WO2019233891A1

WO2019233891A1 - Tbk1 inhibitor compounds

Info

Publication number: WO2019233891A1
Application number: PCT/EP2019/064181
Authority: WO
Inventors: Giovanna Bergamini; James C CLEMENS; Kathleen Johnson; Marcel Muelbaier; Douglas Thomson
Original assignee: Glaxosmithkline Intellectual Property Development Limited
Priority date: 2018-06-04
Filing date: 2019-05-31
Publication date: 2019-12-12

Abstract

The present invention relates to compounds of formula I, and pharmaceutical salts thereof, and their use as TBK1 inhibitors. The invention also provides methods of introducing nucleic acids into a cell, changing the genotype of a cell and animal cells produced from the methods provided.

Description

TBK1 inhibitor compounds

Field of the Invention

The present invention relates to novel compounds and their use as TANK-binding kinase 1 (TBK1 ) inhibitors.

Background to the Invention

Activation of Toll-Like Receptors (TLRs) by Pathogen Associated Molecular Patterns (PAMPs), such as double stranded DNAs (dsDNAs) of viral or bacterial origin, can activate innate immune response pathways. Delivery of double stranded plasmid DNA constructs to cells can also activate an innate immune response.

TANK-binding kinase 1 (TBK1 ) mediates signalling from TLRs, such as TLR3, in response to the detection of PAMPs such as dsDNAs. TBK1 is a noncanonical IkB protein kinase that phosphorylates and activates downstream targets such as IRF3 and c-Rel. Therefore TBK1 signalling also plays a role in innate immune response pathways.

Molecular biology techniques often require the deliberate introduction of dsDNA constructs into cells. In these circumstances, activation of TBK1 and the innate immune responses can decrease the number of dsDNA transformed cells and therefore such activation can be undesirable.

TBK1 can also mediate NF-kB activation in cancer and therefore TBK1 activity may play a role in certain cancers.

Thus, a need exists for compounds and methods to inhibit TBK1.

Summary of the Invention

In a first aspect of the present invention, there is provided a compound of formula I or a salt thereof:

Formula I wherein

R¹ is H, C1 -C6 alkyl, 0-(C1 -C4 alkyl), C(=0)NH₂, C3-C6 cycloalkyl or a halogen;

R² is H or C1 -C6 alkyl;

R³ is (CH₂)_n-CF₃, (CH₂)_n-CHF_2, or (CFI₂)_nCFI₂F wherein n is 1 , 2 or 3; and

R⁴ is -S(0)₂-NH₂, -S(0)₂-(C1 -C6 alkyl), -S0_2-NH-(C1 -C6 alkyl) or -C(=0)-NH-(C1 -C6 alkyl).

In a second aspect, the present invention provides use of the compounds of the invention to inhibit TBK1.

In a third aspect of the invention, the present invention provides use of the compounds of the invention in a method of delivering a nucleic acid into a cell.

In a fourth aspect of the invention, the present invention provides a method of introducing a nucleic acid into a cell, said method comprising a step of exposing the cell to a compound of formula I as defined herein.

In a further aspect, the present invention provides a method of introducing a nucleic acid into an animal cell comprising the steps of: a) providing an animal cell; b) contacting the animal cell with a nucleic acid; c) treating the animal cell to cause the nucleic acid to enter the cell; and d) contacting the animal cell with an amount of a compound of formula I as defined herein effective to inhibit TBK1 activity; whereby the nucleic acid is introduced into the animal cell.

In a still further aspect, the present invention provides a method of changing the genotype of an animal cell comprising the steps of: a) providing an animal cell comprising a target nucleic acid; b) contacting the animal cell with a first nucleic acid encoding a transcription activator effector (TAL) comprising a nuclease, and a targeting nucleic acid; c) treating the animal cell to cause the first nucleic acid and the targeting nucleic acid to enter the cell; and d) contacting the animal cell with an amount of a compound of formula I as defined herein effective to inhibit TBK1 activity; whereby the targeting nucleic acid is introduced into the target nucleic acid in the animal cell and the genotype of the animal cell is changed.

In a still further aspect, the present invention provides a method of changing the genotype of an animal cell comprising the steps of a) providing an animal cell comprising a target nucleic acid; b) contacting the animal cell with a first nucleic acid encoding a CRISPR associated protein 9 (CAS9), a second nucleic acid encoding a guide RNA, and a targeting nucleic acid; c) treating the animal cell to cause the first nucleic acid, the second nucleic acid and the targeting nucleic acid to enter the cell; and d) contacting the animal cell with an amount of a compound of formula I as defined herein effective to inhibit TBK1 activity; whereby the targeting nucleic acid is introduced into the target nucleic acid in the animal cell and the genotype of the animal cell is changed.

The present invention also provides animal cells produced by any of the methods described herein.

The present invention may be advantageous in a number of respects. The compounds of the invention may be used to inhibit TBK1. Further, the compounds of the present invention may supress the cell innate immune response pathway. Suppression of the innate immune response pathway may allow improved introduction of nucleic acid molecules into cells, particularly into the cytosol of a cell. In particular, the compounds of the invention may be used to improve introduction of nucleic acid molecules into a cell in gene editing techniques.

Brief Description of the Drawings

Figure 1 shows a model for CRISPR mediated insertion of Flomology-Directed Repair (FIDR) construct into TREX1 gene.

Figure 2 shows THP1 cell viability post-transfection with 0.5,1 and 2ug plasmid DNA. Figure 3 shows THP1 viable cell count post-transfection with 2 ug plasmid DNA.

Figure 4 shows THP1 viable cell count post-transfection with 1 ug plasmid DNA.

Figure 5 shows THP1 viable cell count post-transfection with 0.5 ug plasmid DNA.

Figure 6 shows GFP expression post-transfection with 0.5, 1 and 2ug plasmid DNA. Figure 7 shows the procedure used to generate of TBK1 inhibitor treated THP1 derived TREX1 KO (knock-out) cell lines.

Figure 8 shows total cell counts for GSK CRISPR plasmid transfected cells treated with a TBK1 inhibitor (Compound 4) and subjected to G418 selection.

Figure 9 shows total cell counts for Santa Cruz CRISPR plasmid transfected cells treated with a TBK1 inhibitor (Compound A) and subjected to puromycin selection.

Figure 10 shows TIDE (Tracking of Indels by Decomposition) analysis for bi-allelic knockout clone 4-7.

Figure 1 1 shows a feature map of the GFP control plasmid pmaxGFP™.

Figure 12 shows a feature map of the Santa Cruz CRISPR/Cas9 Knockout plasmid(s) and a DNA targeted by these plasmid(s).

Figure 13 shows a feature map of the Santa Cruz Flomology-Directed Repair (FIDR) Plasmid and a DNA targeted by this plasmid for knockout/editing using the CRISPR/Cas9 system {e.g., in conjunction with the CRISPR/Cas9 Knockout Plasmids).

Figure 14 shows the guide RNA cut sites for the GSK guide RNA (Guide 1 ) and the Santa Cruz guide RNAs (sc-403875A1 -3) in a map of a TREX1 nucleic acid.

Figure 15 A shows Western blot analysis of inhibition of IRF3 phosphorylation by compound 4 in poly(l:C)-stimulated Ramos cells with an average pICso of 6.0 (n=5). B shows that compound 4 inhibits IFN-a secretion in human PBMC with an average pICso of 6.1 (n=3).

Figure 16 shows that compound 4 inhibits secretion of IFNp from THP-1 cells. A shows that compound 4 inhibits secretion of IFNp (pg/mL) in THP-1 cells stimulated with dsDNA- containing virus (Baculovirus) with a pICso of 5.9 (n=3). B shows compound 4 was able to inhibit secretion of IFNp (pg/mL) in THP-1 cells stimulated with cGAMP with a pICso of 6.3 (n=3).

Terms and Definitions

As used herein, the term“alkyl” refers to a monovalent saturated hydrocarbon chain having the specified number of member atoms. For example, C1 -C6 alkyl refers to an alkyl group having from 1 to 6 carbon atoms. Alkyl groups may be optionally substituted with one or more substituents as defined herein. Alkyl groups may be straight or branched. Representative branched alkyl groups have one, two, or three branches. Examples of alkyl include methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl, and t-butyl), pentyl (n-pentyl, isopentyl, and neopentyl), and hexyl.

As used herein, the term “C3-C6 cycloalkyl” refers to a monocyclic saturated ring containing three to six carbon atoms. Therefore, the term includes cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

As used herein, the term“halogen” refers to refers to the halogen radicals fluoro, chloro, bromo, and iodo.

As used herein, the term“nucleic acid” refers to a nucleotide sequence. DNA, RNA, DNA::RNA hybrids and chemically modified nucleotide sequences are examples of nucleic acids.

As used herein the term“TBK1 inhibitor” refers to a chemical compound that inhibits, either directly or indirectly, TBK1 activity. TBK1 inhibitors may include antagonists and inverse agonists of TBK1.

As used herein, the term“TBK1 activity” means a biological activity of a peptide chain comprising the amino acid sequence shown in SEQ ID NO: 8.

As used herein, the term“animal cell” refers to a eukaryotic cell from an organism in the kingdom Animalia. Animal cells of the invention may be provided in vitro as an isolated cell, as a tissue comprising the cell or as a cell present in a material such as blood or waste from an animal.

As used herein the term“targeting nucleic acid” refers to any nucleic acid that can be directed to a nucleic acid of choice (a“target nucleic acid”). A targeting nucleic acid sequence comprises nucleic acid sequences homologous to a portion of a target nucleic acid sequence and can recombine via homologous recombination, or other mechanisms of recombination or repair, with said target nucleic acid sequence. Alternatively, a targeting nucleic acid sequence can comprise non-homologous sequences and can recombine via non-homologous end joining, or other mechanisms of recombination or repair, with said target nucleic acid sequence. As used herein, singular definite articles, such as, for example,“a” and“an” refers“one or more” and/or otherwise include the plural form of an element or article.

Description of Various Embodiments

In one aspect, the present invention provides a compound of formula I or a salt thereof:

Formula I

wherein:

R² is H or C1 -C6 alkyl;

R⁴ is -S(0)₂-NH₂, -S(0)₂-(C1 -C6 alkyl), -S0_2-NH-(C1 -C6 alkyl) or -C(=0)-NH-(C1 -C6 alkyl). For the avoidance of doubt, in all instances of R⁴ the symbol at the beginning of the group indicates a bond to the phenyl ring in Formula I i.e. it is the sulfur of the S(0)₂ group or the C of the C(=0) group which is bonded to the phenyl ring of Formula I.

In an embodiment of the invention, R¹ is H, methyl, ethyl, O-methyl, C(=0)NFI₂, cyclopropyl, Cl, F, Br or I. In one embodiment R¹ is H. In another embodiment R¹ is methyl. In another embodiment R¹ is ethyl. In another embodiment R¹ is O-methyl. In another embodiment R¹ is C(=0)NFI₂. In another embodiment R¹ is cyclopropyl. In another embodiment R¹ is Cl, F, Br or I. In another embodiment R¹ is Cl, Br, methyl, ethyl, O- methyl, C(=0)NFI₂ or cyclopropyl. In a further embodiment R¹ is Br.

R² is FI or C1 -C6 alkyl. In an embodiment of the invention R² is C1 -C6 alkyl. In a further embodiment R² is methyl, ethyl or propyl. In a still further embodiment R² is methyl. R³ is (CH₂)_n-CF₃, (CH₂)_n-CHF_2, or (CFI₂)_nCFI₂F wherein n is 1 , 2 or 3. In an embodiment of the invention n is 1 or 2. In a further embodiment n is 1. In another embodiment of the invention R³ is (CFI₂)_n-CF₃ or (CFI₂)_n-CFIF₂ wherein n is 1 , 2 or 3. In a further embodiment of the invention R³ is (CFI₂)_n-CF₃ or (CFI₂)_n-CFIF₂ wherein n is 1. In a still further embodiment R³ is CFI₂-CF₃.

R⁴ is -S(0)₂-NH₂, -S(0)₂-(C1 -C6 alkyl), -S0_2-NH-(C1 -C6 alkyl) or -C(=0)-NH-(C1 -C6 alkyl). In an embodiment R⁴ is -S(0)₂-NFI₂, -S(0)₂-(C1 -C6 alkyl) or -C(=0)-NFI-(C1-C6 alkyl). In a further embodiment R⁴ is -S(0)₂-NFI₂, -S(0)₂-(C1 -C6 alkyl) or -C(=0)-NFI-(C1 - C6 alkyl) wherein the C1 -C6 alkyl is methyl, ethyl or propyl. In a still further embodiment R⁴ is -S(0)₂-NFI₂, -S(0)₂-CFl₃, or -C(=0)-NFI-CFl₃. In a still further embodiment R⁴ is -S(0)₂- NH₂ or -S(0)₂-CFl₃. In a still further embodiment R⁴ is -S(0)₂-NFI₂.

In an embodiment of the invention R¹ is FI, methyl, ethyl, O-methyl, C(=0)NFI₂, cyclopropyl, Cl, F, Br or I.; R² is methyl; R³ is (CFI₂)_n-CF₃ or (CFI₂)_n-CFIF₂, wherein n is 1 ; and R⁴ is -S(0)₂-NH₂, -S(0)₂-CH₃,or -C(=0)-NH-CH₃.

In another embodiment of the invention R¹ is Cl, Br, methyl, ethyl, O-methyl, C(=0)NFI₂ or cyclopropyl; R² is methyl; R³ is CFI₂-CF₃; and R⁴ is -S(0)₂-NFI₂ or -S(0)₂-CFl₃.

In an embodiment of the invention, the compound of Formula I is selected from the group consisting of the following compounds or salts thereof:

Compound 2

Compound 8

Compound 10

In another embodiment of the invention, the compound of Formula I is selected from the group consisting of the following compounds or salts thereof:

nd 9

Compound 11

In an embodiment, the compound of Formula is selected from the list consisting of Compound 1 , Compound 4 and Compound 5

In an embodiment the compound of Formula is Compound 1 or a salt thereof In an embodiment the compound of Formula is Compound 2 or a salt thereof In an embodiment the compound of Formula is Compound 3 or a salt thereof In an embodiment the compound of Formula is Compound 4 or a salt thereof In an embodiment the compound of Formula is Compound 5 or a salt thereof In an embodiment the compound of Formula is Compound 6 or a salt thereof In an embodiment the compound of Formula is Compound 7 or a salt thereof In an embodiment the compound of Formula is Compound 8 or a salt thereof In an embodiment the compound of Formula is Compound 9 or a salt thereof. In an embodiment the compound of Formula I is Compound 10 or a salt thereof.

In an embodiment the compound of Formula I is Compound 1 1 or a salt thereof.

In an embodiment the compound of Formula I is Compound 12 or a salt thereof.

In an embodiment the compound of Formula I is Compound 13 or a salt thereof.

In an embodiment the compound of Formula I is Compound 14 or a salt thereof.

It will be understood that phrases such as "a compound of Formula I or a salt thereof" or "compounds of the invention" are intended to encompass the compound of Formula I, a pharmaceutically acceptable salt or solvate of the compound of Formula I, or any pharmaceutically acceptable combination of these. Thus by way of non-limiting example used here for illustrative purposes, "a compound of Formula I or a pharmaceutically acceptable salt thereof encompasses a pharmaceutically acceptable salt of a compound of Formula I which is present as a solvate, and this phrase also encompasses a mixture of a compound of Formula I and a pharmaceutically acceptable salt of a compound of Formula I.

It is to be understood that references herein to a compound of Formula I or a pharmaceutically acceptable salt thereof includes a compound of Formula I as a free base or as a pharmaceutically acceptable salt thereof. Thus, in one embodiment, the invention is directed to a compound of Formula I. In another embodiment, the invention may be directed to a pharmaceutically acceptable salt of a compound of Formula I.

The term “pharmaceutically acceptable” refers to those compounds (including salts), materials, compositions, and dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

Pharmaceutically acceptable salts include, amongst others, those described in Berge, J. Pharm. Sci. , 1977, 66, 1 -19, or those listed in P FI Stahl and C G Wermuth, editors, Handbook of Pharmaceutical Salts; Properties, Selection and Use, Second Edition Stahl/Wermuth: Wiley- VCH/VHCA, 201 1 (see http://www.wilev.com/WilevCDA/WilevTitle/productCd-3906390519.html). Suitable pharmaceutically acceptable salts can include acid addition salts and base addition salts. Such salts can be formed by reaction with the appropriate acid, optionally in a suitable solvent such as an organic solvent, to give the salt which can be isolated by crystallisation and filtration.

Representative pharmaceutically acceptable acid addition salts include, but are not limited to 4-acetamidobenzoate, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate (besylate), benzoate, bisulfate, bitartrate, butyrate, calcium edetate, camphorate, camphorsulfonate (camsylate), caprate (decanoate), caproate (hexanoate), caprylate (octanoate), cinnamate, citrate, cyclamate, digluconate, 2,5-dihydroxybenzoate, disuccinate, dodecylsulfate (estolate), edetate (ethylenediaminetetraacetate), estolate (lauryl sulfate), ethane-1 ,2-disulfonate (edisylate), ethanesulfonate (esylate), formate, fumarate, galactarate (mucate), gentisate (2,5-dihydroxybenzoate), glucoheptonate (gluceptate), gluconate, glucuronate, glutamate, glutarate, glycolate, hexyl resorcin ate, hippurate, hydrabamine (/V,/V'-di(dehydroabietyl)-ethylenediamine), hydrobromide, hydrochloride, hydroiodide, hydroxynaphthoate, isobutyrate, lactate, lactobionate, laurate, malate, maleate, malonate, mandelate, methanesulfonate (mesylate), methylsulfate, naphthalene-1 ,5-disulfonate (napadisylate), naphthalene-2-sulfonate (napsylate), nicotinate, nitrate, oleate, palmitate, p-aminobenzenesulfonate, p-aminosalicyclate, pamoate (embonate), pantothenate, pectinate, persulfate, phenylacetate, phosphate, propionate, p-toluenesulfonate (tosylate), pyroglutamate, pyruvate, salicylate, sebacate, stearate, succinate, sulfamate, sulfate, tannate, tartrate, teoclate (8-chlorotheophyllinate), thiocyanate, triethiodide, undecanoate, undecylenate, and valerate.

Representative pharmaceutically acceptable base addition salts include, but are not limited to aluminium, 2-amino-2-(hydroxymethyl)-1 ,3-propanediol (TRIS, tromethamine), , benethamine (/V-benzylphenethylamine), benzathine (L/,L/’-dibenzylethylenediamine), bis- (2-hydroxyethyl)amine, bismuth, calcium, choline, cyclohexylamine, dibenzylethylenediamine, diethylamine, diethyltriamine, dimethylamine, dimethylethanolamine, ethanolamine, ethylenediamine, iron, isoquinoline, lepidine, lithium, lysine, magnesium, meglumine (/V-methylglucamine), piperazine, piperidine, potassium, quinine, quinoline, sodium, strontium, f-butylamine, and zinc.

In a particular embodiment of the invention, the compound of Formula I is in the form of a formic acid salt. In a second aspect of the invention there is provided the use of a compound of Formula 1 as defined herein as a TBK1 inhibitor. The compounds of Formula 1 may inhibit TBK1 in any suitable way.

In a third aspect, the present invention provides use of a compound of Formula 1 as defined herein in a method of delivering a nucleic acid into a cell. The nucleic acid may be delivered into the cell by any suitable means. The cell may be treated to cause the nucleic acid to enter the cell using any technique permitting a nucleic acid to be delivered into a cell. Such techniques may include chemical treatment {e.g., lipofectamine, calcium chloride treatment), temperature shocks or electroporation based techniques to cause the nucleic acids-such as DNAs or RNAs-to enter a cell. Viral vector treatment may also be used to cause the nucleic acid, such as DNAs or RNA, to enter the cell. In some embodiments, the cell is an animal cell and is naturally DNA competent. The nucleic acid may also be introduced into a cell using particles (e.g., using“gene gun” techniques). Those of ordinary skill in the art will be familiar with a number of different techniques for treating cells to introduce the nucleic acid into the cell. All such techniques are encompassed in the present invention. In one embodiment the nucleic acid molecule is delivered into the cell via transfection.

In an embodiment of the invention the cell is an animal cell, e.g. a non-human animal cell. In an embodiment of the invention the animal cell is a non-human animal cell. In one embodiment the animal cell may be provided in vivo as a cell present in a tissue or other material in an animal (e.g., non-human animal). In an alternative embodiment the animal cell is provided in vitro or ex vivo. Preferably, the animal cell is provided ex vivo. Preferred animal cells include cells originating from Homo sapiens {e.g., THP-1 cells) or Cricetulus griseus cells (e.g., Chinese Flamster Ovary (CFIO) cells. In one embodiment the animal cell is a THP1 cell. In an alternative embodiment, the animal cell is a CFIO cell.

In one embodiment of the invention the nucleic acid molecule is an exogenous nucleic acid molecule. In an embodiment, the exogenous nucleic acid molecule integrates into the chromosomal sequence of the cell.

In one embodiment the nucleic acid molecule encodes at least one nuclease. In a particular embodiment the nuclease is an endonuclease. In another embodiment, the nucleic acid molecule comprises at least one guide RNA. In a further embodiment, the nucleic acid molecule comprises DNA encoding at least one guide RNA. In an embodiment of the invention the at least one endonuclease is an RNA-guided endonuclease. In an embodiment the endonuclease is CRISPR associated protein 9 (CAS9).

In a fourth aspect, the present invention provides a method of introducing a nucleic acid into an animal cell comprising the steps of: a) providing an animal cell; b) contacting the animal cell with a nucleic acid; c) treating the animal cell to cause the nucleic acid to enter the cell; and d) contacting the animal cell with a compound of Formula I as defined herein in an amount effective to inhibit TBK1 activity; whereby the nucleic acid is introduced into the animal cell.

In one embodiment the animal cell is contacted with the nucleic acid either before, simultaneously with or after the animal cell is contacted with a compound of Formula I. In one embodiment, the animal cell is contacted with a compound of Formula I before the animal cell is contacted with the nucleic acid. In another embodiment, the animal cell is contacted with a compound of Formula I simultaneously with the nucleic acid. In another embodiment, the animal cell is contacted with a compound of formula I after the cell is contacted with the nucleic acid.

The animal cell is as described in the above third aspect of the invention. Further the animal cell may be treated to cause the nucleic acid to enter the cell as described above in the third aspect of the invention.

Suitably, the nucleic acid is a DNA, RNA, DNA::RNA hybrid or a chemically modified nucleotide sequence. In an embodiment the nucleic acid is DNA.

The animal cell is contacted with a compound of Formula I, as defined herein, in an amount effective to inhibit TBK1 activity. In the methods of the invention the biological activity inhibited is the kinase activity of TBK1.

In a further embodiment the animal cell comprises a target nucleic acid; a targeting nucleic acid is incorporated into the target nucleic acid in the animal cell. In one embodiment the targeting nucleic acid recombines into the target nucleic acid. The target nucleic acid may be selected from genomic DNA or extra-genomic DNA. In an embodiment the target nucleic acid is genomic DNA. The genomic DNA may be chromosomal DNA. In another embodiment the nucleic acid is extra-genomic DNA. The extra-genomic DNA may comprise a nucleic acid element such as a plasmid. Alternatively, the extra-genomic DNA may comprise other DNA elements that are not present in genomic DNA.

The animal cell is contacted with a compound of Formula I in an amount effective to inhibit TBK1 activity. In one embodiment the amount effective to inhibit TBK1 activity is about 10 mM to about 50 mM e.g 10 pM to 50 pM. Suitably, the amount effective to inhibit TBK1 activity is 15 pM to 30 pM. In another embodiment the amount effective to inhibit TBK1 is 20 pM to 25 pM. In a further embodiment the amount effective to inhibit TBK1 is about 20 pM.

In one embodiment the method is a method of changing the genotype of an animal cell. In this embodiment the animal cell comprises a target nucleic acid; the nucleic acid encodes a transcription activator effector (TAL) comprising a nuclease and a targeting nucleic acid; when the animal cell is treated this causes the first nucleic acid and the targeting nucleic acid to enter the cell. Whereby the targeting nucleic acid is introduced into the target nucleic acid in the animal cell and the genotype of the animal cell is changed.

In another embodiment the method is a method of changing the genotype of an animal cell wherein the animal cell comprises a target nucleic acid and the nucleic acid which contacts the animal cell encodes a CRISPR associated protein 9 (CAS9). The animal cell is also contacted with a second nucleic acid encoding a guide RNA, and a targeting nucleic acid. The animal cell is treated to cause the second nucleic acid and the targeting nucleic acid to enter the cell. Whereby the targeting nucleic acid is introduced into the target nucleic acid in the animal cell and the genotype of the animal cell is changed.

The second nucleic acid may enter the cell before, simultaneously with or after the nucleic acid encoding CAS9 enters the cell. The cell may be treated to cause the second nucleic acid to enter the cell in the same way or a different way as for the first nucleic acid. Ways of treating a cell to cause a nucleic acid to enter are described herein above.

The animal cell is contacted with a compound of Formula I as defined herein. In one embodiment the compound of Formula I is any of Compounds 1 to 14 as defined herein. In another embodiment the compound is selected from Compounds 1 , 4, 5 and 9-14 as defined herein. In another embodiment the compound is selected from Compounds 1 , 4 and 5 as defined herein. In another embodiment the compound is Compound 4 as defined herein. In an embodiment the compound of Formula I is in the form of the formic acid salt. In another embodiment the compound of Formula I is the formic acid salt of Compound 4.

In a fifth aspect the present invention provides a method of changing the genotype of an animal cell comprising the steps of: a) providing an animal cell comprising a target nucleic acid; b) contacting the animal cell with a first nucleic acid encoding a transcription activator effector (TAL) comprising a nuclease and a targeting nucleic acid; c) treating the animal cell to cause the first nucleic acid and the targeting nucleic acid to enter the animal cell; and d) contacting the animal cell with a compound of Formula I as defined herein in an amount effective to inhibit TBK1 activity; whereby the targeting nucleic acid is introduced into the target nucleic acid in the animal cell and the genotype of the animal cell is changed.

The animal cell is contacted with a first nucleic acid encoding a transcription activator effector (TAL) comprising a nuclease and a targeting nucleic acid. In an embodiment the nuclease is Fok I.

The animal cell is provided as described as hereinabove in the third aspect of the invention, and may be treated to cause the nucleic acid to enter as described hereinabove. Further the animal cell is contacted with the cell as described hereinabove in the fourth aspect i.e. before, simultaneously with or after the animal cell with contacted with a compound of Formula I.

The nucleic acid is as described herein above in the fourth aspect.

The animal cell is contacted with a compound of Formula I in an amount effective to inhibit TBK1 activity. The compound of Formula I present in the method is as defined hereinabove. In an embodiment the effective amount of a compound of Formula I is as described hereinabove. In a sixth aspect, the present invention provides a method of changing the genotype of an animal cell comprising the steps of: a) providing an animal cell comprising a target nucleic acid; b) contacting the animal cell with a first nucleic acid encoding a CRISPR associated protein 9 (CAS9), a second nucleic acid encoding a guide RNA and a targeting nucleic acid; c) treating the animal cell to cause the first nucleic acid, the second nucleic acid and the targeting nucleic acid to enter the cell; and d) contacting the animal cell with an amount of a compound of Formula I, as defined herein, in an amount effective to inhibit TBK1 activity; whereby the targeting nucleic acid is introduced into the target nucleic acid in the animal cell and the genotype of the animal cell is changed.

The animal cell is as described as hereinabove in the third aspect of the invention, and may be treated to cause the first and second nucleic acids to enter as described hereinabove. Further the animal cell is contacted with the cell as described hereinabove in the fourth aspect i.e. before, simultaneously with or after the animal cell with contacted with a compound of Formula I.

The nucleic acid is as described herein above in the fourth aspect.

The animal cell is contacted with a compound of Formula I in an amount effective to inhibit TBK1 activity. The compound of Formula I present in the method is as defined hereinabove. In an embodiment the effective amount of a compound of Formula I is as described hereinabove.

In a further aspect, the present invention provides an animal cell produced by any of the methods described herein. In an embodiment the animal cell is a stable transfectant or a transient transfectant comprising a genotypic change.

In a still further aspect, the present invention provides a pharmaceutically acceptable composition comprising a compound of Formula I, as defined herein, and a carrier. In an embodiment the compound of Formula I is Compound 4 as defined herein. In another embodiment the compound of Formula I is the formic acid salt of Compound 4.

It will be understood that any of the embodiments of the invention disclosed herein may be combined with any other embodiments.

The following non-limiting Examples illustrate the present invention.

Examples Example 1 : Preparation of compounds of Formula I

Scheme I

A compound of Formula III (1.0 equiv.) and diisopropylethylamine (DIPEA, 1.2 equiv.) was dissolved in ethanol and the solution was cooled in an ice bath. A compound of Formula II (1.0 equiv.) was added and the reaction mixture was warmed to room temperature over 2h. The reaction mixture was checked for full conversion by high performance liquid chromatography (FIPLC) and if necessary further stirred at room temperature. In some instanced the final product (IV) precipitated and was filtered off. In other instances, no precipitate was formed and the solvent was removed in vacuo and the residue purified by preparative FIPLC or reverse phase column chromatography.

Scheme 2

A compound of Formula IV (1.0 equiv.) was dissolved in sec-butanol. To this mixture, a compound of Formula V (I .Oequiv. or 1.2equiv.) and a catalytical amount of 4M HCI in dioxane (1 to 2 drops) were added. The stirred reaction mixture was heated to 120°C for 2-4h. The solvent was then removed in vacuo. The residue was purified by preparative FIPLC and then, if necessary, further purified by reverse phase column chromatography eluting with acetonitrile in water. This afforded a compound of Formula I.

Compounds were measured via FIPLC/MS, using a Waters X-BRIDGE™ C18-column, 5 pm particle size, 4,6 x 150 mm (diameter x length) at a flow rate of 1 ,75 mL/min with a linear gradient (water to acetonitrile, 0.2% formic acid as modifier) from initially 99:1 to 1 :99 over 9.10min, then hold for 1.80 min. Mass signals were determined using a Waters 3100 Mass Detector. HPLC retention times of Compounds 1 to 14 are shown in Table 1.

Table 1

Example 2: Preparation of Compound 1

Compound 1

4-((2,5-dichloropyrimidin-4-yl)amino)methyl)benzenesulfonamide

2,4,5-Trichloropyrimidine (0.50g, 2.74 mmol, 1.0 equiv.) and DIPEA (0.57 ml, 3.30 mmol, 1.2 equiv.) were dissolved in ethanol (5 ml) and the solution was cooled in an ice water bath. 4-Aminomethyl benzenesulfonamide hydrochloride (0.51 g, 2.74 mmol, 1.0 equiv.) was added to the stirred solution and the reaction mixture was warmed to room temperature over 2h. A precipitate formed and this was then collected by filtration to afford 4-((2,5-dichloropyrimidin-4-yl)amino)methyl) benzenesulfonamide (582 mg, 64% yield). No further purification was necessary. 4-(((5-chloro-2-((3-methyl-1-(2,2,2-trifluoroethyl)-1H-pyrazol-4-yl)amino)pyrimidin-

4-ylamino)methyl)benzenesulfonamide

4-((2,5-dichloropyrimidin-4-yl)arnino)methyl) benzenesulfonamide (150 mg, 0.45 mmol, 1.0 equiv.) was dissolved in sec-butanol (2 ml). To this solution 3-methyl-1 -(2,2,2- trif I u o roethy I )- 1 H-pyrazol-4-amine (117 mg, 0.54 mmol, 1.2equiv.) and 4M HCI in dioxane (2 drops) were added. The reaction mixture was stirred and heated to 120°C for 2h. The solvent was then removed in vacuo. The residue was purified by preparative HPLC and then further purified by reverse phase column chromatography eluting with acetonitrile in water (0-100%) to afford 4-(((5-chloro-2-((3-methyl-1 -(2,2,2-trifluoroethyl)-1 /-/-pyrazol-4- yl)amino)pyrimidin-4-ylamino)methyl)benzenesulfonamide (Compound 1 ) (133 mg, 62% yield). ¹H NMR (400 MHz, DMSO-de) d 8.44 (s, 1 H), 7.90 (s, 1 H), 7.84 - 7.65 (m, 4H), 7.41 (d, J = 7.9 Hz, 2H), 7.27 (s, 2H), 4.89 (q, J = 9.0 Hz, 2H), 4.73 - 4.47 (m, 2H), 2.06 (s, 3H).

Example 3: Preparation of Compound 4

Compound 4

4-((5-bromo-2-chloropyrimidin-4-yl)amino)methyl)benzenesulfonamide

5-Bromo-2,4,-dichloropyrimidine (0.1 Og, 0.438 mmol, 1.0 equiv.) and DIPEA (0.09 ml, 0.526 mmol, 1.2 equiv.) were dissolved in ethanol (1 ml) and the solution was cooled in an ice water bath. 4-Aminomethyl benzenesulfonamide hydrochloride (0.081 g, 0.438 mmol, 1.0 equiv.) was added to the stirred solution and the reaction mixture was warmed to room temperature over 2h. A precipitate formed and this was then collected by filtration to afford 4-((5-bromo-2-chloropyrimidin-4-yl)amino)methyl) benzenesulfonamide ( 103 mg, 62% yield).

4-(((5-bromo-2-((3-methyl-1 -(2,2,2-trifluoroethyl)-1H-pyrazol-4-yl)amino)pyrimidin-

4-ylamino)methyl)benzenesulfonamide 4-((5-bromo-2-chloropyrimidin-4-yl)amino)methyl) benzenesulfonamide (60 mg, 0.159 mmol, 1.0 equiv.) was dissolved in sec-butanol (1 ml). To this solution 3-methyl-1 -(2,2,2- trif I u o roethy I )- 1 H-pyrazol-4-amine (34 mg, 0.159 mmol, I .Oequiv.) and 4M HCI in dioxane (1 drop) were added. The reaction mixture was stirred and heated to 120°C for 2h. The solvent was then removed in vacuo. The residue was purified by preparative HPLC and then further purified by reverse phase column chromatography eluting with acetonitrile in water (20-50%) to afford 4-(((5-bromo-2-((3-methyl-1 -(2,2,2-trifluoroethyl)-1 /-/-pyrazol-4- yl)amino)pyrimidin-4-ylamino)methyl)benzenesulfonamide (Compound 4) (30 mg, 36% yield). ¹H NMR (400 MHz, DMSO-de) d 8.44 (s, 1 H), 7.97 (s, 1 H), 7.78 - 7.65 (m, 3H), 7.58 (t, J = 6.1 Hz, 1 H), 7.40 (s, 2H), 7.26 (s, 2H), 5.00 - 4.80 (q, J = 9.3, 2H), 4.60 (d, J = 6.1 Hz, 2H), 2.06 (s, 3H).

Example 4: Preparation of Compound 5

Compound 5

4-((2-chloro-5-methylpyrimidin-4-yl)amino)methyl) benzenesulfonamide

2,4,-Dichloro-5-methylpyrimidine (0.20g, 1.226 mmol, 1.0 equiv.) and DIPEA (0.25 ml, 1.472 mmol, 1.2 equiv.) were dissolved in ethanol (1.5 ml) and the solution was cooled in an ice bath. 4- Aminomethyl benzenesulfonamide^*HCI (0.228 g, 1.226 mmol, 1.0 equiv.) was added to the stirred solution and the reaction mixture was warmed to room temperature over 2h and then stirred at room temperature for another 18h. A precipitate formed and this was then collected by filtration to afford 4-((2-chloro-5-methylpyrimidin-4- yl)amino)methyl) benzenesulfonamide ( 254 mg, 66% yield).

4-(((5-methyl-2-((3-methyl-1 -(2,2,2-trifluoroethyl)-1H-pyrazol-4-yl)amino)pyrimidin-

4-ylamino)methyl)benzenesulfonamide

4-((2-chloro-5-methylpyrimidin-4-yl)amino)methyl) benzenesulfonamide (60 mg, 0.190 mmol, 1.0 equiv.) was dissolved in sec-butanol (1 ml). To this solution 3-methyl-1 -(2,2,2- trif I u o roethy I )- 1 H-pyrazol-4-amine (41 mg, 0.190 mmol, I .Oequiv.) and 4M HCI in dioxane (1 drop) was added. The reaction mixture was heated to 120°C for 4h. The solvent was then removed in vacuo. The residue was purified by preparative FIPLC and then further purified by reverse phase column chromatography eluting with acetonitrile in water (0- 100%). This afforded 4-(((5-bromo-2-((3-methyl-1 -(2,2,2-trifluoroethyl)-1 /-/-pyrazol-4- yl)amino)pyrimidin-4-ylamino)methyl)benzenesulfonamide (Compound 5) (36 mg, 42% yield). ¹H NMR (400 MHz, DMSO-de) d 7.97 (s, 1 H), 7.77 (s, 1 H), 7.72 (d, J = 8.4, Hz, 2H), 7.63 (1 H,s), 7.43 (d, J = 8.4, 2H), 7.26 (s, 2H), 7.24 - 7.18 (m, 1 H), 4.86 (q, J = 9.3 Hz, 2H), 4.63 (d, J = 6.0 Hz, 2H), 2.08 (s, 3H), 1.95 (s, 3H).

All other compounds were prepared in substantially the same way as Compounds 1 , 4 and 5.

Example 5: TBK1 array assay

The TBK1 assay was based on chemoproteomics affinity-enrichment technology and was performed in a 384-well format using an antibody based read-out. The affinity matrix was prepared with the capturing compound as (Z)-N-(3-aminopropyl)-5-((5-fluoro-2- oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1 H-pyrrole-3-carboxamide, a coupling density of 0.3umol/ml, as a 50% slurry in isopropanol of matrix NHS-activated SEPHAROSE 4 FAST FLOW™ (Amersham Biosciences). For the assays, 25 pi Ramos cell lysate with a protein concentration of 5 mg/ml in DP buffer (50 mM T ris-HCI (pH 7.4), 0.4% (v/v) IGEPAL™-CA630, 5% (v/v) glycerol, 150 mM NaCI, 1.5 mM MgCI₂, 25 mM NaF, 1 mM sodium vanadate, 1 mM dithiothreitol supplemented with Roche protease inhibitor tablets) and 50 pi capturing matrix (as 2.5 % slurry in DP buffer) per well were incubated in the absence or presence of test compound at 4°C for 2h. Compounds were tested in a concentration-response format applying 1 :3 dilution steps. DMSO concentration was kept constant at 2% (v/v). After incubation, the non-bound fraction was removed by washing the beads with DP buffer. Proteins retained on the beads were eluted in SDS sample buffer (100 mM Tris (pH 7.4), 4% (w/v) SDS, 20% (v/v) glycerol, 0.01 % (w/v) bromophenol blue, 50 mM dithiothreitol) and spotted on nitrocellulose membranes (400 nl per spot) using an automated pin-tool liquid transfer (BIOMEK FX™, Beckman). After drying, the membranes were rehydrated in 20% (v/v) ethanol and processed for detection by overnight incubation at 25°C with a specific anti-TBK1 antibody (diluted 1 :6000 or alternatively diluted 1 :1000) in the presence of 0.4% TWEEN™ followed by incubation for 1 h at room temperature with an IRDye™ 800-labeled secondary antibody for visualization (anti-rabbit diluted 1 :5000) in presence of 0.2% TWEEN™. Spot intensities were quantified using a LICOR ODYSSEY™ scanner. The results of the TBK1 assay array are shown in Table 2. Compounds 4, 9, 10, 12, 13 and 14 in Table 2 were tested in the formic acid salt form.

Table 2

Example 6: Inhibition of phosphorylation of IRF3 in Ramos cells

The effects of a compound of the invention in live cells was investigated. Ramos cells were stimulated with the TLR3 ligand poly(l:C) and the phosphorylation of IRF3 was measured by Western blot.

Ramos cells (ATCC™, 10⁶/well) were exposed to Compound 4 for 60 min in cell culture media (RPMI1640, GIBCO™) containing 2% fetal bovine serum (FBS, GIBCO™) and then stimulated with poly(l:C) (30 pg/mL, Invivogen) for 120 min at 37°C, 5% CO2. Cells were then collected and washed once with ice-cold D-PBS (GIBCO™). The cells were lysed in 50 mM T ris-HCI (pH 7.4, Sigma) containing 5% glycerol (VWR), 1.5 mM MgCL (Sigma), 20 mM NaCI (Sigma), 1 mM Na₃V0₄ (Sigma), 0.8% (v/v) IGEPAL™-CA630 (Sigma), 50 nM Calyculin A (Sigma), phosphatase inhibitors mix (PhosStop™, Roche), protease inhibitors mix (aprotinin, bastatin, leupeptin, pepstatin, phosphoramidon, all Sigma), 25 mM NaF (Sigma), 1 mM dithiothreitol (Sigma). Cell debris were removed by centrifugation and the soluble proteins were denatured with NUPAGE™ LDS Sample Buffer (Life Technologies) supplemented with 50 mM dithiothreitol). The samples were analysis by Western blot using 4-10% polyacrylamide gel and antibodies. The relative phosphorylation of IRF3 (Ser396, Cell Signaling #4947) was analysed in SDS lysates by Western blot. Band intensities were quantified using a LICOR ODYSSEY™ scanner. The normalised phospho-IRF3 signal is displayed as percent inhibition, with unstimulated cells giving the minimum signal and vehicle-treated poly(l:C)-stimulated cells giving the maximum signal. The plC50 was derived from a four-parameter sigmoidal curve fit constrained to top (100%) using GRAPHPAD PRISM™ software (V7). Compound 4 inhibited phospho-IRF3 with an average pICso of 6.0 (Figure 15A), confirming effective inhibition of TBK1 kinase activity in live cells.

Example 7: Inhibition of IFNa secretion in human PBMCs

IFNa secretion was measured in poly(l:C)-stimulated human peripheral blood mononuclear cells (PBMCs).

Peripheral blood mononuclear cells (PBMC) were isolated from human whole blood by density centrifugation using a Ficoll-based medium (LSM1077, PAA). PBMCs were seeded in 96-well plates at 50,000 cells per well in 50 mI_ media (RPMI1640, Gibco) with 10% fetal bovine serum (Gibco). Cells were incubated with compound 4 or vehicle (DMSO 0.2%) for 1 h at 37°C, 5% CO2. The cells were then stimulated with 100pg/ml poly(l:C) for 16 h at 37°C, 5% CO2. The supernatant was collected and analysed by multiplex Cytometric Bead Array (CBA) Flex Sets (BD Biosciences) for secreted IFNa (#560379). Percent inhibition of IFNa secretion was calculated based on the mean fluoresence intensities (MFI) measured by flow cytometry (FACSCalibur™, BD Biosciences). The IFNa receptor antagonist (anti-IFNAR2) was purchased from PBL Assay Science (#21385-1 ). IFNp was measured by ELISA (Mesoscale Discoveries) on MSD SI 2400. As IFNp was below the limit of detection of the assay (MSD readout, data not shown), IFNa was measured because it could be robustly detected in supernatant by flow cytometry (CBA assay). Compound 4 inhibited the release of IFNa with a pICso of 6.1 , demonstrating sub micromolar potency in primary immune cells (Figure 15B).

Example 8: Inhibition of IFNB secretion in human THP-1

It is well known that TBK1 propagates biological signalling of IFNp downstream of DNA sensing by cGAS and STING. To test whether compounds of the invention inhibit this pathway, THP-1 cells were stimulated with dsDNA containing virus (BacMam) or the natural STING ligand cGAMP.

THP-1 cells were plated in 96-well tissue culture plates at 100,000 cells per well in 100 pL media (RPMI-140, 10 % heat-inactivated FBS, 1 % penicillin/streptomysin/amphotericin) followed by 45 min incubation at 37 °C with increasing concentrations of compound 4 (0.003-10 mM). Cells were stimulated with 10 pL BacMam virus (7.32 X 10⁸ pfu/mL) or 10 pL of 600 pg/mL cGAMP solution (in water). Levels of secreted IFNp were measured from cell supernatants following a 20 hour incubation by electrochemiluminescence using a MESO SECTOR S 600™ platform following the manufacturer’s instructions for the MSD 96-well MULTI-ARRAY™ Fluman IFN-b assay (K1 11ADB-2). Data from three technical replicate experiments were averaged and plot as a function of compound 4 concentration in GRAPHPAD PRISM™ 4.0 and fit to model of inhibition to determine the IC50. Compound 4 was able to inhibit secretion of IFNp with a measured pICso of 5.9 and 6.3 for the dsDNA virus and cGAMP stimulated cells, respectively (figure 16). The biological activity of compound 4 was demonstrated, resulting in inhibition of IRF3 phosphorylation, IFNa secretion from human PBMCs and secretion of IFNp from THP-1 cells with low micromolar potency.

Example 9: TBK1 Inhibitor Treatment Improves Cell Survival and Growth after

Transfection with Nucleic Acids

The effects of different concentrations of TBK1 inhibitor (Compound 4), different plasmid DNA concentrations and post-transfection media replacement on cell survival after plasmid DNA transfection were examined.

Methods

Cell culture and reagents

THP1 cells were cultured at 37°C in 5% C0₂ atmosphere to a density of <1X10⁶ cells/ml in complete media. Complete media comprised RPMI-1640 medium modified to contain 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/L glucose, and 1500 mg/L sodium bicarbonate and 10% FBS. Cells were split to 0.3x10⁶ cells/ml twice weekly.

The TBK1 inhibitor (Compound 4) was resuspended in DMSO to create a 10mM stock (500x) that was stored at -70°C. The stock was diluted to working concentration with complete media. THP1 cells subjected to 10 passages were incubated with TBK1 inhibitor at a final concentration of 20uM or 40uM for 20 hours prior to transfection with plasmid DNAs. The pmaxGFP™ plasmid (Figure 1 1 ) was a transfection control. This plasmid DNA encodes a green fluorescent protein (GFP) from the copepod Pontellina plumata and expression of this GFP is used to monitor transfection efficiency. Constitutive GFP expression from pmaxGFP™ is driven by a CMV promotor.

Transfection

The pmaxGFP™ plasmid DNA was transfected into THP-1 cells by electroporation with an AMAXA™ device, using the Lonza Transfection Kit for Fluman Monocytes and NUCLEOFECTOR2B™ cuvettes. Transfection efficiencies were typically >65% with this method. For each experiment 1.0x10⁶ cells were resuspended in 0.5ml OPTIMEM™ low serum media in each well of a 12 well culture dish. Cells were transfected with 0.5ug, 1 ug or 2ug GFP plasmid and transferred into media containing 20uM or 40uM TBK1 inhibitor per Table 3. Media was exchanged with complete media containing 20% FBS and 20uM or 40uM TBK1 inhibitor at either 6hr or 20hr post-transfection. For 6hr media replacement ~300ul media was removed with a pipette without removing cells and 1 ml fresh media including TBK1 inhibitor was added. For all experiments with 20hr media exchange, no media was removed until 20hr. Instead 1 ml of complete growth media with TBK1 inhibitor was added to the wells at 20hr. At 48hr post-transfection media was removed from the cells and replaced with complete growth media with TBK1 inhibitor. GFP expression then was measured using an INCUCYTE™ instrument at approximately 7, 48 and 72 hr. On days 3 and 8 post-transfection, 0.5 ml of cell culture was removed for viable cell count and a %viability determination was made using a VIA-CELL™ counter. Experimental parameters are shown in Table 3 (transfection conditions for DNA sensitivity study +/- TBK1 inhibitor).

Table 3

Results

Transfection with DNA concentrations >1 ug/ml had a negative effect on cell viability. The addition of TBK1 inhibitors significantly improved day 3 cell viability at 1 ug/ml and 2ug/ml DNA (Figure 2). A DNA concentration of 2ug/ml also had a negative effect on cell growth and day 8 viable cell count. Importantly, the presence of TBK1 inhibitor significantly improved cell survival and cell growth post-transfection (Figures 3, 4 and 5). In Figures 2, 3, 4, and 5 dashed lines mean TBK1 inihibitor is absent and solid lines mean TBK1 inhibitor is present at the indicated concentration. Beneficial effect from TBK1 inhibitors is believed to be more pronounced at higher plasmid concentration.

Cell growth and %viability were also higher with media replacement at 6hr vs 20hr. GFP expression was significantly higher with 1 & 2 ug/ml DNA transfections (Figure 6). Expression on day 3 was independent of inhibitor addition. Transfection with 2ug of GFP encoding plasmid gave the highest fluorescence, ~50 GFU as measured by INCUCYTE™. Control cells had ~12 GFU background fluorescence, similar to the 0.5ug DNA transfection conditions. Cells were not assayed past day 3. This data illustrates that a DNA concentration >1 ug/ml is desirable for efficient transfection of 1x10⁶ THP1 cells with the AMAXA™ method.

Example 10: Generation of TREX1 Knock-out with CRISPR/Cas9 in THP1 Cells using TBK1 Inhibitor

A 2³ factorial experiment comparing CRISPR mediated gene delivery with two sets of CRISPR CAS9 reagents, two guide RNA concentrations, with and without TBK1 inhibitor was performed. The best conditions from this experiment were carried forward to generate the TREX1 KO cell line per the protocol outlined in Figure 7.

Methods

Growth assessment and antibiotic kill curve

Two thousand THP1 cells were plated into the first column of a 96 well plate and serially diluted to 1 cell/well. G418 or puromycin were serially diluted into the plate in the opposite direction. No antibiotic was added to the last row of the plate to assess the ability of THP1 cells to grow in 96 well plates when seeded at 1 cell/well. Plates were cultured for 8 days at 37°C with antibiotic replacement on day 4. Growth of THP1 cells seeded at a density of 1 cell/well in a 96 well plate was then confirmed. Additionally, the minimum concentration of antibiotic that killed 100% of cells at a density of 2000 cells/well was determined.

Guide RNA (qRNA) construction

Guide RNA

The Cas9 gRNA guide was designed to target the Cas9 nuclease to the TREX1 gene and make a double stranded break to allow insertion of the GSK TREX-1 homology-directed DNA repair (HDR) donor plasmid ( see e.g ., Figure 1 and Figure 14). A plasmid expressing Cas9 and TREX1 guide RNA was generated for this purpose. The 20nt target sequence 5’-AGCTTGTCTACCACACGCGG-3’ (SEQ ID NO: 1 ) (reverse complement) was cloned into the linearized pCas-Guide™ plasmid (Origene) using the pCas-Guide™ pre-cut cloning kit. A circularized plasmid vector was then created by annealing the following oligonucleotides (TREX1 -1 Fb: 5'-gatcgAGCTTGTCTACCACACGCGGg-3' (SEQ ID NO: 2); TREX1 -1 Rb: 5'-aaaacCCGCGTGTGGTAGACAAGCTc-3’ (SEQ ID NO: 3)) and ligating into the pre-linearized pCas-Guide™ plasmid vector arms. The Cas9 expression cassette in the resulting plasmid construct was driven by the human U6 promotor. This construct contained no fluorescence marker to verify transfection of the guide RNA encoding plasmid.

Santa Cruz guide RNA

CRISPR/Cas9 trexl knockout plasmids were obtained from Santa Cruz Biotech (Figure 12). Guides were designed to make a Cas9 induced double stranded break to allow insertion of the Santa Cruz TREX-1 HDR donor plasmid into the targeted sites of the TREX1 gene (Figure 13 and Figure 14). The knockout plasmids were supplied as a mixture of three different Cas9 gRNA plasmids (sc-403875A1 , A2 & A3) with target sequences 5’-AGCAT CT ACACT CGCCT GT A-3’ (SEQ ID NO: 4), 5’- AGTTCCTCCACCACCGCGTG-3’ (SEQ ID NO: 5) and 5’-

CCCTCAGCCGTGTGCGAGTC-3’ (SEQ ID NO: 6), respectively. GFP sequence encoded by these plasmids was used to visually verify the transfection. GFP and Cas9 expression were both driven by the CBh (chicken b-actin hybrid) promotor in these CRISPR/Cas9 knockout plasmid vector construct(s). Guide RNA expression was driven by the U6 promotor in these CRISPR/Cas9 knockout plasmid vector construct(s).

Donor Plasmid Construction

GSK HDR donor

A donor plasmid expressing GFPneo was generated. The plasmid was a homologous recombination vector for inserting a GFPneo fusion protein sequence into the targeted site within the TREX1 gene to monitor cells positive for expression and stable selection with G418 resistance. There was no promoter in this construct and expression was driven by the native TREX1 promoter. The TREX-1 FIDR plasmid contained a FIDR template corresponding to the cut sites generated by the GSK guide RNA. Each FIDR template contained two 800 bp homology arms designed to specifically bind to the genomic DNA surrounding the corresponding Cas9-induced double-strand DNA break site.

Santa Cruz FIDR donor

A donor plasmid preparation expressing RFPpuro was obtained from Santa Cruz Biotech. The plasmid was a homologous recombination vector for inserting RFPpuro fusion protein sequence into the targeted site within the trexl gene to monitor cells positive for expression and stable selection with puromycin resistance. Expression of the fusion protein was driven by the EF1 a promoter. The Santa Cruz TREX-1 FIDR Plasmids consisted of a pool of 3 plasmids, each containing a homology-directed DNA repair (FIDR) template corresponding to the cut sites generated by the TREX-1 CRISPR/Cas9 KO Plasmid: sc-403875. Each HDR template contained two 800 bp homology arms designed to specifically bind to the genomic DNA surrounding the corresponding Cas9-induced double-strand DNA break site. HDR insertion was confirmed by RFP expression in the clonal cell population and insertion into the break site by junction PCR of the homology arms.

Cell Transfection - Guide RNA targeting in cells

The procedure used for generation of TREX1 KO cell lines is outlined in Figure 7. Guide RNA and Donor DNA plasmids were co-transfected into cells via electroporation with an AMAXA™ device and the Lonza Transfection Kit for Human Monocytes using the NUCLEOFECTOR2B™ cuvette. Transfection efficiencies were typically >65%. TBK1 inhibitor (Compound 4) was diluted to 10mg/ml in DMSO for a 500X working stock. All plasmids were resuspended to ~1 ug/ul as verified via NANODROP™ quantitation. 1.5X10⁶ cells for each experimental condition were transfected. Cells were co-transfected with 1 ug or 3ug guide RNA, and 1 ug donor DNA per Table 4 (which shows the transfection conditions for the CRISPR/Cas9 TREX1 KO experiments) and resuspended in 0.5ml OPTIMEM™ low serum media in each well of a 12 well Corning culture dish to give 1 ml total volume. Positive control cells were examined by fluorescence microscopy for GFP expression after 4hr to monitor transfection efficiency. Transfection with the Santa Cruz TREX1 KO plasmid was confirmed via GFP fluorescence after 4hr. Conditioned media in each well was replaced with complete growth media with or without 20uM of TBK1 inhibitor (Compound 4) at minus 24, 0, 6 and 20hr. DMSO was added in experiments 5-12 at 1 :500 dilution on transfection as a solvent control.

Table 4

Cells were then allowed to recover in 12 well plates for 5 days. Cell count and viability determinations were then performed. G418 was added at 0.5 mg/ml and puromycin was added at 0.75 ug/ml. Antibiotic selection was carried out for 10 days with increasing concentrations of antibiotic to a final concentration of 1 mg/ml and 1 ug/ml, respectively. Cultures were sampled for cell count and viability determination. Cells transfected with 3ug GSK or Santa Cruz gRNA were dilution plated at 1 cell/well into 96 well flat bottom plates in RPMI 1640 + 10% FBS growth media without antibiotic selection for cloning. Because editing efficiency is thought to be greater at higher gRNA concentration, experiments 2 and 4 were carried forward. Plates with the fewest # of colonies (<20) were selected for clonal screening. About 75 clones were transferred to duplicate plates for cell culture and screening using the WES™ western analysis system. Three trexl antibodies were screened and the Cell Signaling TREX1 antibody #12215 gave a superior signal.

Amplification of targeted regions

Genomic DNA was extracted from cells using the following protocol. Genomic DNA was isolated from -1X10⁶ cells in a 96 well plate by combining cells from 2 wells. Plates were centrifuged at 3000 RPM for 20min to remove media and cells were washed with PBS. Cells were lysed in the plate with Viagen Biotech DIRECTPCR™ Lysis Reagent (Cell) Cat # 301 -C, 302-C and benzenoase. PCR primer pairs were designed for TIDE analysis for Santa Cruz sc-403875A2 vector. Primers spanned a 600-1 OOObp sequence of trexl genomic DNA with the CRISPR cut site within 50bp of the middle of the sequence. The targeted region was amplified from genomic DNA with PHUSION HOT START™ DNA polymerase.

PCR worked well with all primer pairs using the PHUSION HOT START™ thermocycler protocol optimized for Trexl TIDE. Product was a single band on E-GEL™ 2% agarose 48 well gel (Invitrogen) with 10ul H₂0 + 5ul PCR product load. PCR product was purified with the QIAQUICK™ 96 PCR Purification Kit. 1 ul of sodium, or ammonium, acetate was added for every 10ul of reaction mix since PHUSION HOT™ start polymerase is basic.

Results

Transfection in the presence of TBK1 inhibitor (Compound 4) significantly increased cell survival on day 5, before antibiotic selection (Figure 8 and Figure 9). The TBK1 inhibitor was critical to cell survival after antibiotic selection. In all test conditions, cells transfected without TBK1 inhibitor (see e.g., experiments 5-8) did not survive 10 days of selection in either puromycin or G418. Antibiotic used for selection was dependent on the HDR plasmid used.

NHEJ and HDR (donor insertion) for generation of T rex1 KO

~12 clones edited with Santa Cruz HDR donor and ~75 clones edited with GSK donor vectors were screened for trexl protein expression using the WES™ instrumentation (Cell Signaling antibody #12215 & vinculin control). Clones 4-2, 4-6 and 4-7 were identified as possible knockouts generated using the Santa Cruz transfected clones. Subsequent screening for NHEJ via TIDE analysis at the SC-403875A2 gRNA cut site revealed two edited alleles for clone 4-7, confirming a bi-allelic knock-out via NHEJ as predicted from combined WES™ and PCR analysis (Figure 10). Knock-out clones 4-2 and 4-6 had a higher molecular weight PCR product via PCR analysis with TIDE primers, which was consistent with the insertion of the HDR plasmid. Clones 4-2 and 4-6 both showed some degree of editing at the SC-403875A2 gRNA cut site as assessed via TIDE analysis. However, TIDE analysis specific for the SC-403875A1 & sc-403875A3 gRNA cut sites was performed with different sets of primers since they are not in close proximity to the sc- 403875A2 gRNA cut site. Based on WES™, PCR, partial TIDE analysis and RFP fluorescence, clones 4-2 and 4-6 are predicted to have one allele edited via HDR and one allele edited via NHEJ.

The material in the ASCII text file named“PB66541_FF_Seq_List_29May2019” created on May 29, 2019 and having a size of 1 1 ,000 bytes is incorporated herein by reference in its entirety. INFORMAL SEQUENCE LISTING

SEQ ID NO: 1

Source/Origin: Artificial nucleic acid generated by molecular biology techniques.

AGCTTGTCTACCACACGCGG

SEQ ID NO: 2

GATCGAGCTTGTCTACCACACGCGGG

SEQ ID NO: 3

AAAACCCGCGT GTGGT AGACAAGCT C

SEQ ID NO: 4

AGCATCTACACTCGCCTGTA

SEQ ID NO: 5

AGTTCCTCCACCACCGCGTG

SEQ ID NO: 6

CCCTCAGCCGTGTGCGAGTC

SEQ ID NO: 7

CCCTCAGCCGTGTGCGAGTC

SEQ ID NO: 8

Source/Origin: Homo sapiens amino acid sequence for serine/threonine-protein kinase TBK1 (TANK Binding Protein 1 )

QSTSNHLWLLSDILGQGATANVFRGRHKKTGDLFAIKVFNNISFLRPVDVQMREFEVLK

KLNHKNIVKLFAIEEETTTRHKVLIMEFCPCGSLYTVLEEPSNAYGLPESEFLIVLRDVVG

GMNHLRENGIVHRDIKPGNIMRVIGEDGQSVYKLTDFGAARELEDDEQFVSLYGTEEYL

HPDMYERAVLRKDHQKKYGATVDLWSIGVTFYHAATGSLPFRPFEGPRRNKEVMYKII

TGKPSGAISGVQKAENGPIDWSGDMPVSCSLSRGLQVLLTPVLANILEADQEKCWGFD

QFFAETSDILHRMVIHVFSLQQMTAHKIYIHSYNTATIFHELVYKQTKIISSNQELIYEGRR

LVLEPGRLAQHFPKTTEENPIFVVSREPLNTIGLIYEKISLPKVHPRYDLDGDASMAKAIT

GVVCYACRIASTLLLYQELMRKGIRWLIELIKDDYNETVHKKTEVVITLDFCIRNIEKTVKV

YEKLMKINLEAAELGEISDIHTKLLRLSSSQGTIETSLQDIDSRLSPGGSLADAWAHQEG

THPKDRNVEKLQVLLNCMTEIYYQFKKDKAERRLAYNEEQIHKFDKQKLYYHATKAMTH

FTDECVKKYEAFLNKSEEWIRKMLHLRKQLLSLTNQCFDIEEEVSKYQEYTNELQETLP

QKMFTASSGIKHTMTPIYPSSNTLVEMTLGMKKLKEEMEGVVKELAENNHILERFGSLT

MDGGLRNVDCL

Claims

1. A compound of formula I:

Formula I wherein:

R² is H or C1 -C6 alkyl;

R⁴ is -S(0)₂-NH₂, -S(0)₂-(C1 -C6 alkyl), -S0_2-NH-(C1 -C6 alkyl) or -C(=0)-NH-(C1 -C6 alkyl); or a salt thereof.

2. The compound according to Claim 1 , wherein R¹ is H, methyl, ethyl, O-methyl, C(=0)NFI₂, cyclopropyl, Cl, F, Br or I.

3. The compound according to Claim 2, wherein R¹ is Cl, Br, methyl, ethyl, O-methyl, C(=0)NFI₂ or cyclopropyl.

4. The compound according to Claim 3, wherein R¹ is Br.

5. The compound according to any preceding claim, wherein R² is methyl.

6. The compound according to any preceding claim, wherein R³ is (CFI₂)_n-CF₃ or (CH₂)_n-CHF_2.

7. The compound according to Claim 6, wherein n is 1.

8. The compound according to Claim 7, wherein R³ is CH2-CF3.

9. The compound according to any preceding claim, wherein R⁴ is -S(0)₂-NFl₂, - S(0)₂-CH₃,or -C(=0)-NH-CH₃.

10. The compound according to Claim 9, wherein R⁴ is -S(0)₂-NFl₂ or -S(0)₂-CFl₃.

1 1. The compound according to Claim 10, wherein R⁴ is -S(0)₂-NFl₂.

12. The compound according to Claim 1 , wherein:

R¹ is H, methyl, ethyl, O-methyl, C(=0)NFl₂, cyclopropyl, Cl, F, Br or I;

R² is methyl;

R³ is (CFl2)n-CF3 or (CFl2)n-CFIF2, wherein n is 1 ; and R⁴ is -S(0)₂-NH₂, -S(0)₂-CH₃,or -C(=0)-NH-CH₃.

13. The compound according to Claim 12, wherein:

R¹ is Cl, Br, methyl, ethyl, O-methyl, C(=0)NH₂ or cyclopropyl;

R³ is CH2-CF3; and

R⁴ is -S(0)₂-NH₂ or -S(0)₂-CH₃.

14. The compound according to Claim 1 , wherein the compound is selected from the group consisting of:

Compound 2

Compound 8

12

Compound 14.

15. The compound according to Claim 14, wherein the compound is:

16. The compound according to any preceding claim, wherein the compound is in the form of the formic acid salt.

17. Use of a compound of Formula 1 as defined in any of Claims 1 to 16 as a TBK1 inhibitor.

18. Use of a compound of Formula 1 as defined in any of Claims 1 to 16 in a method of delivering a nucleic acid into a cell.

19. Use according to Claim 18, wherein the nucleic acid integrates into a chromosomal sequence of the cell.

20. Use according to any of Claims 17 to 19 wherein the nucleic acid:

a. encodes at least one nuclease;

b. comprises at least one guide RNA; or

c. comprises DNA encoding at least one guide RNA.

21. Use according to Claim 20 wherein the nucleic acid encodes at least one nuclease and the nuclease is CRISPR associated protein 9 (CAS9).

22. A method of introducing a nucleic acid into an animal cell comprising the steps of: a) providing an animal cell; b) contacting the animal cell with a nucleic acid; c) treating the animal cell to cause the nucleic acid to enter the cell; and d) contacting the animal cell with a compound as defined in any of Claims 1 to 16 in an amount effective to inhibit TBK1 activity; whereby the nucleic acid is introduced into the animal cell.

23. The method according to Claim 22, wherein the method is in vitro or ex vivo.

24. The method according to Claim 22 or Claim 23 wherein the nucleic acid encodes a) a zinc finger protein comprising a nuclease;

b) a transcription activator effector (TAL) comprising a nuclease;

c) a meganuclease; and/or

d) a CRISPR associated protein 9 (CAS9).

25. The method according to Claim 24 wherein the nucleic acid encodes a CRISPR associated protein 9 (CAS9) and the CAS9 is provided with a second nucleic acid encoding a guide RNA.

26. The method according to any of Claims 22 to 25 wherein the animal cell comprises a target nucleic acid; and a targeting nucleic acid is incorporated into the target nucleic acid in the animal cell.

27. The method according to Claim 26 wherein the targeting nucleic acid recombines into the target nucleic acid.

28. The method according to Claim 26 or 27 wherein the target nucleic acid is selected from the group consisting of a genomic DNA or extra-genomic DNA.

29. A method of changing the genotype of an animal cell comprising the steps of: a) providing an animal cell comprising a target nucleic acid; b) contacting the animal cell with a first nucleic acid encoding a transcription activator effector (TAL) comprising a nuclease, and a targeting nucleic acid; c) treating the animal cell to cause the first nucleic acid and the targeting nucleic acid to enter the cell; and d) contacting the animal cell with a compound as defined in any of Claims 1 to 16 in an amount effective to inhibit TBK1 activity; whereby the targeting nucleic acid is introduced into the target nucleic acid in the animal cell and the genotype of the animal cell is changed.

30. The method according to Claim 29 wherein the nuclease is Fok I.

31. The method according to Claim 29 or 30 wherein the target nucleic acid is genomic DNA or extra-genomic DNA.

32. A method of changing the genotype of an animal cell comprising the steps of: a) providing an animal cell comprising a target nucleic acid; b) contacting the animal cell with a first nucleic acid encoding a CRISPR associated protein 9 (CAS9), a second nucleic acid encoding a guide RNA, and a targeting nucleic acid; c) treating the animal cell to cause the first nucleic acid, the second nucleic acid and the targeting nucleic acid to enter the cell; and d) contacting the animal cell with an amount of a compound as defined in any of Claims 1 to 16 in an amount effective to inhibit TBK1 activity; whereby the targeting nucleic acid is introduced into the target nucleic acid in the animal cell and the genotype of the animal cell is changed.

33. The method according to Claim 32 wherein the target nucleic acid is selected from the group consisting of a genomic DNA or extra-genomic DNA.

34. The method according to any one of Claims 22 to 33, wherein the animal cell is a Chinese hamster ovary (CHO) cell or a THP1 cell.

35. The method according to any one of Claims 22 to 34, wherein the compound of Formula I is a compound as defined in Claim 14.

36. The method according to any one of Claims 22 to 34, wherein the compound of Formula I is the compound defined in Claim 15.

37. The method of any one of Claims 22 to 36, wherein the effective amount of the compound of Formula I is about 10 mM to about 50 pM, about 15 pM to 30 pM, and about 20 pM.

38. An animal cell produced by the method according to any one of Claims 22 to 37.