PBS was added, the plates were covered with a black adherent membrane and stored at 4° C. overnight. Nuclear p53 immunofluorescence intensity was measured using a Cellomics ArrayScan VTI™ automated fluorescence imager.


Briefly, images were acquired with a 20× objective in the Hoechst™ and GFP (XF53) channels. Images of 15 fields were acquired for each well, corresponding to ˜2000 cells.


The Compartment Analysis bioapplication was used to identify the nuclei and define their border. The nuclear Alexa 488™ fluorescence intensity was then measured and expressed as average nuclear fluorescence intensity or % positive nuclei, using as a threshold the fluorescence intensity of nuclei from untreated cells (50-75, depending on experiment).


Automated Electrophoresis Western Analysis Assay

HDQ-P1 cells were seeded at 100,000 cells per well of TC-treated 6-well plates. The next day, the medium was replaced with fresh medium containing compounds to be tested and were incubated for 48 to 96 h. The medium was removed by aspiration, cell monolayers were rinsed with 1 ml ice-cold PBS. Cells were lysed in 80 μl lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Triton X100™, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate supplemented with fresh 1 mM Na3VO4, 1 mM dithiothreitol and 1× complete protease inhibitor cocktail (Roche Molecular Biochemicals™)). Lysates were pre-cleared by centrifugation at 18,000 g for 15 min at 4° C. Supernatants were collected, protein was quantitated using the Bradford assay and lysates were adjusted to 1 mg/ml protein. Capillary electrophoresis and western analysis conditions were carried out with manufacturer's reagents according to the user manual (ProteinSimple WES™). Briefly, 5.6 μl of cell lysate was mixed with 1.4 μl fluorescent master mix and heated at 95° C. for 5 min. The samples, blocking reagent, wash buffer, DO-1 p53 antibody (0.5 μg/ml) and vinculin antibody (1:2000, R&D clone 728526), secondary antibody and chemiluminescent substrate were dispensed into the microplate provided by the manufacturer. The electrophoretic separation and immunodetection was performed automatically using default settings. The data was analyzed with inbuilt Compass™ software (Proteinsimple™). The truncated and full-length p53 peak intensities were normalized to that of the vinculin peak, used as a loading control. Results are shown as pseudo blots and as electropherograms.


In some instances, a traditional western blotting procedure was used (e.g. FIG. 4), using the same antibodies.


Compounds Tested

Gentamicin, gentamicin A, B, B1, C1, C1a, C2, C2a, C2b, X2, sisomicin, as well as gentamicin fragments garamine and ring C (see FIG. 1) were obtained from MicroCombiChem. G418 was from Sigma™. Betamethasone, dexamethasone and medroxyprogesterone acetate were from Sigma™. Gentamicin B1 was purchased from MicroCombiChem™ (catalogue # MCC3436). Gentamicin X2 was from TokuoE (catalogue # G036). G418 from Life Technologies (catalogue #11811-023). Gentamicin from Sigma (catalogue # G1264).


Immunofluorescence P53 Testing

Methods for FIG. 5: Panels A to D: Human HDQ-P1 breast carcinoma cells with a homozygous R213× nonsense mutation in the TP53 gene were exposed to three different batches of pharmaceutical gentamicin sulfate or to major and minor gentamicin components purified from pharmaceutical gentamicin, for 72 h. The cells were then fixed, DNA was stained with Hoechst™ 33323 and nuclear p53 was detected by immunofluorescence labeling using Santa Cruz DO-1 p53 antibody. The p53-positive nuclei were determined using a Cellomics™ VTI 96-well imager as described in Baradaran-Heravi et al. (2016). The percent p53-positive nuclei is a measure of the extent of PTC readthrough. Panels E and F: HDQ-P1 cells were exposed to the gentamicin batches, gentamicin B1 or gentamicin X2 for 72 h and subjected to p53 Western analysis using Santa Cruz™ DO-1 p53 antibody as described in Baradaran-Heravi et al. (2016) to measure formation of truncated p53 and full-length p53, where full-length p53 is the PTC readthrough product. The y axis in Panel F shows the full-length p53 signal intensity, expressed as chemiluminescence units.


Premature Stop Codon Testing with Genatamicin B1


Methods for FIG. 6: NCI-H1299 human non-small cell lung carcinoma cells were transiently transfected with p53 expression constructs bearing a TGA, TAG or TAA nonsense mutation at amino acid position 213. Cells exposed to transfection reagent only (mock) or transiently transfected with a WT p53 expression were included as controls. The cells were exposed to the indicated concentrations of gentamicin B1 or USP gentamicin sulfate (Sigma™) for 48 h and the formation of truncated p53 and full-length p53 (readthrough product) was determined as described in Baradaran-Heravi et al. (2016). The amounts of full-length p53 and truncated p53 are expressed relative to the amount of full-length (for WT) or truncated p53 in untreated cells.


Premature Stop Codon Testing with Genatamicin B1


Methods for FIG. 7: Different human cancer cell lines with homozygous TP53 nonsense mutations (i.e. SW900; NCI-H1688; ESS-1; SK-MES-1; HCC1937; H1299; and HCT116) were exposed to the indicated concentrations of gentamicin B1 or G418 for 3 days, 6 days or 13 days, as indicated and the formation of truncated p53 (lower arrowhead) and full length p53 (upper arrowhead, readthrough product) was determined as described in Baradaran-Heravi et al. (2016). The nonsense mutations are indicated under the cell line names. Vinculin, which migrates around 116 kDa, was used as a protein loading control.


Induction of PTC Readthrough In Vivo

Methods for FIG. 8: Two million NCI-H1299 human non-small cell lung carcinoma cells stably expressing a TP53 expression construct bearing the R213X (TGA) nonsense mutation were implanted subcutaneously on the lower back of immunocompromised NRG (NOD-Rag1null IL2rgnull) mice. Panel A: When the tumour xenografts reached a size of approximately 0.2-0.5 cubic centimeters, the mice were injected intraperitoneally with saline, gentamicin B1 or USP gentamicin sulfate at the indicated doses for 5 consecutive days. 72 hours after the last injection, the mice were sacrificed and the amounts of truncated p53 (TR-p53) and full-length p53 (FL-p53) were determined by western analysis as described in Baradaran-Heravi et al. (2016). Panel B: When the tumour xenografts reached a size of approximately 0.2-0.5 cubic centimeters, the mice were injected intraperitoneally once with saline, gentamicin B1 or USP gentamicin sulfate. 48 hours after the last injection, the mice were sacrificed and the amounts of truncated p53 and full-length p53 were determined by western analysis. The amounts of full-length p53 relative to saline-treated mice are indicated under each lane. Vinculin was used as a protein loading control.


Induction of PTC Readthrough by Gentamicin B1 in Cells Derived from Patients with Rare Genetic Diseases.


Methods for FIG. 9: Panels A and B: GM16485 primary fibroblasts derived from a Neuronal Ceroid Lipofuscinosis patient with compound heterozygous nonsense mutations in the TPP1 (tripeptidylpeptidase I) gene (R127X/R208X) were exposed to 25 μg/ml gentamicin B1 or 100 μg/ml gentamicin for up to 10 days. Cell lysates were prepared and TPP1 enzyme activity was determined as in Lojewski et al. (2014) with modifications: Lysates were diluted 1:5 in 50 mM sodium acetate pH 4.0 and pre-incubated at 37° C. for 1 h. After pre-incubation, 2014 of total protein from GM16485 lysates or 5 μg of total protein from lysates of fibroblasts from unaffected individuals (WT) was incubated in 150 μl of 50 mM sodium acetate pH 4.0 containing a final concentration of 62.5 μM Ala-Ala-Phe-7-amido-4-methylcoumarin for 2 h at 37° C. Fluorescence was measured using a TECAN Infinite M200™ spectrophotometer with an excitation wavelength of 360 nm and an emission wavelength of 460 nm. Assays were carried out under conditions where product formation was linear with respect to protein concentration and time. TPP1 activity was expressed relative to the average activity of untreated primary fibroblasts from two unaffected individuals (WT) (Panel A). For panel B, the same cell extracts were analysed for formation of TPP1 by automated capillary electrophoresis western analysis using the Abcam™ ab54685 α-TPP1 antibody as in Baradaran-Heravi et al (2016). Extracts from WT fibroblasts were also analysed, using 20% of the amount of protein used for GM16485.


Panel C: HSK001 myoblasts derived from a Duchenne Muscular Dystrophy patient with nonsense mutation (DMD: E2035X) were differentiated into myotubes and exposed to the indicated concentrations of gentamicin B1 or gentamicin for 3 days and dystrophin expression level was determined by automated capillary electrophoresis western analysis using Abeam™ ab1527 α-dystrophin antibody. Extracts from WT myotubes were also analyzed, using 5% of the amount of protein used for DMD cells. Beta-actin was used as a loading control.


Panel D: SD123 fibroblasts from a patient with Schimke Immuno-Osseous Dysplasia, with a homozygous SMARCAL1 nonsense mutation (R17X) were exposed to the indicated concentrations of gentamicin B1 or gentamicin for 6 days and SMARCAL1 levels were determined by western blotting using an anti SMARCAL1 antibody provided by Dr. Cornelius Boerkoel (University of British Columbia). Extracts from WT fibroblasts were also analyzed, using 10% of the amount of protein used for SIOD cells. Beta-actin was used as a loading control.


Panel E: EB14 keratinocytes from a patient with Recessive Dystrophic Epidermolysis Bullosa, with a homozygous Q251X nonsense mutation on the COL7A1 gene were incubated with the indicated concentrations of gentamicin B1 or gentamicin for 72 h and cellular collagen 7 was measured by western blotting using EMD Millipore 234192 collagen 7 antibody. Extracts from WT keratinocytes were also analyzed, using 10% of the amount of protein used for EB14 cells.


Proposed Synthesis of Compounds

Synthesis of the gentamicin analogues (i.e. see Table B) is proposed via α-glycosylation of the pseudo-disaccharide comprising garosamine linked to deoxystreptamine, either chemically or enzymatically. This would require access to the selectively protected disaccharide in which the alcohol to be glycosylated is free while the other alcohols and amines are protected. One route to this disaccharide would involve first protection of all amines with a suitable blocking group known to one skilled in the art (Cbz, Boc etc), then protection of the syn-diol within the streptamine moiety using Ley's reagent. Subsequent protection of the remaining alcohols and selective removal of the Ley protecting group would leave the pseudo-disaccharide with two free alcohols. Glycosylation of this under conditions for generating 1,2-syn linked product (α-gluco in this case) would likely generate a mixture of the two glycosides from which the one of interest could be separated and protecting groups removed.


Alternatively the direct enzymatic glycosylation of the pseudo-disaccharide comprising garosamine linked to deoxystreptamine may be carried out using a variety of α-glycoside phosphorylases, α-glucosidases (run in trans-glycosylation mode) or available α-glucosyl transferases may prove successful. Large libraries of such enzymes are being assembled making such “screening approaches” feasible. If successful this synthesis may provide a remarkably simple and scalable synthetic route.


EXAMPLES
Example 1: p53 Read-Through Assay

Gentamicin, gentamicin A, B, B1, C1, C1a, C2, C2a, C2b, X2, sisomicin, as well as gentamicin fragments garamine and ring C (see FIG. 1) were tested for PTC read-through using the 96-well plate assay. For comparison, G418, a related aminoglycoside that is known to be potent inducer of PTC read-through was used as a positive control. G418 is not an approved drug.


As shown in FIG. 2 Gentamicin did not induce PTC read-through at the concentrations tested, which did not exceed 200 μM. However, it is active at 3 mM as shown in FIG. 3A. Gentamicin A, B, C1, C1a, C2, C2a, C2b, sisomicin, garamine and ring C showed no activity whatsoever (data not shown). G418 showed activity in the 25-200 μM concentration range. Gentamicin X2 showed activity, but it was less potent than G418. Gentamicin B1 showed strong activity, slightly more potent than G418. Therefore, the PTC read-through activity of gentamicin drug is due mostly to the presence of the minor components B1 and X2. Similarly, FIG. 3B shows the induction of PTC read-through by G418, gentamicin, gentamicin B1 and gentamicin X2 using western analysis, wherein the amount of full-length p53 observed in FIG. 3A was plotted versus the concentration of the different compounds on a log scale.


The 96-well plate assay results were confirmed using western analysis as shown in FIG. 3A, wherein HDQ-P1 cells contain very small amounts of p53 protein truncated at R213, and no full-length p53. Induction of PTC read-through causes the appearance of full length p53. Western analysis was performed using an automated quantitative capillary electrophoresis western system. The results confirm the 96-well plate assays and show that gentamicin B1 induces the appearance of full length p53 and that is more potent than G418 or X2. The activity of gentamicin at 3 mM is shown for comparison.


This result is important for medical applications of PTC read-through. Gentamicin is known to be nephrotoxic and ototoxic. (Kohlhepp S. J. et al. 1984) have examined the nephrotoxicity of the major gentamicins C, C1a and C2 and found that nephrotoxicity was caused mainly by C2. Although it is not yet know to what extent gentamicin B1 might be nephrotoxic or ototoxic, it is anticipated that treatment of patients with gentamicin B1 would induce PTC read-through at lower doses than treatment with gentamicin, which typically contains only 0.5-3% B1 (MicroCombiChem™, personal communication). Treatment with gentamicin B1 instead of gentamicin should achieve both higher PTC read-through and lower toxicity via omission of toxic gentamicin C2.


Monitoring of gentamicin plasma concentrations is recommended to avoid toxicity. A cursory search indicates that plasma levels of gentamicin are typically between 1 and 12 μg/ml (2-24 μM) and that concentrations above about 10 μM should be avoided during long-term treatment. The concentrations of gentamicin B1 showing read-through (3 μM and higher) are within this range.


Example 2: p53 Read-Through Assay with Steroids

As shown in FIG. 4, G418 showed much improved PTC read-through at a concentration of 25 μM in combination with Dexamethasone (5 Betamethazone (5 μM) or Medroxyprogesterone acetate (Medroxy pro)(5 μM), whereas Dexamethasone, Betamethazone alone and Medroxy pro alone showed no read-through activity.


Example 3: p53 Read-Through Assay with Steroids

The results presented in FIG. 5 show that two gentamicin batches display low PTC readthrough activity at 1 mg/ml while a third batch was inactive (see FIGS. 5A, B, E and F—batch 2). The results also show that gentamicin B1 and gentamicin X2 display potent PTC readthrough activity (see FIGS. 5C-F).


Example 4: Read-Through Assay Comparing Stop Codons

The results in FIG. 6 show that gentamicin B1 at 50 μg/ml and 100 μg/ml are induce PTC readthrough at all three premature termination codons (i.e. TGA, TAG and TAA). However, there appears to be a slight decrease in readthrough with the TAA stop codon.


Example 5: Read-Through Assays Comparing Cell Types


FIG. 7 shows that gentamicin B1 can induce PTC readthrough in a variety of cancer cell lines having nonsense mutations at different positions in the TP53 gene (i.e. SW900; NCI-H1688; ESS-1; SK-MES-1; HCCl937; H1299; and HCT116). Gentamicin B1 consistently showed readthrough of the stop codons in various cancer cell lines.


Example 6: In Vivo Read-Through Assays

As shown in FIG. 8 gentamicin B1 can induce premature termination codon readthrough in a tumour xenograft in vivo. Gentamicin B1 showed readthrough as low as 50 mg/kg (see FIG. 8A), at 200 mg/kg and at 400 mg/kg (see FIG. 8B), whereas no readthrough was detected for gentamicin. No toxicity was observed for B1 but 400 mg/kg gentamicin induced acute toxicity and the mice had to be sacrificed shortly after administration, as denoted by the asterisks.


Example 7: Induction of PTC Readthrough by Gentamicin B1 in Cells Derived from Patients with Rare Genetic Diseases


FIGS. 9A and B show GM16485 primary fibroblasts derived from a Neuronal Ceroid Lipofuscinosis patient with heterozygous nonsense mutations in the TPP1 (tripeptidylpeptidase I) gene (R127X/R208X) where the fibroblasts were exposed to 25 μg/ml gentamicin B1 or 100 μg/ml gentamicin for up to 10 days and before the fluorescence of cell extracts were measured for TPP1 activity was expressed relative to the average activity of untreated primary fibroblasts from two unaffected individuals (WT) (A). FIG. 9B, shows the same cell extracts analysed for formation of TPP1 by automated capillary electrophoresis western analysis. FIG. 9C shows HSK001 myoblasts derived from a Duchenne Muscular Dystrophy patient with nonsense mutation (DMD: E2035X) were differentiated into myotubes and exposed to the indicated concentrations of gentamicin B1 or gentamicin for 3 days and subsequent dystrophin expression levels were determined by automated capillary electrophoresis western analysis as compared to WT myotubes and loading control. FIG. 9D shows SD123 fibroblasts from a patient with Schimke Immuno-Osseous Dysplasia, with a homozygous SMARCAL1 nonsense mutation (R17X) exposed to the indicated concentrations of gentamicin B1 or gentamicin for 6 days before the SMARCAL1 levels were determined by western blotting as compared to WT fibroblasts and loading control. FIG. 9E shows EB14 keratinocytes from a patient with Recessive Dystrophic Epidermolysis Bullosa, with a homozygous Q251X nonsense mutation on the COL7A1 gene incubated with the indicated concentrations of gentamicin B1 or gentamicin for 72 h prior to cellular collagen 7 measurement by western blotting as compared to WT keratinocytes. In all of the tested genetic diseases gentamicin B1 induced readthrough.


Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. The word “comprising” is used herein as an open-ended term, substantially equivalent to the phrase “including, but not limited to”, and the word “comprises” has a corresponding meaning. As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a thing” includes more than one such thing. Citation of references herein is not an admission that such references are prior art to an embodiment of the present invention. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings.


REFERENCES



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Claims
  • 1.-13. (canceled)
  • 14. A method of treating or ameliorating a medical condition associated with premature termination codons (PTCs) in RNA, the method comprising administering a compound, or a pharmaceutically acceptable salt thereof, in an amount effective for treating or ameliorating a medical condition associated with a PTC in RNA, wherein the compound has the structure of Formula I:
  • 15. The method of claim 14, wherein the compound selected from one or more of the following:
  • 16. The method of claim 14, wherein the compound is selected from one or more of the following:
  • 17. The method of claim 14, wherein the medical condition is selected from TABLE 1 or TABLE 2.
  • 18. The method of claim 14, wherein the medical condition is selected from the group consisting of: central nervous system disease; peripheral nervous system disease; neurodegenerative disease; autoimmune disease; DNA repair disease; inflammatory disease; collagen disease; kidney disease; pulmonary disease; eye disease; cardiovascular disease; blood disease; metabolic disease; neuromuscular diseases; neoplastic disease; and any genetic disorder caused by nonsense mutation(s).
  • 19. The method of claim 18, wherein the medical condition is selected from the group consisting of: ataxia-telangiectasia; muscular dystrophy; Duchenne muscular dystrophy; Dravet syndrome; myotonic dystrophy; multiple sclerosis; infantile neuronal ceroid lipofuscinosis; Alzheimer's disease; Tay-Sachs disease; neural tissue degeneration; Parkinson's disease; chronic rheumatoid arthritis; lupus erythematosus; graft-versus-host disease; primary immunodeficiencies; severe combined immunodeficiency; DNA Ligase IV deficiency; Nijmegen breakage disorders; xeroderma pigmentosum (XP); rheumatoid arthritis; hemophilia; von Willebrand disease; thalassemia (for example; β-thalassemia); familial erythrocytosis; nephrolithiasis; osteogenesis imperfecta; cirrhosis; neurofibroma; bullous disease; lysosomal storage diseases; Hurler's disease; familial cholesterolemia; cerebellar ataxia; tuberous sclerosis; immune deficiency; cystic fibrosis; familial hypercholesterolemia; pigmentary retinopathy; retinitis pigmentosa; amyloidosis; atherosclerosis; giantism; dwarfism; hypothyroidism; hyperthyroidism; aging; obesity; diabetes mellitus; familial polycythemia; Niemann-Pick disease; epidermolysis bullosa; Marfan syndrome; Becker muscular dystrophy (BMD); spinal muscular atrophy; cancer; and any genetic disorder caused by nonsense mutation(s).
  • 20. The method of claim 19, wherein the cancer is of the head and neck, eye, skin, mouth, throat, esophagus, chest, bone, blood, lung, colon, sigmoid, rectum, stomach, prostate, breast, ovaries, kidney, liver, pancreas, brain, intestine, heart or adrenals.
  • 21. The method of claim 19, wherein the cancer is sarcoma, carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, Kaposi's sarcoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, retinoblastoma, a blood-born tumor or multiple myeloma.
  • 22. The method of claim 19, wherein the cancer is acute lymphoblastic leukemia, acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute monoblastic leukemia, acute erythroleukemic leukemia, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acute nonlymphocyctic leukemia, acute undifferentiated leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, or multiple myeloma.
  • 23. The method of claim 14, wherein the premature termination codon is UGA or UAG.
  • 24. The method of claim 14, wherein the premature termination codon is UGA.
  • 25. The method of claim 14, wherein the premature termination codon is UAG.
  • 26. The method of claim 14, wherein the premature termination codon is UAA.
  • 27.-28. (canceled)
  • 29. A compound, wherein the compound has the structure:
  • 30.-60. (canceled)
PCT Information
Filing Document Filing Date Country Kind
PCT/CA2016/000240 9/23/2016 WO 00
Provisional Applications (1)
Number Date Country
62232789 Sep 2015 US