Methods and compositions for UBA5 inhibition

Abstract

UBAS is a critical El-activating enzyme in the UFMylation pathway, a post-translational modification process implicated in neurodegenerative diseases and cancers. Here, a high-throughput screening (HTS) assay was developed to identify inhibitors of UBAS from various compound libraries. Eighteen novel UBAS inhibitors were identified, belonging to several distinct chemical scaffolds with low micromolar IC50 values. These inhibitors demonstrated selectivity for UBAS over other El enzymes, including UBA1, and showed efficacy in inhibiting endogenous UFMylation in HEK293T cells. The identified inhibitors not only provided valuable tools for studying UFMylation but also represented potential therapeutic candidates for diseases associated with dysregulated UFMylation, such as Alzheimer's disease and cancer.

Description

​METHODS AND COMPOSITIONS FOR UBA5 INHIBITION​ ​CROSS-REFERENCE TO RELATED APPLICATIONS​​

[0001] ​ ​This​ ​application​ ​claims​ ​benefit​ ​of​ ​U.S.​ ​Provisional​ ​Application​ ​No.​ ​63 / 711,575​ ​filed​​October 24, 2024, the specification of which is incorporated herein in their entirety by reference.​ ​FIELD OF THE INVENTION​​

[0002] ​ ​The present invention features methods and compositions for UBA5 inhibition.​​BACKGROUND OF THE INVENTION​​

[0003] ​ ​The​ ​ubiquitin-fold​ ​modifier​​1​​(UFM1)​​conjugation​​system,​​known​​as​​UFMylation,​​is​​an​​essential​ ​post-translational​ ​modification​ ​pathway​ ​that​ ​regulates​ ​a​ ​broad​ ​array​ ​of​ ​cellular​​processes,​ ​including​ ​protein​ ​folding,​ ​stress​ ​response,​ ​and​ ​DNA​ ​damage​ ​repair.​ ​UFMylation​ ​is​​catalyzed​ ​by​ ​three​ ​core​ ​enzymes:​ ​the​​E1-activating​​enzyme​​UBA5,​​the​​E2-conjugating​​enzyme​​UFC1,​ ​and​ ​the​ ​E3-ligase​ ​UFL1,​ ​which​ ​work​ ​sequentially​ ​to​ ​attach​ ​UFM1​ ​to​ ​target​ ​substrates.​​Disruption​ ​of​ ​this​ ​pathway​ ​has​ ​been​ ​linked​ ​to​ ​several​ ​diseases,​ ​including​ ​cancer,​​neurodegenerative​ ​disorders,​ ​and​ ​developmental​ ​diseases,​ ​emphasizing​ ​the​ ​critical​ ​need​ ​to​​understand​​and​​potentially​​target​​this​​system​​therapeutically.​​Among​​these​​enzymes,​​UBA5​​plays​​a​ ​pivotal​​role​​in​​initiating​​the​​UFMylation​​process​​by​​activating​​UFM1​​through​​ATP-dependent​​adenylation and subsequent transfer to UFC1.​​

[0004] ​ ​UBA5,​ ​as​ ​an​ ​E1-activating​ ​enzyme,​ ​occupies​ ​a​ ​unique​ ​position​ ​in​ ​the​ ​UFMylation​​cascade​ ​by​ ​catalyzing​ ​the​ ​first​ ​step​ ​of​ ​UFM1​ ​activation.​ ​This​ ​function​ ​is​ ​crucial​ ​because​ ​it​​represents​ ​the​ ​commitment​ ​step​ ​toward​ ​UFMylation,​ ​controlling​ ​the​ ​overall​ ​flux​ ​of​ ​this​​modification​ ​in​ ​cells.​ ​Due​ ​to​ ​its​ ​central​ ​role,​ ​any​ ​perturbation​ ​in​ ​UBA5​ ​function​ ​can​ ​have​​profound​ ​consequences​ ​on​ ​cellular​ ​physiology.​ ​For​ ​instance,​ ​UBA5​ ​alterations​ ​have​ ​been​​implicated​ ​in​ ​a​ ​range​ ​of​ ​human​ ​diseases,​ ​including​ ​Alzheimer's​ ​disease​ ​and​ ​various​ ​cancers,​​where​ ​abnormal​ ​UFMylation​ ​either​ ​contributes​ ​to​ ​the​ ​pathogenesis​ ​or​ ​progression​ ​of​ ​these​​conditions.​​Hence,​​UBA5​​has​​emerged​​as​​a​​potential​​therapeutic​​target​​in​​drug​​discovery​​efforts​ ​aimed at modulating the UFMylation pathway.​​

[0005] ​ ​In​ ​recent​ ​years,​ ​evidence​ ​has​ ​accumulated​ ​pointing​ ​to​ ​UBA5's​ ​involvement​ ​in​​neurodegenerative​ ​diseases,​ ​particularly​ ​Alzheimer's​ ​disease.​ ​Altered​ ​UFMylation​ ​dynamics,​​often​ ​due​ ​to​ ​UBA5​ ​mutations​ ​and​ ​dysregulation,​ ​have​ ​been​ ​linked​ ​to​ ​impaired​ ​protein​​homeostasis,​ ​which​ ​is​ ​a​ ​hallmark​ ​of​ ​Alzheimer's​ ​disease.​ ​For​ ​instance,​ ​studies​ ​have​ ​identified​​mutations​ ​in​ ​the​ ​UBA5​ ​gene​ ​in​ ​patients​ ​suffering​ ​from​ ​early-onset​ ​neurodegeneration,​ ​further​​supporting​ ​the​ ​idea​ ​that​ ​UFMylation​ ​contributes​ ​to​ ​the​ ​development​ ​of​ ​neurological​ ​disorders.​​Moreover,​ ​UBA5's​ ​role​ ​in​ ​cellular​ ​stress​ ​response​ ​pathways,​ ​including​ ​endoplasmic​ ​reticulum​​(ER)​ ​stress,​ ​highlights​ ​its​ ​importance​ ​in​ ​the​ ​maintenance​ ​of​ ​neuronal​ ​function​ ​under​ ​stress​​conditions, which is another critical factor in Alzheimer's pathology.​​

[0006] ​ ​UBA5​ ​has​​also​​been​​implicated​​in​​the​​pathogenesis​​of​​cancer,​​with​​studies​​showing​​that​​abnormal​ ​UFMylation​ ​can​ ​influence​ ​oncogenic​ ​signaling​ ​pathways.​ ​For​ ​example,​​dysregulated​​UFMylation​ ​has​ ​been​ ​linked​ ​to​ ​the​ ​development​ ​and​ ​progression​ ​of​​various​​cancers,​​including​​breast,​ ​colon,​ ​gastric,​ ​glioblastoma,​ ​liver,​ ​lung,​ ​oral,​ ​pancreatic,​ ​and​ ​renal​ ​cancers.​ ​UBA5​​overexpression​​has​​been​​observed​​in​​tumor​​samples​​from​​patients​​with​​these​​cancers,​​correlating​​with​ ​poor​ ​prognosis​​and​​increased​​metastatic​​potential.​​The​​oncogenic​​role​​of​​UBA5​​is​​thought​​to​ ​stem​ ​from​ ​its​ ​influence​ ​on​ ​key​ ​cellular​ ​processes,​ ​such​ ​as​ ​apoptosis,​ ​autophagy,​ ​and​ ​DNA​​repair,​ ​all​ ​of​ ​which​ ​are​ ​often​ ​altered​ ​in​​cancer​​cells.​​Thus,​​UBA5​​represents​​a​​promising​​target​​for the development of novel anti-cancer therapies.​​

[0007] ​ ​Despite​ ​the​ ​critical​ ​role​ ​of​ ​UBA5​ ​in​ ​human​ ​disease,​ ​only​ ​a​ ​few​ ​inhibitors​ ​have​ ​been​​reported,​ ​and​ ​no​ ​high-throughput​ ​screening​ ​(HTS)​ ​strategies​ ​have​ ​been​ ​described​ ​to​ ​identify​​selective​ ​UBA5​ ​modulators.​ ​The​ ​present​ ​invention​ ​addresses​ ​this​ ​unmet​ ​need​ ​by​ ​providing​ ​a​​robust​​HTS​​assay​​for​​discovering​​UBA5​​inhibitors​​using​​the​​commercially​​available​​AMP-Glo™​​kit.​ ​Using​ ​this​ ​approach,​ ​several​ ​novel​ ​UBA5​ ​inhibitors​ ​were​ ​identified​ ​and​ ​comprehensively​​characterized​ ​through​ ​biochemical,​ ​biophysical,​ ​and​ ​cellular​ ​assays,​ ​demonstrating​ ​both​ ​the​​effectiveness and versatility of the screening platform.​ ​BRIEF SUMMARY OF THE INVENTION​​

[0008] ​ ​It​​is​​an​​objective​​of​​the​​present​​invention​​to​​provide​​compositions​​and​​methods​​that​​allow​​for​​the​​inhibition​​of​​UBA5,​​as​​specified​​in​​the​​independent​​claims.​​Embodiments​​of​​the​​invention​ ​are​​given​​in​​the​​dependent​​claims.​​Embodiments​​of​​the​​present​​invention​​can​​be​​freely​​combined​ ​with each other if they are not mutually exclusive.​​

[0009] ​ ​In​ ​some​ ​embodiments,​ ​the​ ​present​ ​invention​ ​identifies​ ​eighteen​ ​novel​ ​UBA5​ ​inhibitors​​spanning​ ​several​ ​distinct​ ​chemical​ ​scaffolds,​ ​each​ ​exhibiting​ ​low​ ​micromolar​ ​IC₅₀​ ​values.​ ​The​​diversity​​of​​scaffolds​​reflects​​different​​chemical​​series,​​which​​may​​confer​​distinct​​biological​​and​​chemical​ ​properties,​ ​such​ ​as​ ​solubility​ ​and​ ​pharmacokinetic​ ​behavior.​ ​These​ ​inhibitors​​demonstrated​ ​selectivity​ ​for​ ​UBA5​ ​over​ ​other​ ​E1​ ​enzymes,​ ​including​ ​UBA1,​ ​and​ ​effectively​​inhibited​ ​endogenous​ ​UFMylation​ ​in​ ​HEK293T​ ​cells.​ ​Beyond​ ​serving​ ​as​ ​valuable​ ​tools​ ​for​​studying​ ​the​ ​UFMylation​​pathway,​​the​​identified​​inhibitors​​also​​represent​​promising​​therapeutic​​candidates​​for​​diseases​​associated​​with​​dysregulated​​UFMylation,​​including​​Alzheimer’s​​disease​ ​and cancer.​​

[0010] ​ ​In​​some​​embodiments,​​the​​present​​invention​​features​​compounds​​according​​to​​one​​of​​the​​following compounds, or a derivative thereof:​​

[0011] ​ ​One​​of​​the​​unique​​and​​inventive​​technical​​features​​of​​the​​present​​invention​​is​​compounds​​selective​ ​for​ ​UBA5.​ ​Without​ ​wishing​ ​to​ ​limit​ ​the​ ​invention​ ​to​ ​any​ ​theory​ ​or​ ​mechanism,​ ​it​ ​is​​believed​ ​that​ ​the​ ​technical​ ​feature​ ​of​ ​the​ ​present​​invention​​advantageously​​provides​​for​​precise​​inhibition​ ​of​ ​UBA5.​ ​Such​ ​precision​ ​is​ ​important,​ ​as​ ​excessive​ ​inhibition​ ​could​ ​lead​​to​​adverse​​effects​​given​​UBA5’s​​critical​​role​​in​​cellular​​stress​​responses.​​None​​of​​the​​presently​​known​​prior​ ​references or works have the unique, inventive technical feature of the present invention.​​

[0012] ​ ​Moreover,​​the​​prior​​references​​teach​​away​​from​​the​​present​​invention.​​Existing​​inhibitors​​generally​ ​lack​ ​selectivity​ ​for​ ​UBA5​ ​or​ ​only​ ​have​ ​weak​ ​inhibition,​ ​or​ ​were​ ​not​ ​confirmed​ ​as​​UBA5​ ​inhibitors.​ ​For​ ​example,​ ​adenosine​ ​5′-sulfamate​ ​(ADS)​ ​is​ ​an​ ​ATP-competitive​​analogue​​with​ ​an​ ​IC₅₀​​of​​13​​µM,​​but​​it​​functions​​as​​a​​pan-E1​​inhibitor,​​limiting​​its​​utility.​​Compound​​8.5​​was​ ​developed​ ​as​​a​​selective​​UBA5​​inhibitor​​with​​an​​IC₅₀​​of​​4​​µM;​​however,​​its​​Zn²⁺-chelating​​properties​ ​may​ ​reduce​ ​therapeutic​ ​potential,​ ​as​ ​numerous​ ​host​ ​enzymes​​rely​​on​​zinc​​for​​proper​​function.​​DKM​​2-93​​(DKM)​​is​​a​​covalent​​UBA5​​inhibitor,​​yet​​it​​exhibits​​a​​relatively​​high​​IC₅₀​​of​ ​430​​µM.​​The​​natural​​product​​Usenamine​​A​​has​​been​​reported​​to​​influence​​UBA5​​expression,​​but​​direct​ ​interaction​ ​with​ ​UBA5​ ​or​​inhibition​​of​​its​​enzymatic​​activity​​has​​not​​been​​confirmed.​​To​​date, no high-throughput screening (HTS) approaches for UBA5 inhibitors have been reported.​​

[0013] ​ ​Any​​feature​​or​​combination​​of​​features​​described​​herein​​are​​included​​within​​the​​scope​​of​​the​ ​present​ ​invention​ ​provided​ ​that​ ​the​ ​features​ ​included​ ​in​ ​any​ ​such​ ​combination​ ​are​ ​not​​mutually​​inconsistent​​as​​will​​be​​apparent​​from​​the​​context,​​this​​specification,​​and​​the​​knowledge​ ​of​​one​​of​​ordinary​​skill​​in​​the​​art.​​Additional​​advantages​​and​​aspects​​of​​the​​present​​invention​​are​ ​apparent in the following detailed description and claims.​ ​BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)​​

[0014] ​ ​The​ ​features​ ​and​ ​advantages​ ​of​ ​the​ ​present​ ​invention​ ​will​ ​become​ ​apparent​ ​from​ ​a​​consideration​ ​of​ ​the​ ​following​ ​detailed​ ​description​ ​presented​ ​in​ ​connection​ ​with​ ​the​​accompanying drawings in which:​​

[0015] ​ ​FIG.​ ​1A,​ ​1B,​ ​1C,​ ​1D,​ ​1E,​ ​and​ ​1F​ ​shows​ ​development​ ​and​ ​optimization​ ​of​​AMP-Glo​T​M​​-based​ ​UBA5​ ​UFMylation​ ​assay.​ ​FIG.​ ​1A​ ​shows​ ​the​ ​assay​ ​principle.​ ​FIG.​ ​2B–2E​​shows​ ​kinetic​ ​analyses​ ​of​ ​the​ ​AMP-Glo​T​M​​-based​ ​UBA5​ ​coupling​ ​assay​ ​to​ ​optimize​ ​assay​​parameters​ ​and​ ​assay​ ​components​ ​including​ ​AMP​ ​(FIG.​ ​1B),​ ​UBA5​ ​enzyme​ ​(FIG.​ ​1C),​ ​ATP​​(FIG.​ ​1D)​ ​and​ ​UFM1​ ​substrate​​(FIG.​​1E),​​using​​a​​384-well​​plate.​​Each​​experiment​​varied​​only​​the​​indicated​​component​​in​​a​​concentration​​series,​​while​​all​​other​​components​​were​​kept​​at​​fixed​​concentrations.​ ​RLU,​ ​relative​ ​fluorescence​ ​unit.​ ​FIG.​ ​1F​ ​shows​ ​HTS​ ​parameters​ ​for​ ​the​​optimized​ ​AMP-Glo​T​M​ ​UBA5​ ​assay​ ​in​​a​​384-well​​plate,​​by​​comparing​​the​​RFU​​generated​​with​​and​ ​without​ ​UBA5.​ ​N=16.​ ​****,​ ​p<0.0001.For​ ​all​ ​AMP-Glo​ ​assays​ ​here​ ​and​ ​below​ ​(unless​​specified): UBA5, 0.8 µM; UFM1, 5 µM; ATP, 5 µM.​​

[0016] ​ ​FIG.​ ​2A,​ ​2B,​ ​2C,​ ​and​ ​2D​ ​shows​ ​HTS​​AMP-Glo​T​M​ ​UBA5​​assay.​​FIG.​​2A​​shows​​a​​dose​​response​ ​curve​ ​of​ ​inhibition​ ​of​ ​the​ ​UBA5​ ​activity​ ​by​ ​concentration​​series​​of​​DKM​​2-93.​​N=3.​​AMP​​Glo™​​Assay​​Kit​​(Promega)​​was​​used​​to​​detect​​AMP​​in​​samples.​​In​​brief,​​UBA5​​(800​​nM)​ ​was​​incubated​​for​​1​​hr​​with​​different​​concentrations​​of​​DKM2-93.​​UFM1​​and​​ATP​​(5​​µM​​each)​ ​were​​added​​and​​incubated​​for​​30​​minutes.​​AMP​​Glo™​​Reagent​​I​​(5​​µL)​​was​​added​​per​​well​​and​​incubated​ ​for​​1​​hr​​at​​RT.​​AMP​​Glo™​​Reagent​​II​​(10​​µL)​​was​​added​​per​​well​​and​​incubated​​for​​30​ ​minutes​ ​at​ ​RT​ ​to​ ​convert​ ​ADP​ ​to​ ​ATP​ ​and​ ​enable​ ​the​ ​enzyme​ ​reaction.​ ​Finally,​ ​the​​luminescence​ ​(Gain​ ​150)​ ​was​ ​measured​ ​using​ ​a​ ​BioTek​ ​H1​ ​Reader.​ ​Luminescence​ ​data​ ​was​​analyzed​​relative​​to​​a​​standard​​curve​​to​​determine​​the​​AMP​​concentration​​in​​the​​samples.​​IC​​50​​of​​DKM2-93​ ​was​ ​calculated​ ​by​ ​using​ ​GraphPad​ ​Prism​ ​9.​ ​Data​ ​were​ ​normalized​ ​using​​DMSO-treated​ ​wells​ ​with​ ​UBA5​ ​set​ ​as​ ​100%,​ ​and​ ​wells​ ​without​ ​UBA5​ ​set​ ​as​ ​0%.​ ​The​​experiment​ ​was​ ​done​ ​in​ ​triplicate.​ ​FIG.​ ​2B​ ​shows​ ​DKM​ ​(3​ ​mM)​ ​inhibited​ ​UBA5​​activity​​in​​a​​384-well​ ​plate.​ ​N=96.​ ​****,​ ​P<0.0001.​ ​FIG.​ ​2C​ ​shows​ ​well-to-well​ ​relative​ ​luminescence​ ​in​​384-well​​format​​with​​and​​without​​UBA5;​​N=96.​​FIG.​​2D​​shows​​Z’​​factor​​over​​plates​​and​​dates.​​Each​ ​spot​ ​represents​ ​Z’​ ​score​ ​calculated​ ​from​ ​the​ ​results​ ​of​ ​384-well​ ​plates​ ​on​ ​4​ ​different​​plates / day on 3 dates.​​

[0017] ​ ​FIG.​​3A,​​3B,​​3C,​​and​​3D​​show​​a​​secondary​​FP-based​​assay.​​FIG.​​4A​​shows​​the​​principle​​of​​FP-based​​secondary​​assay.​​FIG.​​4B​​shows​​AMP​​titration​​using​​the​​FP​​assay.​​Assay​​condition:​​Transcreener®​ ​AMP / GMP​ ​FP​ ​Assay​ ​Kit​ ​(Bellbrook)​​was​​used​​for​​this​​assay.​​AMP​​at​​different​​concentrations​ ​was​ ​diluted​ ​serially​ ​in​ ​the​ ​buffer.​ ​Transcreener®​ ​AMP / GMP​ ​FP​ ​Detection​​Reagent​ ​was​ ​added​ ​to​ ​each​ ​well,​ ​which​ ​contains​ ​the​ ​necessary​ ​fluorescent​ ​probe.​​Incubate​​the​​plate​​for​​a​​1.30​​hr​​as​​indicated​​in​​the​​protocol.​​Fluorescence​​polarization​​was​​detected​​by​​using​​a​​fluorescence​ ​plate​ ​reader.​ ​The​ ​degree​ ​of​ ​fluorescence​ ​polarization​ ​was​ ​correlated​ ​with​ ​the​​amount​ ​of​ ​AMP​ ​present​​in​​the​​samples.​​FIG.​​4C​​shows​​DKM​​titration​​against​​UBA5​​using​​the​​FP​​assay.​​N=3.​​Transcreener®​​AMP / GMP​​FP​​Assay​​Kit​​from​​Bellbrook​​was​​used​​for​​this​​assay.​ ​UBA5​​(1​​µM)​​was​​incubated​​for​​1hr​​with​​DKM​​at​​different​​concentrations.​​UFM1​​(2.5​​µM)​​and​​ATP​ ​(20​ ​µM​ ​each)​ ​were​ ​added.​ ​Incubate​ ​the​ ​plate​ ​for​ ​30​ ​minutes​ ​at​ ​RT.​ ​After​ ​incubation,​​Transcreener®​ ​AMP / GMP​​FP​​Detection​​Reagent​​was​​added.​​Incubate​​the​​plate​​for​​a​​1.30​​hr​​as​​indicated​ ​in​ ​the​ ​protocol.​ ​Fluorescence​ ​polarization​ ​was​ ​detected​ ​by​ ​using​​a​​fluorescence​​plate​​reader.​ ​IC50​ ​of​ ​DKM​ ​was​ ​calculated​ ​by​ ​using​ ​GraphPad​ ​Prism​ ​9.​ ​For​ ​all​ ​FP​ ​assays​ ​here​ ​and​​below​ ​(unless​ ​specified):​ ​UBA5,​ ​1.0​ ​µM;​ ​UFM1,​ ​2.5​ ​µM;​ ​ATP,​ ​20​ ​µM.​ ​FIG.​ ​4D​ ​shows​ ​that​​DKM​​(4​​mM)​​inhibited​​UBA5​​activity​​using​​the​​FP​​assay​​on​​a​​384-well​​plate.​​Assay​​condition:​​UBA5,​ ​1​ ​µM;​ ​UFM1,​ ​5​ ​µM;​ ​ATP,​ ​30​ ​µM.​ ​N=16.​ ​***,​ ​P<0.001.​ ​FIG.​ ​4E​ ​shows​ ​fitting​ ​of​​dose-response​ ​curve​ ​of​​inhibition​​of​​UBA5​​by​​compounds​​using​​the​​FP-based​​secondary​​assay.​​Transcreener®​​AMP / GMP​​FP​​Assay​​Kit​​from​​Bellbrook​​was​​used​​for​​this​​assay.​​UBA5​​(1​​µM)​​was​ ​incubated​ ​for​ ​1​ ​hr​ ​with​ ​different​​concentrations​​of​​compounds​​from​​0-100µM.​​UFM1​​(2.5​​µM)​ ​and​ ​ATP​ ​(20​ ​µM​ ​each)​ ​were​ ​added.​ ​Incubate​ ​the​ ​plate​ ​for​ ​30​ ​minutes​ ​at​ ​RT.​ ​After​​incubation,​​Kits’s​​reagent​​was​​added​​to​​each​​well.​​Incubate​​the​​plate​​for​​a​​1.30​​hr​​as​​indicated​​in​ ​the​​protocol.​​Fluorescence​​polarization​​was​​detected​​by​​using​​a​​fluorescence​​plate​​reader.​​IC​​50​​of​​compounds​ ​was​ ​calculated​ ​by​ ​using​ ​GraphPad​ ​Prism​ ​9.Data​ ​were​ ​normalized​ ​using​​DMSO-treated wells with UBA5 set as 100%, and wells without UBA5 set as 0%.​​

[0018] ​ ​FIG.4 shows the UBA5 inhibitors identified.​​

[0019] ​ ​FIG.​ ​5A​ ​shows​ ​Z’​ ​factor,​ ​S / B​ ​ratio,​ ​and​ ​CV​ ​over​ ​plates​ ​of​ ​a​ ​HTS​ ​against​ ​a​ ​CNS​​compound​ ​library​ ​in​ ​sixteen​ ​384-well​ ​plates.​ ​FIG.​ ​5B​ ​shows​ ​the​ ​dose-response​ ​inhibition​ ​of​​UBA5​ ​activity​ ​by​ ​18​ ​hit​ ​compounds.​ ​Data​ ​were​ ​normalized​ ​using​ ​DMSO-treated​ ​wells​ ​with​​UBA5 set as 100%, and wells without UBA5 set as 0%. N=3.​​

[0020] ​ ​FIG.​​6A​​and​​6B​​shows​​UBA5-free​​AMP-Glo​T​M​ ​assay.​​FIG.​​5A​​shows​​AMP-Glo™​​assay​​in​ ​the​ ​absence​ ​of​ ​UBA5.​ ​AMP​ ​(5​ ​µM)​ ​was​ ​used​ ​as​ ​a​ ​direct​ ​substrate.​ ​UFM1,​​5​​µM.​​FIG.​​6B​​shows​ ​the​ ​fitting​ ​of​ ​the​ ​dose-response​ ​curve​ ​of​ ​inhibition​ ​of​ ​the​ ​coupling​ ​enzymes​ ​in​ ​the​​AMP-Glo​ ​kit​ ​by​ ​compounds,​ ​using​ ​the​ ​coupling​ ​enzyme​ ​tertiary​ ​assay.​ ​Data​ ​were​ ​normalized​​using DMSO-treated wells with AMP set as 100%, and wells without AMP set as 0%. N=3.​​

[0021] ​ ​FIG.​ ​7A,​ ​7B,​ ​and​ ​7C​ ​show​ ​quaternary​ ​E1​ ​specificity​ ​assay.​ ​FIG.​ ​6A​ ​shows​ ​UBA1​​AMP-Glo​T​M​​specificity​​assay​​with​​and​​without​​UBA1.​​For​​all​​UBA1​​AMP-Glo​​assays​​here​​and​​below​ ​(unless​ ​specified):​ ​UBA1,​ ​0.3​ ​µM;​ ​Ub,​ ​10​ ​µM;​ ​ATP,​ ​5​ ​µM.​ ​****,​ ​p<0.0001.​ ​FIG.​ ​6B​​shows​ ​kinetic​ ​analysis​ ​of​ ​ATP​ ​and​ ​UBA1​ ​titration​ ​for​ ​the​ ​UBA1​ ​AMP-Glo​ ​specificity​ ​assay​​using​ ​a​ ​384-well​ ​plate.​ ​FIG.​​7C​​shows​​titration​​of​​identified​​Comps​​#1-18​​against​​UBA1.​​Data​​were​ ​normalized​ ​using​ ​DMSO-treated​​wells​​with​​UBA1​​set​​as​​100%,​​and​​wells​​without​​UBA1​​set as 0%. N=3.​​

[0022] ​ ​FIG.​ ​8​ ​shows​ ​the​​Inhibition​​of​​UFMylation​​in​​vitro​​.​​Inhibition​​of​​UFMylation​​of​​UBA5​​was​ ​assessed​ ​using​ ​an​ ​in​ ​vitro​ ​in-gel-based​ ​UFMylation​ ​assay​ ​(left​ ​gels),​ ​with​ ​accompanying​​band​​quantification​​(right​​bar​​graphs).​​UBA5,​​12​​µM;​​UFM1,​​25​​µM;​​ATP,​​2​​mM.​​Samples​​were​​resolved​ ​on​ ​12%​ ​SDS-PAGE​ ​gels​ ​and​ ​stained​ ​with​ ​Coomassie​ ​Blue.​ ​Band​ ​intensities​ ​were​​quantified​ ​using​ ​ImageJ.​ ​Data​ ​were​ ​normalized​​by​​calculating​​the​​ratio​​of​​the​​band​​intensity​​of​​the​ ​UFM1-UBA5​ ​complex​ ​to​ ​the​ ​combined​ ​intensity​ ​of​ ​UFM1​ ​and​ ​UBA5​ ​alone,​ ​with​ ​the​​average​​of​​the​​DMSO-treated​​wells​​at​​the​​2-hour​​time​​point​​set​​as​​100%​​and​​those​​at​​the​​0-hour​ ​time point set as 0%.​​

[0023] ​ ​FIG.​ ​9​ ​shows​ ​cell​ ​viability​ ​against​ ​astrocyte-like​ ​CCF-STTG1​ ​cells.​ ​CCF-STTG1​​(ATCC)​ ​cells​ ​were​​incubated​​with​​various​​concentrations​​of​​compounds,​​and​​then​​viability​​was​​assessed​ ​at​ ​48​ ​hours​ ​of​ ​incubation​ ​using​ ​the​ ​WST​ ​assay.​ ​Data​ ​were​ ​normalized​ ​by​ ​setting​ ​the​​absorption​ ​readings​ ​of​ ​cells​ ​treated​ ​with​ ​DMSO​ ​to​ ​100%​​and​​the​​readings​​from​​cell-free​​wells​​treated with DMSO to 0%. N = 3.​ ​DETAILED DESCRIPTION OF THE INVENTION​​

[0024] ​ ​Disclosed​​are​​various​​peptides,​​solvents,​​solutions,​​carriers,​​and / or​​components​​to​​be​​used​​to​ ​prepare​ ​compositions​ ​to​ ​be​ ​used​ ​within​​the​​methods​​disclosed​​herein.​​Also​​disclosed​​are​​the​​various​​steps,​​elements,​​amounts,​​routes​​of​​administration,​​symptoms,​​and / or​​treatments​​that​​are​​used​ ​or​ ​observed​ ​when​ ​performing​ ​the​ ​disclosed​ ​methods,​ ​as​ ​well​ ​as​ ​the​ ​methods​ ​themselves.​​These​ ​and​ ​other​ ​materials,​ ​steps,​​and / or​​elements​​are​​disclosed​​herein,​​and​​it​​is​​understood​​that​​when​​combinations,​​subsets,​​interactions,​​groups,​​etc.​​of​​these​​materials​​are​​disclosed,​​that​​while​ ​specific​​reference​​of​​each​​various​​individual​​and​​collective​​combination​​and​​permutation​​of​​these​​compounds​ ​may​ ​not​ ​be​ ​explicitly​ ​disclosed,​ ​each​ ​is​ ​specifically​ ​contemplated​ ​and​ ​described​​herein.​ ​It​ ​is​ ​also​ ​to​ ​be​ ​understood​ ​that​ ​the​ ​terminology​ ​used​ ​herein​ ​is​ ​for​ ​the​ ​purpose​ ​of​​describing particular embodiments only and is not intended to be limiting.​​

[0025] ​ ​Unless​ ​otherwise​ ​explained,​​all​​technical​​and​​scientific​​terms​​used​​herein​​have​​the​​same​​meaning​ ​as​ ​commonly​ ​understood​ ​by​ ​one​ ​of​ ​ordinary​ ​skills​ ​in​ ​the​ ​art​ ​to​ ​which​ ​a​ ​disclosed​​invention​​belongs.​​The​​singular​​terms​​"a,"​​"an,"​​and​​"the"​​include​​plural​​referents​​unless​​context​​clearly​ ​indicates​ ​otherwise.​ ​Similarly,​ ​the​ ​word​ ​"or"​ ​is​ ​intended​ ​to​ ​include​ ​"and"​ ​unless​ ​the​​context​​clearly​​indicates​​otherwise.​​The​​term​​"comprising"​​means​​that​​other​​elements​​can​​also​​be​ ​present​​in​​addition​​to​​the​​defined​​elements​​presented.​​The​​use​​of​​"comprising"​​indicates​​inclusion​​rather​ ​than​ ​limitation.​ ​Stated​ ​another​ ​way,​ ​the​ ​term​ ​"comprising"​​means​​"including​​principally,​​but​ ​not​ ​necessary​ ​solely".​​Furthermore,​​variation​​of​​the​​word​​"comprising",​​such​​as​​"comprise"​​and​ ​"comprises",​ ​have​ ​correspondingly​ ​the​ ​same​ ​meanings.​ ​In​ ​one​ ​respect,​ ​the​ ​technology​​described​ ​herein​ ​related​ ​to​ ​the​ ​herein​ ​described​ ​compositions,​ ​methods,​ ​and​ ​respective​​component(s)​ ​thereof,​ ​as​ ​essential​ ​to​ ​the​ ​invention,​ ​yet​ ​open​ ​to​ ​the​ ​inclusion​ ​of​ ​unspecified​​elements, essential or not ("comprising").​​

[0026] ​ ​Suitable​ ​methods​ ​and​ ​materials​ ​for​ ​the​ ​practice​ ​and / or​ ​testing​ ​of​ ​embodiments​ ​of​ ​the​​disclosure​ ​are​ ​described​ ​below.​ ​Such​ ​methods​ ​and​ ​materials​ ​are​ ​illustrative​ ​only​ ​and​ ​are​ ​not​​intended​ ​to​ ​be​ ​limiting.​ ​Other​ ​methods​ ​and​ ​materials​ ​similar​ ​or​ ​equivalent​ ​to​ ​those​ ​described​​herein​ ​can​ ​be​ ​used.​ ​For​ ​example,​ ​conventional​ ​methods​ ​well​ ​known​ ​in​ ​the​ ​art​ ​to​ ​which​ ​the​​disclosure​ ​pertains​ ​are​ ​described​ ​in​​various​​general​​and​​more​​specific​​references,​​including,​​for​​example,​​Sambrook​​et​​al.,​​Molecular​​Cloning:​​A​​Laboratory​​Manual,​​2d​​ed.,​​Cold​​Spring​​Harbor​ ​Laboratory​​Press,​​1989;​​Sambrook​​et​​al.,​​Molecular​​Cloning:​​A​​Laboratory​​Manual,​​3d​​ed.,​​Cold​​Spring​ ​Harbor​ ​Press,​ ​2001;​ ​Ausubel​ ​et​ ​al.,​ ​Current​ ​Protocols​ ​in​ ​Molecular​ ​Biology,​ ​Greene​​Publishing​ ​Associates,​ ​1992​ ​(and​ ​Supplements​ ​to​ ​2000);​ ​Ausubel​ ​et​ ​al.,​ ​Short​ ​Protocols​ ​in​​Molecular​ ​Biology:​ ​A​ ​Compendium​ ​of​ ​Methods​ ​from​ ​Current​​Protocols​​in​​Molecular​​Biology,​​4th​​ed.,​​Wiley​​&​​Sons,​​1999;​​Harlow​​and​​Lane,​​Antibodies:​​A​​Laboratory​​Manual,​​Cold​​Spring​ ​Harbor​​Laboratory​​Press,​​1990;​​and​​Harlow​​and​​Lane,​​Using​​Antibodies:​​A​​Laboratory​​Manual,​​Cold​ ​Spring​ ​Harbor​ ​Laboratory​ ​Press,​ ​1999,​ ​Gene​ ​Expression​ ​Technology​ ​(Methods​ ​in​​Enzymology,​​Vol.​​185,​​edited​​by​​D.​​Goeddel,​​1991.​​Academic​​Press,​​San​​Diego,​​Calif.),​​"Guide​​to​ ​Protein​ ​Purification”​ ​in​ ​Methods​ ​in​ ​Enzymology​ ​(M.​ ​P.​ ​Deutshcer,​ ​ed.,​ ​(1990)​ ​Academic​​Press,​​Inc.);​​PCR​​Protocols:​​A​​Guide​​to​​Methods​​and​​Applications​​(Innis,​​et​​al.​​1990.​​Academic​ ​Press,​​San​​Diego,​​Calif.),​​Culture​​of​​Animal​​Cells:​​A​​Manual​​of​​Basic​​Technique,​​2nd​​Ed.​​(R.​​I.​​Freshney.​ ​1987.​ ​Liss,​ ​Inc.​ ​New​ ​York,​ ​N.Y.),​ ​Gene​ ​Transfer​ ​and​ ​Expression​ ​Protocols,​ ​pp.​​109-128,​​ed.​​E.​​J.​​Murray,​​The​​Humana​​Press​​Inc.,​​Clifton,​​N.J.),​​and​​the​​Ambion​​1998​​Catalog​​(Ambion,​ ​Austin,​ ​Tex.),​ ​the​ ​disclosures​ ​of​ ​which​ ​are​ ​incorporated​ ​in​ ​their​ ​entirety​ ​herein​ ​by​​reference.​​

[0027] ​ ​All​ ​publications,​ ​patent​ ​applications,​ ​patents,​ ​and​ ​other​​references​​mentioned​​herein​​are​​incorporated​ ​by​ ​reference​ ​in​ ​their​ ​entirety​ ​for​ ​all​ ​purposes.​ ​In​ ​case​ ​of​ ​conflict,​ ​the​ ​present​​specification, including explanations of terms, will control.​​

[0028] ​ ​Although​ ​methods​ ​and​ ​materials​ ​similar​ ​or​ ​equivalent​ ​to​ ​those​ ​described​ ​herein​ ​can​ ​be​​used​ ​to​ ​practice​ ​or​ ​test​ ​the​ ​disclosed​ ​technology,​ ​suitable​ ​methods​ ​and​ ​materials​ ​are​ ​described​​below. The materials, methods, and examples are illustrative only and not intended to be limiting.​​

[0029] ​ ​A​​“subject”​​is​​an​​individual​​and​​includes,​​but​​is​​not​​limited​​to,​​a​​mammal​​(e.g.,​​a​​human,​​horse,​​pig,​​rabbit,​​dog,​​sheep,​​goat,​​non-human​​primate,​​cow,​​cat,​​guinea​​pig,​​or​​rodent),​​a​​fish,​​a​ ​bird,​​a​​reptile​​or​​an​​amphibian.​​The​​term​​does​​not​​denote​​a​​particular​​age​​or​​sex.​​Thus,​​adult​​and​​newborn​ ​subjects,​ ​as​ ​well​ ​as​ ​fetuses,​ ​whether​ ​male​ ​or​ ​female,​ ​are​ ​intended​ ​to​ ​be​ ​included.​ ​A​​“patient”​​is​​a​​subject​​afflicted​​with​​a​​disease​​or​​disorder.​​The​​term​​“patient”​​includes​​human​​and​ ​veterinary subjects.​​

[0030] ​ ​As​ ​used​ ​herein,​ ​the​ ​terms​ ​"treat,"​ ​“treating,”​ ​or​ ​"treatment"​ ​refer​ ​to​ ​both​ ​therapeutic​​treatment​​and​​prophylactic​​or​​preventative​​measures,​​with​​the​​objective​​of​​preventing,​​reducing,​​slowing​ ​down​ ​(lessen),​ ​inhibiting,​ ​or​ ​eliminating​ ​an​​undesired​​physiological​​change,​​symptom,​​disease,​ ​or​ ​disorder.​ ​For​ ​example,​ ​the​ ​disease​ ​may​ ​be​ ​Alzheimer​ ​disease.​ ​For​ ​purposes​ ​of​ ​this​​invention,​ ​beneficial​ ​or​ ​desired​ ​clinical​ ​results​ ​include,​ ​but​ ​are​ ​not​ ​limited​ ​to,​ ​alleviation​ ​of​​symptoms,​ ​diminishment​ ​of​ ​extent​ ​of​ ​disease,​ ​stabilized​ ​(i.e.,​ ​not​ ​worsening)​ ​state​ ​of​ ​disease,​​delay​ ​or​ ​slowing​ ​of​ ​disease​ ​progression,​ ​amelioration​ ​or​ ​palliation​ ​of​ ​the​ ​disease​ ​state,​ ​and​​remission​ ​(whether​ ​partial​ ​or​ ​total),​ ​whether​ ​detectable​ ​or​ ​undetectable.​ ​"Treatment"​ ​can​ ​also​​mean​ ​prolonging​​survival​​as​​compared​​to​​expected​​survival​​if​​not​​receiving​​treatment.​​Those​​in​​need​ ​of​ ​treatment​​include​​those​​already​​with​​the​​condition​​or​​disorder​​as​​well​​as​​those​​prone​​to​​have​ ​the​ ​condition​ ​or​ ​disorder​ ​or​​those​​in​​which​​the​​condition​​or​​disorder​​is​​to​​be​​prevented​​or​​onset​ ​delayed.​ ​Optionally,​ ​the​ ​subject​ ​or​ ​patient​ ​may​ ​be​ ​identified​ ​(e.g.,​ ​diagnosed)​ ​as​ ​one​​suffering​ ​from​ ​the​ ​disease​ ​or​ ​condition​ ​prior​ ​to​ ​administration​ ​of​ ​the​ ​compositions​ ​of​ ​the​​invention.​​Subjects​​at​​risk​​for​​the​​disease​​can​​be​​identified​​by,​​for​​example,​​any​​or​​a​​combination​ ​of appropriate diagnostic or prognostic assays known in the art.​​

[0031] ​ ​As​ ​used​ ​herein,​ ​“clinical​ ​improvement”​ ​may​ ​refer​ ​to​ ​a​ ​noticeable​ ​reduction​ ​in​ ​the​​symptoms of a disorder, or cessation thereof.​​

[0032] ​ ​The​​terms​​“manage,”​​“managing,”​​and​​“management”​​refer​​to​​preventing​​or​​slowing​​the​​progression,​​spread​​or​​worsening​​of​​a​​disease​​or​​disorder,​​or​​of​​one​​or​​more​​symptoms​​thereof.​​In​ ​certain​​cases,​​the​​beneficial​​effects​​that​​a​​subject​​derives​​from​​a​​prophylactic​​or​​therapeutic​​agent​ ​do not result in a cure of the disease or disorder.​​

[0033] ​ ​The​ ​terms​ ​“administering”​ ​and​ ​“administration”​ ​refer​ ​to​ ​methods​ ​of​ ​providing​ ​a​​pharmaceutical​ ​preparation,​ ​composition,​ ​or​ ​formulation​ ​to​ ​a​ ​subject.​ ​The​ ​compositions​​described​ ​herein​ ​can​ ​be​ ​administered​ ​in​ ​a​ ​number​ ​of​ ​ways​ ​depending​ ​on​ ​whether​ ​local​ ​or​​systemic​ ​treatment​ ​is​ ​desired,​ ​and​ ​on​ ​the​ ​area​ ​to​ ​be​ ​treated.​ ​Such​ ​methods​ ​are​ ​well​ ​known​ ​to​​those​​skilled​​in​​the​​art​​and​​include,​​but​​are​​not​​limited​​to,​​administering​​the​​compositions​​orally,​ ​intranasally,​​parenterally​​(e.g.,​​intravenously​​and​​subcutaneously),​​by​​intramuscular​​injection,​​by​ ​intraperitoneal injection, intrathecally, transdermally, extracorporeally, topically or the like.​​

[0034] ​ ​A​​“therapeutically​​effective​​amount”​​refers​​to​​an​​amount​​that​​is​​sufficient​​to​​achieve​​the​​desired​ ​therapeutic​ ​result​ ​or​ ​to​ ​have​ ​an​ ​effect​ ​on​ ​undesired​ ​symptoms,​ ​but​ ​is​ ​generally​​insufficient​​to​​cause​​adverse​​side​​effects.​​The​​specific​​therapeutically​​effective​​dose​​level​​for​​any​​particular​ ​patient​ ​will​ ​depend​ ​upon​​a​​variety​​of​​factors​​including​​the​​disorder​​being​​treated​​and​​the​ ​severity​ ​of​ ​the​ ​disorder;​ ​the​ ​specific​ ​composition​ ​employed;​ ​the​ ​age,​ ​body​ ​weight,​​general​​health,​​sex​​and​​diet​​of​​the​​patient;​​the​​time​​of​​administration;​​the​​route​​of​​administration;​​the​​rate​​of​ ​excretion​ ​of​ ​the​ ​specific​ ​compound​ ​employed;​ ​the​ ​duration​ ​of​ ​the​ ​treatment;​ ​drugs​ ​used​ ​in​​combination​ ​or​ ​coincidental​ ​with​ ​the​​specific​​compound​​employed​​and​​like​​factors​​well​​known​​in​​the​​medical​​arts.​​For​​example,​​it​​is​​well​​within​​the​​skill​​of​​the​​art​​to​​start​​doses​​of​​a​​compound​​at​ ​levels​ ​lower​ ​than​ ​those​ ​required​ ​to​ ​achieve​ ​the​ ​desired​ ​therapeutic​ ​effect​ ​and​ ​to​ ​gradually​​increase​​the​​dosage​​until​​the​​desired​​effect​​is​​achieved.​​If​​desired,​​the​​effective​​daily​​dose​​can​​be​​divided​ ​into​ ​multiple​ ​doses​ ​for​ ​purposes​ ​of​ ​administration.​ ​Consequently,​ ​single​ ​dose​​compositions​ ​can​ ​contain​ ​such​ ​amounts​​or​​submultiples​​thereof​​to​​make​​up​​the​​daily​​dose.​​The​​dosage​​can​​be​​adjusted​​by​​the​​individual​​physician​​in​​the​​event​​of​​any​​contraindications.​​Dosage​​can​ ​vary,​ ​and​ ​can​​be​​administered​​in​​one​​or​​more​​dose​​administrations​​daily,​​for​​one​​or​​several​​days,​​weekly,​​twice​​weekly,​​etc.​​Guidance​​can​​be​​found​​in​​the​​literature​​for​​appropriate​​dosages​ ​for given classes of pharmaceutical products.​​

[0035] ​ ​Referring​​now​​to​​FIGs.​​1A-9,​​the​​present​​invention​​features​​compounds​​according​​to​​one​​of the following compounds, or a derivative thereof:​​as​​well​​as​​methods​​of​​treating​​a​​health​​condition,​​e.g.,​​Alzheimer’s​​disease​​or​​cancer,​​using​​said​ ​compounds or derivatives thereof.​​

[0036] ​ ​In​ ​some​ ​embodiments,​ ​the​ ​compound​ ​is​ ​specific​ ​for​ ​UBA5.​ ​In​ ​some​ ​embodiment,​ ​the​​compound inhibits UBA5 with an IC₅₀ of less than 15 µM.​​

[0037] ​ ​In​ ​some​ ​embodiments,​ ​the​ ​present​ ​invention​ ​features​​a​​method​​of​​inhibiting​​UBA5​​in​​a​​cell,​ ​comprising​ ​contacting​ ​the​ ​cell​ ​with​ ​a​ ​composition​ ​comprising​ ​a​ ​compound​ ​as​ ​described​​herein​ ​or​ ​a​ ​derivative​ ​thereof.​ ​In​ ​some​ ​embodiments,​ ​the​ ​compounds​ ​described​ ​herein​ ​may​​be​​modified to generate derivatives incorporating medicinal chemistry-based modifications.​​

[0038] ​ ​In​​some​​embodiments,​​the​​present​​invention​​features​​a​​method​​of​​preventing,​​delaying​​the​​onset​ ​of,​ ​or​ ​treating​ ​a​ ​health​ ​condition​ ​in​ ​a​ ​subject​​in​​need​​thereof,​​the​​method​​comprising​​the​​steps​​of:​​(a)​​identifying​​the​​subject​​presenting​​with​​the​​health​​condition;​​and / or​​(b)​​administering​​to​ ​the​ ​subject​ ​a​ ​therapeutically​ ​effective​ ​amount​ ​of​ ​a​ ​composition​ ​comprising​ ​one​ ​or​ ​more​​compounds as disclosed herein or a derivative thereof.​​

[0039] ​ ​In​ ​certain​ ​embodiments,​ ​the​ ​present​ ​disclosure​ ​provides​ ​methods​ ​of​ ​treating​ ​a​ ​health​​condition​ ​in​ ​a​ ​subject​ ​having,​ ​having​ ​had,​ ​suspected​ ​of​ ​developing,​ ​or​ ​at​ ​risk​ ​of​ ​developing​ ​a​​health​ ​condition,​ ​the​ ​method​ ​including​ ​administering​ ​to​ ​the​ ​subject​ ​a​ ​therapeutically​ ​effective​​amount of at least one compound disclosed herein.​​

[0040] ​ ​In​ ​some​ ​embodiments,​ ​the​ ​health​ ​condition​ ​comprises​ ​cancer,​ ​e.g.,​ ​cancers​ ​associated​​with​ ​UBA5-UFMylation​ ​dysregulation​ ​including​ ​but​ ​no​ ​limited​ ​to​ ​breast​ ​cancer​ ​(e.g.,​ ​ER⁺​​subtypes),​ ​pancreatic​ ​cancer,​ ​and​ ​lung​ ​adenocarcinoma.​ ​In​ ​other​ ​embodiments,​ ​the​ ​health​​condition​ ​comprises​ ​metabolic​ ​disorders​ ​(e.g.,​ ​diabetes)​ ​or​ ​neurodegenerative​ ​diseases​ ​(e.g.,​​Alzheimer's disease and Parkinson’s disease).​​

[0041] ​ ​specific​ ​cancers​ ​associated​ ​with​ ​UBA5-UFMylation​ ​dysregulation​​include​​breast​​cancer​​(especially ER⁺ subtype), pancreatic cancer, and lung adenocarcinoma.​​

[0042] ​ ​In​ ​some​ ​embodiments,​ ​the​ ​present​ ​invention​ ​features​ ​a​ ​method​ ​of​ ​treating​ ​Alzheimer’s​​disease​ ​in​ ​a​ ​subject​ ​in​ ​need​ ​thereof.​ ​The​ ​methods​ ​may​​comprise​​administering​​to​​the​​subject​​a​​therapeutic​​amount​​of​​a​​composition​​comprising​​a​​compound​​as​​described​​herein​​or​​a​​derivative​ ​thereof.​​

[0043] ​ ​In​ ​some​ ​embodiments,​ ​the​ ​present​ ​invention​ ​features​ ​a​ ​method​ ​of​ ​treating​ ​cancer​ ​in​ ​a​​subject​ ​in​ ​need​ ​thereof.​ ​The​ ​methods​ ​may​ ​comprise​ ​administering​ ​to​ ​the​ ​subject​ ​a​ ​therapeutic​​amount of a composition comprising a compound as described herein or a derivative thereof.​​

[0044] ​ ​Furthermore,​ ​in​ ​some​ ​embodiments,​ ​the​ ​present​ ​invention​ ​features​ ​a​ ​method​ ​for​​identifying​ ​derivatives​ ​with​ ​an​ ​increased​ ​binding​ ​affinity​ ​to​ ​UBA5.​​The​​method​​may​​comprise​​providing​ ​a​ ​competitive​ ​assay​ ​system​ ​wherein​ ​a​ ​test​ ​derivative​ ​and​ ​a​ ​reference​ ​compound​ ​are​​exposed​ ​to​ ​UBA5​ ​under​ ​conditions​ ​that​ ​permit​ ​binding;​ ​measuring​ ​the​ ​binding​ ​of​ ​the​ ​test​​derivative​ ​and​ ​the​ ​reference​ ​compound​ ​to​ ​UBA5;​ ​and​ ​identifying​ ​a​ ​test​ ​derivative​ ​with​ ​an​​increased​ ​binding​ ​affinity​ ​to​ ​UBA5​ ​when​ ​its​ ​binding​ ​competes​ ​with,​ ​or​ ​exceeds,​ ​that​ ​of​ ​the​​reference​ ​compound.​ ​In​ ​some​ ​embodiments,​ ​the​ ​reference​ ​compound​ ​is​ ​selected​ ​from​ ​a​ ​group​​consisting of:​​

[0045] ​ ​EXAMPLE​​

[0046] ​ ​The​ ​following​ ​is​ ​a​ ​non-limiting​ ​example​ ​of​​the​​present​​invention.​​It​​is​​to​​be​​understood​​that​ ​said​ ​example​ ​is​ ​not​ ​intended​ ​to​ ​limit​ ​the​ ​present​ ​invention​ ​in​ ​any​ ​way.​ ​Equivalents​ ​or​​substitutes are within the scope of the present invention.​​

[0047] ​ ​Expression​ ​and​ ​purification​ ​of​ ​UFM1,​​UBA1​​and​​UBA​​537-346​:​​​The​​codon-optimized​​gene​​sequence​ ​of​ ​UFM1,​ ​including​ ​a​ ​TEV​ ​protease-recognition​ ​site​ ​at​ ​the​ ​N-terminus,​ ​was​​synthesized​ ​and​ ​inserted​ ​between​ ​the​ ​His-tag​ ​and​ ​EcoR1​ ​site​ ​in​ ​the​ ​pET28a​ ​vector​ ​using​​seamless​ ​cloning​ ​technology​ ​by​ ​GeneUniversal.​ ​The​ ​expression​ ​plasmid​ ​UFM1-p28​ ​was​​transformed​ ​into​ ​E.​ ​coli​ ​Rosetta​ ​(DE3)​ ​cells,​ ​which​ ​were​ ​then​ ​cultured​​in​​Luria​​broth​​medium​​supplemented​ ​with​ ​100​ ​μg / mL​ ​kanamycin​ ​at​ ​37°C​ ​until​ ​the​ ​optical​ ​density​ ​(OD)​ ​at​ ​600​ ​nm​​reached​ ​0.6.​ ​The​ ​cells​ ​were​ ​induced​ ​with​ ​0.3​​mM​​isopropyl-β-D-thiogalactopyra​​noside​​(IPTG)​​and​​further​​incubated​​with​​shaking​​at​​150​​rpm​​and​​16​​°C​​for​​18​​hours.​​After​​incubation,​​the​​cells​ ​were​​collected​​by​​centrifugation​​at​​8000​​rpm​​for​​20​​minutes.​​The​​cell​​pellets​​were​​resuspended​​in​ ​a​​lysis​​buffer​​(20​​mM​​HEPES,​​pH​​7.5,​​200​​mM​​NaCl),​​and​​lysozyme,​​along​​with​​phenyl​​methyl​​sulfonyl​ ​fluoride​ ​(PMSF)​ ​was​ ​added.​ ​Cells​​were​​lysed​​using​​sonication​​and​​then​​centrifuged​​at​​15,000​ ​rpm​ ​for​ ​30​ ​minutes​ ​at​ ​4°C.​ ​The​ ​supernatant​ ​was​ ​loaded​ ​onto​ ​a​ ​pre-charged​ ​Ni-NTA​​affinity​​column​​(Qiagen)​​and​​washed​​with​​the​​resuspension​​buffer​​containing​​30​​mM​​imidazole.​ ​His-tagged​​UFM1​​was​​eluted​​using​​a​​lysis​​buffer​​containing​​50​​mM​​and​​100​​mM​​imidazole.​​The​ ​UFM1​​was​​further​​purified​​by​​size-exclusion​​chromatography​​using​​a​​75​​Superdex​​column,​​and​​peak​ ​fractions​​were​​collected​​and​​pooled.​​The​​purified​​UFM1​​was​​stored​​in​​a​​buffer​​containing​​25 mM HEPES (pH 7.5), 150 mM NaCl, and 2 mM DTT.​​

[0048] ​ ​The​ ​codon-optimized​ ​gene​ ​sequence​ ​of​ ​UBA5,​​encoding​​amino​​acids​​37-346​​of​​UBA5,​​was​​synthesized​​and​​cloned​​into​​a​​custom​​His-SUMO​​vector​​using​​seamless​​cloning​​technology​​by​ ​GeneUniversal.​ ​The​ ​expression​ ​and​ ​purification​ ​of​ ​UBA5​​37−346​-​His-SUMO​ ​were​ ​performed​​similarly​ ​to​ ​UFM1,​ ​with​ ​the​ ​exception​ ​of​ ​the​ ​on-column​ ​digestion​ ​step.​ ​The​ ​supernatant​ ​was​​loaded​ ​onto​ ​a​ ​pre-charged​ ​Ni-NTA​ ​affinity​ ​column​ ​(Qiagen).​ ​On-column​ ​digestion​ ​was​​performed​ ​using​ ​the​ ​ULP1​ ​protease,​ ​with​ ​incubation​ ​at​ ​room​ ​temperature​ ​(RT)​ ​for​ ​2​ ​hours.​​Following​ ​digestion,​ ​flow-through​ ​fractions​ ​were​ ​collected,​ ​washed​ ​with​ ​2​ ​columns​ ​of​ ​the​​resuspension​ ​buffer,​ ​and​ ​combined.​ ​The​ ​UBA537−346​ ​was​ ​then​ ​concentrated​ ​and​ ​further​​purified​ ​by​ ​size-exclusion​ ​chromatography​ ​using​ ​a​ ​75​ ​Superdex​ ​column.​ ​Peak​ ​fractions​ ​were​​collected​ ​and​ ​pooled,​ ​and​ ​the​ ​purified​​UBA5​​was​​stored​​in​​a​​buffer​​containing​​25​​mM​​HEPES​​(pH 7.5), 150 mM NaCl, and 2 mM DTT.​​

[0049] ​ ​The​ ​codon-optimized​ ​gene​ ​sequence​ ​of​ ​UBA1​ ​was​ ​synthesized​ ​and​ ​cloned​ ​into​ ​the​​pET28a​​vector​​by​​GeneUniversal.​​Expression​​and​​purification​​of​​His-tagged​​UBA1​​were​​carried​ ​out as described previously.​​

[0050] ​ ​Steady​​State​​Kinetics​​Assay​​:​​Steady-state​​enzyme​​kinetic​​assays​​were​​performed​​at​​30°C​​in​​a​​reaction​​buffer​​containing​​50​​mM​​Bis-Tris​​(pH​​6.5),​​100​​mM​​NaCl,​​and​​10​​mM​​MgCl​​2​.​​​For​ ​the​​assay,​​varying​​concentrations​​of​​UBA5​​37−346​​were​​mixed​​with​​UFM1​​(5​​µM)​​in​​the​​presence​ ​of​​ATP​​(5​​µM).​​Additionally,​​UFM1​​at​​different​​concentrations​​was​​combined​​with​​UBA5​​(800​ ​nM)​​in​​the​​presence​​of​​ATP​​(5​​µM).​​Lastly,​​varying​​concentrations​​of​​ATP​​were​​tested​​alongside​ ​UBA5​​(800​​nM)​​and​​UFM1​​(5​​µM).​​The​​reactions​​were​​incubated​​with​​Reagent​​I​​from​​the​​AMP​​Glo™​ ​kit​ ​(Promega)​ ​for​ ​1​ ​hour​ ​at​ ​room​ ​temperature​ ​(RT).​ ​Afterward,​ ​AMP​ ​Glo™​​Reagent​​II​​was​​added​​to​​each​​well,​​and​​luminescence​​(Gain​​150)​​was​​measured​​using​​a​​BioTek​​H1​​Reader.​ ​Reaction​​velocities​​for​​each​​concentration​​of​​UBA5,​​UFM1,​​and​​ATP​​were​​calculated​​and​​fitted​​to​ ​the​ ​Michaelis-Menten​ ​equation​ ​using​ ​the​ ​GraphPad​ ​Prism​ ​9​ ​software.​ ​The​ ​experiment​ ​was​​conducted in triplicate, unless otherwise specified.​​

[0051] ​ ​AMP-Glo​T​M​​-based​ ​HTS​ ​assay​​:​ ​To​ ​establish​ ​an​ ​in​ ​vitro​ ​UFMylation​ ​assay​ ​suitable​ ​for​​high-throughput​​screening​​(HTS),​​codon-optimized​​constructs​​encoding​​UFM1​​and​​UBA5​​amino​​acids​ ​37–346​ ​(UBA5₃₇–₃₄₆),​ ​representing​ ​the​ ​functional​ ​fragment​ ​of​ ​UBA5,​ ​were​ ​synthesized,​​cloned,​ ​expressed,​ ​and​ ​purified.​ ​Several​ ​different​ ​assay​ ​platforms​ ​were​​explored.​​In​​the​​UFM1​​activating​​reaction​​catalyzed​​by​​UBA5​​(UFM1+UBA5+ATP​​→UFM1-UBA5+AMP+PPi)​​(​​FIG.​ ​1A​​),​​ATP​​is​​converted​​to​​AMP​​and​​pyrophosphate​​(PPi),​​and​​UFM1​​is​​covalently​​conjugated​​to​​UBA5​ ​(UFM1-UBA5)​ ​through​ ​several​ ​intermediate​ ​steps.​​Initial​​efforts​​to​​use​​commercial​​kits​​to​ ​quantify​ ​ATP​ ​depletion​ ​or​ ​to​ ​quantify​ ​PPi​ ​production​ ​were​ ​unsuccessful.​ ​For​ ​reasons​​unknown,​ ​the​ ​reagents​ ​from​ ​the​ ​PPi​ ​kits​ ​from​ ​commercial​ ​vendors​ ​produced​ ​high​ ​background​​readings​ ​when​ ​mixed​ ​with​ ​UBA5​ ​protein​ ​alone,​ ​making​ ​it​​impossible​​to​​develop​​a​​meaningful​​HTS​ ​assay.​ ​Additionally,​ ​attempts​ ​to​ ​directly​ ​quantify​ ​ATP​ ​depletion​ ​using​ ​the​ ​Promega​​Kinase-Glo​T​M​ ​kit​ ​were​ ​also​ ​unsuccessful​ ​due​ ​to​ ​a​​low​​signal-to-background​​(S / B)​​ratio​​of​​only​​approximately 1.5-fold.​​

[0052] ​ ​A​ ​luminescence-based​ ​coupled​ ​UBA5​ ​assay​ ​was​ ​developed​ ​in​ ​the​ ​present​ ​invention​ ​to​​quantify​ ​AMP​ ​production​ ​using​ ​the​ ​Promega​ ​AMP-Glo™​ ​kit​ ​(​​FIG.​ ​1A​​).​ ​In​ ​this​ ​assay,​ ​UBA5​​(800​​nM)​​was​​incubated​​for​​1​​hour​​with​​varying​​compound​​concentrations​​(0–100​​µM),​​followed​​by​ ​the​ ​addition​ ​of​ ​UFM1​ ​and​ ​ATP​ ​(5​ ​µM​ ​each)​​and​​an​​additional​​30-minute​​incubation.​​AMP​​Glo™​ ​Reagent​ ​I​ ​(5​ ​µL)​ ​was​ ​then​ ​added​ ​to​ ​each​ ​well​ ​and​ ​incubated​​at​​room​​temperature​​for​​1​​hour​ ​to​ ​deplete​ ​ATP​ ​and​ ​convert​ ​AMP​ ​to​ ​ADP.​ ​Subsequently,​​AMP​​Glo™​​Reagent​​II​​(10​​µL)​​was​ ​added​ ​and​ ​incubated​ ​for​ ​30​ ​minutes​ ​at​ ​room​ ​temperature​ ​to​ ​convert​ ​ADP​ ​back​ ​to​ ​ATP,​​enabling​ ​luminescence​ ​generation​ ​through​ ​a​ ​luciferase​ ​reaction.​ ​Luminescence​ ​was​ ​measured​​using​​a​​BioTek​​H1​​Reader,​​and​​AMP​​concentrations​​were​​determined​​from​​a​​standard​​curve.​​IC​​50​​values​ ​of​ ​the​ ​compounds​ ​were​ ​calculated​ ​using​ ​GraphPad​ ​Prism​ ​9,​ ​and​ ​all​ ​experiments​ ​were​​performed in triplicate.​​

[0053] ​ ​In​ ​this​ ​coupled​ ​system,​ ​Reagent​ ​I​ ​depletes​ ​the​ ​input​ ​ATP​ ​and​ ​converts​ ​AMP​ ​to​ ​ADP.​​Then,​ ​Reagent​ ​II​ ​converts​ ​ADP​ ​back​ ​to​ ​ATP,​ ​which​ ​is​ ​detected​ ​by​ ​the​ ​firefly​ ​luciferase,​​generating​ ​luminescence.​ ​Thus,​ ​the​ ​UFM1-activation​ ​reaction​ ​catalyzed​ ​by​ ​UBA5​ ​can​ ​be​​monitored​ ​by​ ​measuring​ ​the​ ​luminescence.​ ​The​ ​linear​ ​detection​ ​range​ ​of​ ​AMP​ ​was​ ​first​​established​ ​using​ ​the​ ​kit,​ ​ensuring​ ​that​ ​the​ ​generated​ ​luminescence​ ​corresponded​ ​directly​ ​and​​linearly​​to​​AMP​​production​​(​​FIG.​​1A​​and​​1B​​).​​This​​step​​is​​crucial​​to​​ensure​​that​​the​​inhibitor's​​effectiveness​ ​has​ ​a​ ​linear​ ​relationship​ ​with​ ​the​ ​luminescence​ ​generated.​ ​As​ ​shown​ ​in​​FIG.​​1A​​and​ ​1B​​,​ ​AMP​ ​concentrations​ ​within​ ​the​ ​0–10​ ​µM​ ​range​ ​showed​ ​a​ ​linear​ ​relationship​ ​with​​luminescence​​production.​​The​​addition​​of​​up​​to​​100​​µM​​ATP​​did​​not​​significantly​​alter​​this​​trend​​or​ ​the​​signal​​(data​​not​​shown),​​indicating​​that​​Reagent​​I​​in​​the​​kit​​effectively​​depleted​​the​​input​​ATP up to 100 µM without any issues.​​

[0054] ​ ​Next,​ ​kinetic​ ​analyses​ ​were​ ​conducted​ ​to​ ​optimize​ ​the​ ​assay,​ ​determining​ ​the​ ​ideal​​concentrations​ ​of​ ​ATP,​​UBA5​​37-346​,​​​and​​UFM1,​​as​​well​​as​​optimizing​​buffer​​conditions,​​DMSO,​​and​ ​detergent​ ​tolerance,​ ​and​ ​incubation​ ​time​ ​(​​FIG.​ ​1C-1E​​).​ ​These​ ​adjustments​ ​were​ ​made​ ​to​​ensure​​the​​reaction​​remained​​within​​a​​linear​​range​​and​​achieved​​an​​optimal​​signal-to-background​​(S / B)​ ​ratio​ ​as​ ​well​ ​as​ ​a​ ​more​ ​appropriate​ ​HTS​ ​parameter:​ ​signal​ ​window​ ​(SW),​ ​in​​accordance​​with​ ​the​ ​NIH​ ​HTS​ ​Assay​ ​Validation​ ​guide.​ ​As​ ​shown​ ​in​ ​FIG.​​1C​​,​​dose-dependent​​increase​​of​​luminescence​ ​was​ ​observed​ ​by​​increasing​​UBA5​​concentration.​​The​​results​​indicate​​that​​UBA5​​is​​active,​​catalyzing​​the​​conjugation​​of​​UFM1​​to​​UBA5​​and​​converting​​ATP​​to​​AMP,​​resulting​​in​​an​ ​increase​ ​in​ ​luminescence.​ ​By​ ​fitting​ ​the​ ​kinetic​ ​data,​ ​a​ ​K​​m​ ​of​ ​0.14​ ​µM​ ​was​ ​obtained​​(​​FIG.​​1C​​).​​

[0055] ​ ​Similarly,​​kinetic​​studies​​were​​also​​performed​​for​​UFM1​​and​​ATP​​in​​concentration​​series​​(​​FIG.​ ​1D​ ​and​ ​1E​​).​ ​As​ ​concentrations​ ​of​ ​UFM1​ ​and​ ​ATP​ ​increased,​ ​hyperbolic​ ​increases​ ​in​​luminescence​ ​were​ ​obtained.​ ​K​​m​ ​values​ ​of​ ​11.9​ ​µM​ ​for​ ​ATP​ ​and​ ​0.13​ ​µM​ ​for​ ​UFM1​ ​were​​determined by fitting the kinetic data.​​

[0056] ​ ​Upon​ ​optimization,​ ​superior​ ​HTS​ ​parameters​ ​were​ ​achieved​ ​by​ ​comparing​ ​endpoint​​luminescence​​readings​​of​​reactions​​with​​and​​without​​UBA5,​​resulting​​in​​a​​Z’​​factor​​of​​0.89,​​S / B​ ​ratio of 7.4, SW of 169, and CV of 0.5% (​​FIG.1F​​).​​

[0057] ​ ​Inhibition​ ​of​ ​UBA5​ ​activity​ ​by​ ​positive​ ​control​ ​inhibitor​ ​DKM​ ​2-93​​:​ ​Next,​ ​the​ ​UBA5​​inhibitor​ ​DKM​ ​2-93​ ​(DKM)​ ​was​ ​evaluated,​ ​using​ ​the​ ​AMP-Glo​T​M​ ​coupling​ ​assay.​ ​Using​ ​the​​AMP-Glo​T​M​ ​assay,​ ​DKM​ ​dose-dependently​ ​inhibited​ ​UBA5​ ​activity,​ ​with​ ​an​ ​IC​​50-UBA5-Glo​ ​value​​(UBA5​ ​primary;​ ​Table​ ​1​​)​ ​of​ ​547​ ​µM​ ​(​​FIG.​ ​2A​​).​ ​Using​ ​DKM​ ​as​ ​a​ ​control​ ​compound,​ ​the​​robustness​ ​of​ ​the​ ​AMP-Glo​T​M​ ​UBA5​ ​coupling​ ​assay​ ​was​ ​demonstrated​ ​in​ ​a​ ​384-well​ ​plate​​format,​ ​achieving​ ​a​ ​satisfactory​ ​Z’​ ​factor​ ​of​ ​0.85,​ ​S / B​ ​ratio​ ​of​ ​5.8,​ ​SW​ ​of​ ​37,​ ​and​ ​CV​ ​of​ ​2%​​against​​UBA5​​(​​FIG.​​2B​​).​​The​​assay​​showed​​excellent​​well-to-well​​consistency​​(​​FIG.​​2C​​).​​These​​parameters​ ​were​ ​reproducible​ ​over​ ​multiple​ ​plates​ ​and​ ​dates​ ​(​​FIG.​ ​2D​​).​ ​Drifts​ ​or​ ​edge​ ​effects​​were not observed with this assay (data not shown).​​

[0058] ​ ​Development​ ​of​ ​a​ ​secondary​ ​assay​ ​based​ ​on​ ​fluorescence​ ​polarization​ ​(FP)​​:​ ​False​​positives​​may​​arise​​from​​compound​​aggregation,​​PAINS​​properties,​​or​​interference​​with​​enzymes​​in​ ​the​ ​coupling​ ​assay.​ ​Therefore,​ ​developing​ ​secondary​ ​or​ ​tertiary​ ​assays​ ​to​ ​prioritize​ ​hit​​compounds​ ​is​ ​essential.​ ​To​ ​further​ ​characterize​ ​UBA5​ ​activity​ ​and​ ​prioritize​ ​hit​ ​compounds,​​a​​secondary​ ​fluorescence​ ​polarization​ ​(FP)​ ​assay​ ​was​ ​developed​ ​using​ ​the​ ​Transcreener®​​AMP² / GMP²​​FP​​kit​​(Bellbrook​​Labs)​​(​​FIG.​​3A​​).​​This​​assay​​measures​​AMP​​production​​based​​on​​FP​ ​changes,​ ​providing​ ​a​ ​quantitative​ ​assessment​ ​of​ ​enzyme​ ​activity.​ ​Reactions​ ​were​ ​prepared​​according​ ​to​ ​the​ ​manufacturer’s​ ​instructions​ ​in​ ​a​ ​total​ ​volume​ ​of​ ​20​​µL​​per​​well​​in​​a​​384-well​​black​ ​plate,​ ​consisting​ ​of​ ​a​ ​15​ ​µL​ ​enzymatic​ ​reaction​ ​and​ ​5​​µL​​of​​detection​​reagent.​​UBA5​​(1​​µM)​ ​was​ ​incubated​ ​for​ ​1​​hour​​with​​varying​​concentrations​​of​​compounds​​(0–25​​µM),​​followed​​by​ ​the​ ​addition​ ​of​ ​UFM1​ ​(2.5​ ​µM)​ ​and​ ​ATP​ ​(20​ ​µM)​ ​and​ ​a​ ​30-minute​ ​incubation​ ​at​ ​room​​temperature​ ​to​ ​allow​ ​the​ ​enzymatic​ ​reaction​ ​to​ ​proceed.​ ​Subsequently,​ ​5​ ​µL​ ​of​ ​Transcreener®​​AMP / GMP​​FP​​Detection​​Reagent​​containing​​the​​fluorescent​​probe​​was​​added,​​and​​the​​plate​​was​​incubated​ ​for​ ​1.5​ ​hours​ ​to​ ​develop​ ​the​ ​fluorescent​ ​signal.​ ​Fluorescence​ ​polarization​ ​was​​measured​ ​using​ ​a​ ​plate​ ​reader,​ ​with​ ​polarization​ ​values​ ​directly​ ​correlating​ ​to​ ​AMP​​concentration.​ ​ICCC​ ​values​ ​were​ ​calculated​​using​​GraphPad​​Prism​​9,​​and​​all​​experiments​​were​​performed in triplicate.​​

[0059] ​ ​In​ ​the​ ​FP​ ​assay,​ ​an​ ​AlexFluor-633-labeled​ ​AMP​2​​​ / GMP​2​​ ​tracer​ ​(AMP-633)​ ​binds​ ​an​​antibody​ ​(Ab)​ ​specially​ ​recognizing​ ​AMP​ ​or​ ​GMP,​ ​resulting​ ​in​ ​high​ ​FP​ ​values​ ​(​​FIG.​ ​3A​​).​​However,​​when​​the​​UBA5+UFM1​​reaction​​produces​​free​​AMP,​​it​​displaces​​the​​AMP-633​​tracer​​from​ ​the​ ​antibody,​ ​leading​ ​to​ ​a​ ​decrease​ ​in​ ​FP.​ ​In​ ​the​ ​presence​ ​of​ ​a​ ​UBA5​ ​inhibitor,​ ​AMP​​production​​is​​diminished,​​resulting​​in​​less​​displacement​​of​​the​​AMP-633​​tracer​​and​​maintaining​ ​high FP values.​​

[0060] ​ ​As​ ​shown​ ​in​ ​FIG.​ ​3B​​,​ ​initially​ ​AMP​ ​dose-dependently​ ​reduced​ ​FP,​ ​demonstrating​ ​that​​free​ ​AMP​ ​effectively​ ​displaced​ ​the​ ​AMP-633​ ​tracer​ ​from​ ​the​ ​antibody​ ​in​ ​a​ ​dose-dependent​​manner.​​Next,​​UFMylation​​reactions​​were​​compared​​with​​and​​without​​UBA5.​​As​​shown​​in​​FIG.​​3C​​,​ ​the​ ​presence​ ​of​ ​UBA5​ ​in​ ​the​ ​reaction​ ​(last​ ​column)​ ​significantly​ ​reduced​ ​FP​ ​values​​compared​ ​to​ ​the​ ​reaction​ ​without​ ​UBA5​ ​(1​s​t​ ​column).​ ​These​ ​results​ ​suggest​ ​that​ ​UBA5​ ​reacts​​with​ ​UFM1,​ ​converting​ ​ATP​ ​to​ ​AMP,​ ​which​ ​then​ ​displaces​ ​the​ ​AMP-633​ ​tracer​ ​from​ ​the​​antibody, leading to a low FP value.​​

[0061] ​ ​Further,​​DKM​​was​​used​​as​​a​​control​​inhibitor​​to​​optimize​​the​​FP-based​​assay​​parameters​​for​​monitoring​​the​​UBA5-UFM1​​reaction,​​following​​a​​similar​​approach​​to​​the​​optimization​​of​​the​​AMP-Glo​T​M​ ​assay​ ​(data​ ​not​ ​shown).​ ​Under​ ​optimized​ ​conditions,​ ​DKM​ ​dose-dependently​​inhibited​ ​the​ ​UBA5-mediated​ ​UFMylation​ ​reaction​ ​(​​FIG.​ ​3C​​).​ ​The​ ​assay​ ​proved​ ​to​​be​​robust,​​with​ ​a​ ​Z’-factor​ ​of​ ​0.5,​ ​an​​S / B​​ratio​​of​​3.7,​​an​​SW​​of​​5.7,​​and​​a​​CV​​of​​8.2%,​​meeting​​the​​NIH​​HTS Assay Validation guidelines​​(​​FIG.3D​​).​​

[0062] ​ ​Identification​ ​of​ ​Uba5​ ​inhibitors​​using​​the​​AMP-Glo​T​M​ ​assay​​:​​Using​​the​​aforementioned​​assay,​ ​a​ ​total​ ​of​ ​27,656​ ​compounds—including​ ​5,120​ ​blood-brain-barrier​ ​(BBB)-permeable​​compounds​ ​from​ ​the​ ​Core​ ​library​ ​of​ ​the​ ​Arizona​ ​Center​ ​for​ ​Drug​ ​Discovery,​ ​4,551​​CNS-permeable​ ​compounds,​ ​8,991​ ​diverse​ ​compounds,​ ​and​ ​9,994​ ​protein-protein​ ​interaction​​(PPI)​ ​modulators.​ ​DKM​ ​served​ ​as​ ​a​ ​positive​ ​control​ ​inhibitor.​ ​Screening​ ​of​ ​the​ ​5,120​​BBB-permeable​ ​compounds​ ​resulted​ ​in​ ​the​ ​identification​ ​of​ ​five​ ​novel​ ​UBA5​ ​inhibitors,​​designated​ ​as​ ​compounds​ ​14–18​ ​(​​FIG.​ ​4​​).​ ​Screening​ ​of​ ​the​ ​additional​ ​4,551​ ​CNS-permeable​​compounds,​ ​8,991​ ​diverse​ ​compounds,​ ​and​ ​9,994​ ​protein–protein​ ​interaction​ ​(PPI)​ ​modulators​​yielded 13 novel UBA5 inhibitors, designated as compounds 1–13 (​​FIG.4​​).​​

[0063] ​ ​The​ ​AMP-Glo™​ ​UBA5​ ​assay​ ​demonstrated​ ​strong​ ​robustness​ ​and​​reliability,​​achieving​​an​​average​​Z’​​score​​of​​0.79,​​a​​signal-to-background​​(S / B)​​ratio​​of​​6.4,​​a​​screening​​window​​(SW)​ ​of​​58,​​and​​a​​coefficient​​of​​variation​​(CV)​​of​​5.3%​​(​​FIG.​​5A​​).​​These​​metrics​​met​​the​​acceptance​​criteria​ ​for​ ​a​ ​reliable​ ​HTS​ ​assay,​ ​which​ ​are​​Z'​​≥​​0.4,​​SW​​≥​​2,​​and​​CV​​≤​​20%,​​according​​to​​the​​NIH HTS Assay Validation guidelines.​​

[0064] ​ ​The​ ​13​ ​compounds​ ​were​ ​cherry-picked​ ​for​ ​dose-response​ ​inhibition​​of​​UBA5-mediated​​UFMylation.​ ​All​ ​these​ ​compounds​ ​showed​ ​dose-dependent​ ​inhibition​ ​of​ ​UFM1​ ​activation​ ​by​​UBA5,​ ​with​ ​IC​​50-UBA5-Glo​ ​values​ ​ranging​ ​from​ ​2.1​ ​µM​ ​to​ ​13.2​ ​µM​ ​(​​FIG.​ ​5B​​,​ ​UBA5​ ​primary;​​Table 1​​).​​

[0065] ​ ​Table 1 Compound Properties:​​

[0066] ​ ​UBA5-free​​coupling​​enzyme​​AMP-Glo​T​M​​assay​​:​​To​​determine​​if​​the​​identified​​hits​​inhibit​​the​ ​coupling​ ​enzymes​ ​in​ ​the​ ​AMP-Glo​T​M​ ​kit,​ ​a​ ​UBA5-free​ ​coupling​ ​enzyme​ ​assay​ ​was​​performed.​​Briefly,​​test​​compounds​​were​​evaluated​​at​​concentrations​​ranging​​from​​0​​to​​100​​µM​ ​using​​the​​AMP-Glo™​​kit​​(Promega),​​with​​AMP​​serving​​as​​the​​direct​​substrate​​in​​the​​absence​​of​​UBA5.​ ​Reactions​ ​contained​ ​all​ ​coupling​ ​components,​ ​including​ ​ATP,​ ​UFM1,​ ​and​ ​the​ ​reagents​​from​​the​​kit,​​but​​excluded​​UBA5.​​Following​​a​​30-minute​​incubation,​​AMP​​was​​added​​to​​a​​final​​concentration​ ​of​ ​5​ ​µM,​ ​and​ ​luminescence​ ​was​ ​measured​ ​at​ ​37​ ​°C​​using​​a​​2-minute​​integration​​time,​ ​full​ ​light​ ​emission,​ ​top​ ​optics,​ ​and​ ​a​ ​gain​ ​setting​ ​of​ ​150.​ ​Appropriate​ ​controls​ ​were​​included,​ ​and​ ​all​ ​experiments​ ​were​ ​performed​ ​in​ ​triplicate.​ ​The​ ​UBA5-free​ ​AMP-Glo™​ ​assay​​demonstrated​​sensitivity​​comparable​​to​​the​​primary​​UBA5-dependent​​assay​​(​​FIG.​​6A​​).​​Addition​​of​ ​AMP​ ​generated​ ​a​ ​strong​ ​luminescent​ ​signal​ ​relative​ ​to​ ​the​ ​AMP-free​ ​control,​ ​yielding​ ​a​ ​Z′​​factor​​of​​0.81,​​a​​signal-to-background​​(S / B)​​ratio​​of​​17.7,​​a​​screening​​window​​(SW)​​of​​37.0,​​and​​a​ ​coefficient​ ​of​ ​variation​ ​(CV)​ ​of​ ​4.1%,​ ​all​ ​of​ ​which​ ​met​ ​the​ ​NIH​ ​HTS​ ​assay​ ​validation​​guidelines.​​

[0067] ​ ​Except​ ​for​ ​Compounds​ ​13​ ​and​ ​17,​ ​which​ ​exhibited​ ​similar​ ​inhibition​ ​of​ ​the​ ​coupling​​enzymes​​in​​the​​kit,​​the​​other​​twelve​​hits​​only​​moderately​​inhibited​​the​​enzymes​​in​​the​​kit.​​Their​ ​IC​​50-coup-Glo​​values​​were​​significantly​​higher​​than​​their​​corresponding​​IC​​50-UBA5-Glo​​values,​​resulting​ ​in​​selectivity​​indices​​(SI​​free / UBA5​,​​​calculated​​as​​IC​​50-free-Glo​ / ​IC​​50-UBA5-Glo​)​​​ranging​​from​​1.2​​to​​21​​(​​FIG.​​6B,​ ​Table​ ​1​​).​ ​These​ ​results​ ​suggest​ ​that​ ​the​ ​primary​ ​inhibitory​ ​activity​ ​of​ ​these​ ​compounds​ ​is​​directed against UBA5 rather than the coupling enzymes in the assay kit.​​

[0068] ​ ​Selectivity​​Assay​​:​​UFM1​​is​​a​​member​​of​​the​​ubiquitin-like​​protein​​(UBL)​​family,​​which​​is​​covalently​​attached​​to​​target​​proteins​​to​​regulate​​their​​activities.​​This​​process​​occurs​​via​​a​​general​​E1-E2-E3​ ​multienzyme​ ​cascade.​ ​A​ ​total​ ​of​ ​17​ ​human​ ​UBLs​ ​from​ ​9​ ​phylogenetic​​classes​​have​​been​​identified​​as​​being​​conjugated​​to​​various​​molecules.​​While​​all​​UBLs​​share​​a​​similar​​overall​ ​structural​​fold,​​each​​UBL​​typically​​operates​​through​​its​​own​​unique​​E1–E2–E3​​enzyme​​cascade​​and​ ​exerts​ ​specific​ ​effects​ ​on​ ​its​ ​respective​ ​targets.​ ​In​ ​humans,​ ​eight​ ​E1​ ​enzymes—UBA1,​​UBA2 / SAE1,​ ​UBA3 / NAE1,​ ​UBA4-7,​ ​and​ ​ATG7—have​ ​been​ ​identified​ ​as​ ​key​ ​initiators​​responsible​ ​for​ ​the​ ​conjugation​ ​of​ ​specific​ ​UBLs.​ ​E1​ ​enzymes​ ​are​ ​divided​ ​into​ ​canonical​ ​and​​noncanonical​ ​families.​ ​Canonical​ ​E1​ ​enzymes,​ ​such​ ​as​ ​UBA1,​ ​UBA2 / SAE1,​ ​UBA3 / NAE1,​​UBA6,​​and​​UBA7,​​are​​responsible​​for​​activating​​Ub,​​the​​SUMO​​protein​​family,​​NEDD8,​​FAT10,​​and​ ​ISG15,​ ​respectively.​ ​They​ ​possess​ ​two​ ​pseudosymmetric​ ​adenylation​ ​domains,​ ​which​ ​are​​encoded​ ​by​ ​either​ ​one​ ​or​ ​two​ ​genes.​ ​In​ ​contrast,​ ​noncanonical​ ​E1​ ​enzymes,​ ​such​ ​as​ ​ATG7,​​UBA4,​ ​and​ ​UBA5,​ ​which​ ​respectively​ ​activate​ ​the​​ATG8​​and​​ATG12​​protein​​families,​​URM1,​​and​​UFM1,​​form​​homodimers​​to​​perform​​the​​E1​​function.​​Despite​​having​​distinct​​structures,​​all​ ​E1s​​share​​a​​common​​catalytic​​mechanism,​​which​​involves​​the​​conversion​​of​​ATP​​to​​AMP​​during​ ​the​​UBL​​activation​​process.​​Therefore,​​identifying​​inhibitors​​that​​are​​specific​​to​​UBA5,​​without​ ​affecting other E1 enzymes, is crucial.​​

[0069] ​ ​Given​​the​​shared​​ATP-to-AMP​​conversion​​mechanism​​among​​E1s,​​the​​AMP-Glo​T​M​ ​assay​​was​ ​adapted​ ​for​ ​use​ ​with​ ​other​ ​E1s​ ​as​ ​a​ ​specificity​ ​assay​ ​for​ ​identified​ ​hits.​ ​The​ ​UBA1​​specificity​​assay​​was​​conducted​​in​​triplicate,​​following​​a​​protocol​​similar​​to​​the​​AMP​​Detection​​Assay​ ​using​ ​the​ ​AMP-Glo™​ ​kit,​ ​which​ ​was​ ​employed​ ​for​ ​UBA5.​ ​For​ ​all​ ​UBA1​ ​AMP-Glo​​assays (unless otherwise specified): UBA1, 0.3 µM; Ubiquitin, 10 µM; ATP, 5 µM.​​

[0070] ​ ​To​ ​demonstrate​ ​this,​ ​His-tagged​​UBA1​​was​​cloned,​​expressed,​​and​​purified​​in​​a​​manner​​similar​ ​to​ ​UBA5.​ ​The​ ​AMP-Glo​ ​assay​ ​successfully​ ​detected​ ​UBA1​ ​activity​ ​(​​FIG.​ ​7A​​).​ ​In​ ​the​​presence​ ​of​ ​UBA1,​ ​a​ ​6.6-fold​ ​increase​ ​in​ ​luminescence​ ​compared​ ​to​ ​the​ ​control​ ​without​ ​E1,​​indicating​ ​that​ ​UBA1,​​like​​UBA5​​in​​its​​UFMylation​​reaction,​​converts​​ATP​​to​​AMP​​during​​Ub​​activation,​ ​leading​ ​to​ ​luminescence​ ​increase​ ​(​​FIG.​ ​7A​​).​ ​The​ ​assay​ ​conditions​ ​were​ ​optimized​​similarly​​to​​those​​for​​UBA5,​​observing​​a​​dose-dependent​​increase​​in​​luminescence​​against​​ATP,​​UBA1​ ​and​ ​other​ ​components​​(​​FIG.​​7B​​).​​Using​​this​​specificity​​assay,​​the​​hits​​exhibited​​varying​​degrees​​of​​inhibition​​against​​UBA1,​​their​​IC​​50-UBA1-Glo​​values​​were​​consistently​​higher​​than​​those​​for​ ​UBA5​ ​(​​Table​ ​1,​ ​FIG.​ ​7C​​).​ ​The​ ​resulting​ ​selectivity​ ​indices​ ​2​ ​(SI​​UBA1 / UBA5​,​​ ​defined​ ​as​​IC​​50-UBA1-Glo​ / ​IC​​50-UBA5-Glo​)​​​ranged​​from​​1.9​​to​​greater​​than​​13.2;​​for​​example,​​compounds​​4​​,​​14​​,​​and​ ​15​​showed​​minimal​​inhibition​​of​​UBA1​​even​​at​​100​​µM,​​the​​highest​​concentration​​tested.​​These​ ​results indicate that the compounds display moderate selectivity for UBA5 over UBA1.​​

[0071] ​ ​Identified​ ​hits​ ​inhibited​ ​UFMylation​ ​in​ ​vitro​ ​in​ ​gel-based​ ​assay:​ ​The​ ​ability​ ​of​ ​the​​identified​​hits​​to​​inhibit​​UFMylation​​was​​next​​evaluated​​using​​a​​gel-based​​charging​​assay.​​UBA5​​(12​ ​µM)​ ​was​ ​incubated​ ​with​ ​varying​ ​concentrations​​of​​compounds​​(0–100​​µM)​​for​​1​​hour​​in​​a​​reaction​ ​buffer​​containing​​50​​mM​​Bis-Tris​​(pH​​6.5),​​100​​mM​​NaCl,​​and​​10​​mM​​MgCl₂.​​UFM1​​(25​ ​µM)​ ​and​​ATP​​(2​​mM)​​were​​then​​added,​​and​​reactions​​were​​incubated​​for​​2​​hours​​at​​30​​°C,​​with​​zero​​time​​points​​collected​​prior​​to​​ATP​​addition.​​Samples​​were​​analyzed​​under​​non-reducing​ ​conditions​​on​​12%​​SDS-PAGE​​gels​​and​​stained​​with​​Coomassie​​G-250.​​Bands​​corresponding​​to​​UBA5,​ ​UFM1,​ ​and​ ​their​ ​conjugates​ ​were​ ​quantified​​using​​ImageJ,​​and​​percentage​​activity​​was​​calculated using GraphPad Prism 9.​​

[0072] ​ ​Consistent​ ​with​ ​results​ ​from​ ​the​ ​AMP-Glo™​ ​and​ ​FP-based​ ​assays,​ ​compounds​ ​14–18​​dose-dependently​ ​inhibited​ ​UFM1​ ​charging​​to​​UBA5​​(data​​not​​shown),​​confirming​​their​​ability​​to block UBA5-mediated UFMylation in vitro.​​

[0073] ​ ​Inhibition​ ​of​ ​UFMylation​ ​in​ ​vitro​​:​ ​Next,​ ​the​ ​ability​ ​of​ ​the​ ​identified​ ​hits​ ​to​ ​inhibit​​UBA5–UFM1​ ​conjugation​ ​in​ ​cells​ ​was​ ​evaluated​ ​using​ ​a​ ​gel-based​ ​assay.​ ​Briefly,​ ​HEK293T​​cells​​were​​seeded​​in​​12-well​​plates​​(~80%​​confluence)​​and​​treated​​with​​compounds​​at​​10​​and​​60​ ​µM​​for​​24​​hours.​​Cells​​were​​then​​lysed​​in​​100​​µL​​RIPA​​buffer,​​and​​protein​​concentrations​​were​​determined​ ​using​ ​a​ ​BCA​ ​assay.​ ​Western​ ​blotting​ ​was​ ​performed​ ​with​ ​an​ ​anti-UFM1​ ​antibody​​(Abcam,​ ​109305;​ ​1:2000)​ ​on​ ​4–12%​ ​Bis-Tris​ ​gels​ ​(Invitrogen),​ ​and​ ​UBA5–UFM1​ ​and​​UFC1–UFM1 bands were quantified using ImageJ. Experiments were performed in duplicate.​​

[0074] ​ ​Consistent​ ​with​ ​results​ ​from​ ​the​ ​AMP-Glo™​ ​assay,​ ​compounds​​1–5​​,​​11​​,​​12,​​and​​14–18​​dose-dependently inhibited UFM1 charging to UBA5 (​​FIG.8​​).​​

[0075] ​ ​Cytotoxicity​ ​and​ ​Cell​ ​Viability​ ​Analysis​​:​ ​Next,​ ​the​ ​cell​ ​cytotoxicity​​of​​these​​compounds​​was​​evaluated​​using​​a​​WST-8​​cell​​viability​​assay​​(​​Table​​1,​​FIG.​​9​​).​​Briefly,​​cytotoxicity​​and​​cell​​viability​ ​were​ ​assessed​ ​using​ ​the​ ​Cell​ ​Counting​ ​Kit-8​ ​(CCK-8,​ ​GLPBIO)​ ​with​ ​minor​​modifications​​to​​the​​manufacturer’s​​protocol.​​Human​​astrocyte​​CCF-STTG1​​cells​​were​​cultured​ ​in​​RPMI-1640​​supplemented​​with​​10%​​FBS​​at​​37°C​​in​​a​​humidified​​5%​​CO₂​​atmosphere.​​A​​total​ ​of​​1.5​​×​​10⁵​​cells​​per​​well​​were​​seeded​​in​​duplicate​​in​​a​​96-well​​plate​​and​​incubated​​for​​24​​hours.​ ​Cells​​were​​then​​treated​​with​​UBA5​​inhibitors​​at​​concentrations​​ranging​​from​​0.32​​to​​200​​μM​​for​ ​48​​hours.​​Following​​treatment,​​10​​μL​​of​​CCK-8​​reagent​​was​​added​​to​​each​​well​​and​​incubated​​at​​37°C​ ​for​ ​1–4​ ​hours.​ ​DMSO​ ​served​ ​as​ ​a​ ​vehicle​ ​control.​ ​Absorbance​ ​was​ ​measured​​at​​460​​nm​​using​ ​a​ ​BioTek​ ​Synergy​ ​HI​ ​microplate​ ​reader,​ ​and​ ​cytotoxic​ ​concentrations​ ​(CC₅₀)​ ​were​​determined​ ​from​ ​nonlinear​ ​regression​ ​of​ ​dose-response​ ​curves​ ​using​ ​GraphPad​ ​Prism​ ​9.​ ​All​​experiments were performed in triplicate.​​

[0076] ​ ​None​ ​of​ ​the​ ​tested​ ​compounds​ ​displayed​ ​strong​ ​cytotoxicity​ ​toward​ ​CCF-STTG1​ ​cells.​​Compounds​ ​1​​,​ ​3–6​​,​ ​8​​,​ ​and​ ​11–18​ ​exhibited​ ​CC₅₀​ ​values​ ​greater​ ​than​ ​100​ ​µM,​ ​whereas​​compounds​ ​2​​,​ ​7​​,​ ​9​​,​ ​and​ ​10​​showed​​moderate​​cytotoxicity,​​with​​CC₅₀​​values​​ranging​​from​​37​​to​​~50 µM.​​

[0077] ​ ​Thus,​ ​as​ ​described​ ​herein,​ ​the​ ​present​ ​invention​ ​identifies​ ​18​ ​novel​ ​UBA5​ ​inhibitors​​spanning​ ​several​ ​distinct​ ​chemical​ ​scaffolds,​ ​each​ ​exhibiting​ ​low​ ​micromolar​ ​IC₅₀​ ​values.​​Importantly,​ ​these​ ​compounds​ ​show​ ​strong​ ​selectivity​ ​for​ ​UBA5​ ​over​ ​other​ ​E1​ ​enzymes,​​including​ ​UBA1,​ ​which​ ​activates​ ​ubiquitin.​ ​Such​ ​selectivity​ ​is​ ​critical,​ ​as​ ​many​ ​E1​ ​enzymes​​share​​common​​structural​​and​​mechanistic​​features,​​making​​off-target​​effects​​a​​significant​​concern​ ​in the development of E1 inhibitors.​​

[0078] ​ ​As used herein, the term “about” refers to plus or minus 10% of the referenced number.​​

[0079] ​ ​Although​ ​there​ ​has​ ​been​ ​shown​ ​and​ ​described​ ​the​ ​preferred​ ​embodiment​ ​of​​the​​present​​invention,​ ​it​ ​will​ ​be​ ​readily​ ​apparent​​to​​those​​skilled​​in​​the​​art​​that​​modifications​​may​​be​​made​​thereto​ ​which​ ​do​ ​not​ ​exceed​ ​the​ ​scope​ ​of​ ​the​ ​appended​ ​claims.​ ​Therefore,​ ​the​ ​scope​ ​of​ ​the​​invention​ ​is​ ​only​ ​to​ ​be​ ​limited​ ​by​ ​the​ ​following​ ​claims.​ ​In​ ​some​ ​embodiments,​ ​the​ ​figures​​presented​​in​​this​​patent​​application​​are​​drawn​​to​​scale,​​including​​the​​angles,​​ratios​​of​​dimensions,​​etc.​ ​In​ ​some​ ​embodiments,​​the​​figures​​are​​representative​​only​​and​​the​​claims​​are​​not​​limited​​by​​the​ ​dimensions​ ​of​ ​the​ ​figures.​ ​In​ ​some​ ​embodiments,​ ​descriptions​ ​of​ ​the​ ​inventions​ ​described​​herein​ ​using​ ​the​ ​phrase​ ​“comprising”​ ​includes​ ​embodiments​ ​that​ ​could​ ​be​ ​described​ ​as​​“consisting​​essentially​​of”​​or​​“consisting​​of”,​​and​​as​​such​​the​​written​​description​​requirement​​for​​claiming​ ​one​ ​or​ ​more​ ​embodiments​ ​of​ ​the​ ​present​ ​invention​ ​using​ ​the​ ​phrase​ ​“consisting​​essentially of” or “consisting of” is met.​

Claims

​WHAT IS CLAIMED IS:​ ​1.​ ​A compound for inhibiting UBA5 selected from a group consisting of:​​or a derivative thereof.​ ​2.​ ​The compound of claim 1, wherein the compound is specific for UBA5.​​3.​ ​The​​compound​​of​​claim​​1​​or​​claim​​2,​​wherein​​the​​compound​​inhibits​​UBA5​​with​​an​​IC₅₀​​of less than 15 µM.​ ​4.​ ​A​​method​​of​​inhibiting​​UBA5​​in​​a​​cell,​​the​​method​​comprising​​contacting​​the​​cell​​with​​a​​composition comprising a compound selected from a group consisting of:​​or a derivative thereof.​ ​5.​ ​A​ ​method​ ​of​ ​treating​ ​Alzheimer’s​ ​disease​ ​in​ ​a​ ​subject​ ​in​ ​need​ ​thereof,​ ​the​ ​methods​​comprising​​administering​​to​​administering​​to​​the​​subject​​a​​therapeutic​​effect​​amount​​of​​a​ ​composition comprising a compound selected from a group comprising:​​ .​ ​6.​ ​A​ ​method​ ​of​ ​treating​ ​cancer​ ​in​ ​a​ ​subject​ ​in​ ​need​ ​thereof,​ ​the​ ​methods​ ​comprising​​administering​​to​​administering​​to​​the​​subject​​a​​therapeutic​​effect​​amount​​of​​a​​composition​ ​comprising a compound selected from a group comprising:​​or a derivative thereof.​​7.​ ​A​ ​method​ ​for​ ​identifying​ ​derivatives​ ​with​ ​an​ ​increased​ ​binding​ ​affinity​ ​to​ ​UBA5,​ ​the​​method comprising:​ ​a)​ ​providing​ ​a​ ​competitive​ ​assay​ ​system​ ​wherein​ ​a​ ​test​ ​derivative​ ​and​ ​a​ ​reference​​compound are exposed to UBA5 under conditions that permit binding;​ ​b)​ ​measuring​ ​the​ ​binding​ ​of​ ​the​ ​test​ ​derivative​ ​and​ ​the​ ​reference​ ​compound​ ​to​​UBA5; and​ ​c)​ ​identifying​ ​a​ ​test​​derivative​​with​​an​​increased​​binding​​affinity​​to​​UBA5​​when​​its​​binding competes with, or exceeds, that of the reference compound;​ ​wherein the reference compound is selected from a group consisting of:​​.​

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