Methods and preparations for modulating defective in cullin neddylation 1 (DCN1)

Abstract

Methods and preparations to identify compounds useful for the treatment of hemoglobin-related disorders including sickle cell disorders, diseases, and conditions, and thalassemia are described. The disclosure also provides methods for the treatment of hemoglobin-related disorders including sickle cell disorders, diseases, and conditions, and thalassemia, that include administering DCN-1 modulating compounds, such as DCN-1 inhibitors, including covalent DCN-1 inhibitors.

Description

METHODS AND PREPARATIONS FOR MODULATING DEFECTIVE IN CULLIN NEDDYLATION 1 (DCN1)CROSS-REFERENCE TO RELATED APPLICATIONSThis application claims the benefit of United States Provisional Patent Application Nos. 63 / 661 ,514, filed June 18, 2024; 63 / 618,803, filed January 8, 2024; and 63 / 547,284, filed November 3, 2023; the entire contents each of which are hereby incorporated by reference.TECHNICAL FIELD

[0001] In aspects, this specification describes technologies relating to methods and preparations used, inter alia, to identify compounds useful in the treatment of hemoglobin-related disorders including sickle cell disorders, diseases, and conditions, and thalassemia.BACKGROUND

[0002] Hemoglobinopathies are diseases that affect hemoglobin that include sickle cell disease and thalassemia. Sickle cell disease or disorder is a group of inherited red blood cell disorders that affect hemoglobin and can block blood flow to the body. Specifically, a defective beta hemoglobin chain in sickle cell patients twists and changes the shape of each red blood cell from a doughnut-like shape into a “sickled” or croissant shape that can clog small blood vessels and prevent the delivery of oxygen around the body. Sickle-cell disease is characterized by various acute and chronic complications, which are associated with significant morbidity and mortality in an afflicted subject. Thalassemia is also an inherited red blood cell disorder that is caused by a defect in the beta-globin gene, controlling the production of the beta-globin chains of hemoglobin. Accordingly, a patient suffering from thalassemia cannot make enough normal hemoglobin and thus has relatively fewer red blood cells and lower blood oxygen levels than people who do not suffer from the disease. Thalassemia patients may not make enough of either or both of the alpha or beta proteins in hemoglobin.

[0003] Neural precursor cell expressed developmentally down-regulated protein 8 (NEDD8) is a ubiquitin-like protein (UBL) that is post-translationally appended to eukaryotic proteins in a process termed neddylation. Defective in Cullin Neddylation 1 (DCN1) is part of a dynamic signaling system that regulates ligation of both NEDD8 and ubiquitin (UB), which are among more than a dozen human UBLs that dynamically post-translationally modify and regulate the functions of thousands of different eukaryotic proteins. Although the FDA has approved drugs that target the ubiquitin-proteasome system (UPS), bortezomib and carfilzomi b completely block proteasome activity. There are no currently available therapeutic agents that specifically target components of the UBL system, including modulation of the activity of DCN1 .

[0004] Given the above, methods useful for identifying and preparing compounds for the treatment of hemoglobin-related disorders including sickle cell disorders, diseases, and conditions, and thalassemia are needed.SUMMARY

[0005] The present disclosure addresses the drawbacks identified above by, inter alia, providing improved methods and preparations useful for identifying modulators of DCN1 . It should be appreciated that the present disclosure also provides new therapeutic uses of compounds that bind DCN1.

[0006] In aspects, the disclosure provides a method for identifying a candidate compound, comprising:(a) obtaining a test compound having the ability to covalently bind to Defective in Cullin Neddylation 1 (DCN1) or is suspected to have the ability to bind to DCN1 ;(b) contacting the test compound with DCN1;(c) assaying for modulation of DCN1 and / or a biological target associated with DCN1; and(d) classifying the test compound as a candidate compound if modulation of the DCN1 and / or a biological target associated with DCN1 is detected.

[0007] In aspects, the disclosure provides a method for identifying a candidate compound, comprising:(a) obtaining a test compound having the ability to covalently bind to Defective in Cullin Neddylation 1 (DCN1) or is suspected to have the ability to bind to DCN1 ;(b) contacting the test compound with DCN1;(c) assaying for changes in levels of fetal hemoglobin (HbF) protein and / or function; and(d) classifying the test compound as a candidate compound if the change in levels of fetal hemoglobin (HbF) protein and / or function is detected.

[0008] In aspects, the disclosure provides a method for making a therapeutic composition, comprising:(a) identifying a candidate compound by:(i) obtaining a test compound having the ability to covalently bind to Defective in Cullin Neddylation 1 (DCN1) or is suspected to have the ability to bind to DCN1 ;(ii) contacting the test compound with DCN1 ;(iii) assaying for modulation of DCN1 and / or a biological target associated with DCN1 ; and(iv) classifying the test compound as a candidate compound if modulation of DCN1 and / or the biological target associated with DCN1 is detected; and(b) formulating the candidate compound for use in a therapy.

[0009] In aspects, the disclosure provides a method for making a therapeutic composition, comprising:(a) identifying a candidate compound by:(i) obtaining a test compound having the ability to covalently bind to Defective in Cullin Neddylation 1 (DCN1) or is suspected to have the ability to bind to DCN1 ;(ii) contacting the test compound with DCN1 ;(iii) assaying for changes in levels of fetal hemoglobin (HbF) protein and / or function; and(iv) classifying the test compound as a candidate compound if the change in levels of fetal hemoglobin (HbF) protein and / or function is detected; and(b) formulating the candidate compound for use in a therapy.

[0010] In aspects, the disclosure provides method of preparing a solution comprising one or more Defective in Cullin Neddylation 1 (DCN1) covalent adducts useful for analyzing modulation of DCN1 and / or a biological target associated with DCN1 , comprising:(a) obtaining a solution comprising DCN1 ;(b) contacting the DCN1 of the solution of (a) with a test compound having the ability to covalently bind to DCN1 or is suspected to have the ability to bind to DCN1 to form one or more DCN1 covalent adducts,(c) analyzing modulation of DCN1 and / or a biological target associated produced in the solution of (b).

[0011] In aspects, the disclosure provides a method for making a therapeutic composition, comprising:(a) contacting a test compound having the ability to covalently bind to Defective in Cullin Neddylation 1 (DCN1) or is suspected to have the ability to bind to DCN1 with DCN1 , and quantifying modulation of DCN1 and / or a biological target associated with DCN1 by the test compound;(b) selecting the test compound that modulates DCN1 and / or a biological target associated with DCN1 ;(c) classifying the test compound of (b) as a candidate compound; and(d) formulating the candidate compound for administration to a subject, thereby making the therapeutic composition.

[0012] In aspects, the disclosure provides a method for making a therapeutic composition, comprising:(a) contacting a test compound having the ability to covalently bind to Defective in Cullin Neddylation 1 (DCN1) or is suspected to have the ability to bind to DCN1 with DCN1 , and quantifying change in levels of fetal hemoglobin (HbF) protein and / or function by the test compound;(b) selecting the test compound that changes levels of fetal hemoglobin (HbF) protein and / or function;(c) classifying the test compound of (b) as a candidate compound; and(d) formulating the candidate compound for administration to a subject, thereby making the therapeutic composition.

[0013] In aspects, the disclosure provides a method of preparing a non-natural cell-based preparation, comprising:(a) combining Defective in Cullin Neddylation 1 (DCN1), an electrophile, and cells (e.g., CD34+cells) to form one or more DCN1 covalent adducts, and(b) measuring an increase in fetal hemoglobin (HbF) induction above baseline, the increase indicating the formation of the preparation.

[0014] In aspects, the disclosure provides method of preparing non-natural biochemical preparation of Defective in Cullin Neddylation 1 (DCN1) covalent adducts, comprising:(a) combining DCN1 and an electrophile (e.g. via biochemical assay) to form a non-natural biochemical preparation comprising DCN1 covalent adducts;(b) measuring a percent of DCN1 covalent adducts in the preparation of (a) above a baseline;(c) comparing the measurement from (b) and with a reference measurement obtained by combining DCN1 and an electrophile (e.g. via a cell based (e.g. CD34+ cells) assay) to form a non- natural biochemical preparation comprising DCN1 covalent adducts and measuring an increase in fetal hemoglobin (HbF) induction in the preparation above a baseline; and(d) obtaining a positive correlation among a percent of DCN1 covalent adduct formation and an increase in fetal hemoglobin (HbF) induction above baseline.

[0015] In aspects, the disclosure provides a non-natural cell-based preparation, comprising:(a) increased levels of Defective in Cullin Neddylation (DCN1) covalent adducts, above baseline (e.g. baseline adduct levels), wherein the adducts derive from DCN1 and an electrophile, and(b) increased levels of induction of fetal hemoglobin (HbF) above baseline (e.g. baseline induction levels).

[0016] In aspects, the disclosure provides a pharmaceutical composition comprising a candidate compound identified using the methods of the disclosure and a pharmaceutically acceptable carrier.

[0017] In aspects, the disclosure provides a pharmaceutical composition comprising a solution prepared by the methods of the disclosure and a pharmaceutically acceptable carrier.

[0018] In aspects, the disclosure provides a method of treating a hemoglobin-related disorder, the method comprising administering a candidate compound identified using the methods of the disclosure to a subject afflicted with the hemoglobin-related disorder and / or is at risk of developing the hemoglobin-related disorder.

[0019] In aspects, the disclosure provides a method of treating a hemoglobin-related disorder, the method comprising administering a solution prepared by the methods of the disclosure to a subject afflicted with the hemoglobin-related disorder and / or is at risk of developing the hemoglobin-related disorder.

[0020] In aspects, the disclosure provides a method of treating a hemoglobin-related disorder, the method comprising administering a pharmaceutical composition of the disclosure to a subject afflicted with the hemoglobin-related disorder and / or is at risk of developing the hemoglobin-related disorder.

[0021] In aspects, the disclosure provides a method of treating a sickle cell disorder, disease, or condition, the method comprising administering a candidate compound identified using the methods of the disclosure to a subject afflicted with the sickle cell disorder, disease, or condition and / or at risk of developing the sickle cell disorder, disease, or condition.

[0022] In aspects, the disclosure provides a method of treating a sickle cell disorder, disease, or condition, the method comprising administering a solution prepared by the methods of the disclosure to a subject afflicted with the sickle cell disorder, disease, or condition and / or at risk of developing the sickle cell disorder, disease, or condition.

[0023] In aspects, the disclosure provides a method of treating a sickle cell disorder, disease, or condition, the method comprising administering a pharmaceutical composition of the disclosure to a subject afflicted with the sickle cell disorder, disease, or condition and / or at risk of developing the sickle cell disorder, disease, or condition.

[0024] In embodiments, the sickle cell disorder, disease, and / or condition is a sickle cell anemia.

[0025] In aspects, the disclosure provides a method of treating thalassemia, the method comprising administering a candidate compound identified using the methods of the disclosure to a subject afflicted with thalassemia and / or is at risk of developing thalassemia.

[0026] In aspects, the disclosure provides a method of treating thalassemia, the method comprising administering a solution prepared by the methods of the disclosure to a subject afflicted with thalassemia and / or is at risk of developing thalassemia.

[0027] In aspects, the disclosure provides a method of treating thalassemia, the method comprising administering a pharmaceutical composition of the disclosure to a subject afflicted with thalassemia and / or is at risk of developing thalassemia.

[0028] In aspects, the disclosure provides a method of inducing fetal hemoglobin (HbF) in a subject, the method comprising administering to a subject a compound that binds Defective in Cullin Neddylation 1 (DCN1) in an amount sufficient to induce HbF in the subject.

[0029] In aspects, the disclosure provides a method of treating a hemoglobin-related disorder, the method comprising administering to a subject a compound that binds Defective in Cullin Neddylation 1 (DCN1) in an amount sufficient to treat the hemoglobin-related disorder.

[0030] In aspects, the disclosure provides a method of treating sickle cell disease, the method comprising administering to a subject a compound that binds Defective in Cullin Neddylation 1 (DCN1) in an amount sufficient to treat sickle cell disease.

[0031] In aspects, the disclosure provides a method of treating thalassemia, the method comprising administering to a subject a compound that binds Defective in Cullin Neddylation 1 (DCN1) in an amount sufficient to treat thalassemia.

[0032] In some embodiments of the aspect provided herein a compound that binds Defective in Cullin Neddylation 1 (DCN1) covalently binds Defective in Cullin Neddylation 1 (DCN1).

[0033] In aspects, the disclosure provides a pharmaceutical composition comprising a compound that binds Defective in Cullin Neddylation 1 (DCN1) in an amount sufficient to treat a hemoglobin-related disorder, and a pharmaceutically acceptable carrier.

[0034] In aspects, the disclosure provides a pharmaceutical composition comprising a compound that binds Defective in Cullin Neddylation 1 (DCN1) in an amount sufficient to treat sickle cell disease, and a pharmaceutically acceptable carrier.

[0035] In aspects, the disclosure provides a pharmaceutical composition comprising a compound that binds Defective in Cullin Neddylation 1 (DCN1) in an amount sufficient to treat thalassemia, and a pharmaceutically acceptable carrier.

[0036] In aspects, the disclosure provides a method of inducing fetal hemoglobin (HbF) in a subject, the method comprising modifying a reversible inhibitor of Defective in Cullin Neddylation 1 (DCN1) with a warhead, thereby generating a covalent inhibitor of DCN1 , and administering the covalent inhibitor of DCN1 to a subject in an amount sufficient to induce HbF in the subject.

[0037] Various embodiments of methods within the scope of the appended claims each have several aspects, no single one of which is solely responsible for the desirable attributes described herein. Without limiting the scope of the appended claims, some prominent features are described herein. After considering this discussion, and particularly after reading the section entitled “Detailed Description” one will understand how the features of various embodiments are used.BRIEF DESCRIPTION OF FIGURES

[0038] FIG. 1 A, 1 B, and 1C depict graphs showing the induction of fetal hemoglobin in humanized mice by treatment with hydroxyurea. FIG. 1A depicts a graph showing fetal hemoglobin protein (HbF) expression. FIG. 1B depicts a graph showing HBG1 gene expression. FIG. 1C depicts a graph showing the ratio of fetal to adult hemoglobin gene (HBB) expression.

[0039] FIG. 2 depicts a graph showing the expression of fetal hemoglobin (HbF) in CD34+ humanized mouse models.

[0040] FIG. 3 shows genome tracks of transcriptional activity of in the fetal hemoglobin locus after exposure of CD34 cells to DI-1548 (L2) as evaluated by CUT AND RUN.

[0041] FIG. 4 shows the genome tracks of the accessibility of chromatin at the fetal hemoglobin locus in CD34 cells after exposure to DI-1548 (L2), as evaluated by ATAC-sequencing.

[0042] FIG. 5 shows that DI-1548 (L2) can induce fetal hemoglobin associated genes in CD34 cells.DETAILED DESCRIPTION

[0043] Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings. In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the present disclosure. However, it will be apparent to one of ordinary skill in the art that the present disclosure may be practiced without these specific details. In other instances, well-known methods have not been described in detail so as not to unnecessarily obscure aspects of the embodiments.Methods for identifying compounds

[0044] In aspects, the disclosure provides methods useful for, inter alia, identifying compounds capable of modulating DCN1 and / or a biological target associated with DCN1.

[0045] In aspects, the disclosure provides a method for identifying a candidate compound, comprising:(a) obtaining a test compound having the ability to covalently bind to Defective in Cullin Neddylation 1 (DCN1) or is suspected to have the ability to bind to DCN1 ;(b) contacting the test compound with DCN1;(c) assaying for modulation of DCN1 and / or a biological target associated with DCN1 ; and(d) classifying the test compound as a candidate compound if modulation of the DCN1 and / or a biological target associated with DCN1 is detected.

[0046] In aspects, the disclosure provides a method for identifying a candidate compound, comprising:(a) obtaining a test compound having the ability to covalently bind to Defective in Cullin Neddylation 1 (DCN1) or is suspected to have the ability to bind to DCN1 ;(b) contacting the test compound with DCN1;(c) assaying for changes in levels of fetal hemoglobin (HbF) protein and / or function; and(d) classifying the test compound as a candidate compound if the changes in levels of fetal hemoglobin (HbF) protein and / or function are detected.

[0047] In aspects, the disclosure provides a method for making a therapeutic composition, comprising:(a) identifying a candidate compound by:(i) obtaining a test compound having the ability to covalently bind to Defective in Cullin Neddylation 1 (DCN1) or is suspected to have the ability to bind to DCN1 ;(ii) contacting the test compound with DCN1 ;(iii) assaying for modulation of DCN1 and / or a biological target associated with DCN1 ; and(iv) classifying the test compound as a candidate compound if modulation of DCN1 and / or the biological target associated with DCN1 is detected; and(b) formulating the candidate compound for use in a therapy.

[0048] In aspects, the disclosure provides a method for making a therapeutic composition, comprising:(a) identifying a candidate compound by:(i) obtaining a test compound having the ability to covalently bind to Defective in Cullin Neddylation 1 (DCN1) or is suspected to have the ability to bind to DCN1 ;(ii) contacting the test compound with DCN1 ;(iii) assaying for changes in levels of fetal hemoglobin (HbF) protein and / or function; and(iv) classifying the test compound as a candidate compound if changes in levels of fetal hemoglobin (HbF) protein and / or function are detected; and(b) formulating the candidate compound for use in a therapy.

[0049] In aspects, the disclosure provides method of preparing a solution comprising one or more Defective in Cullin Neddylation 1 (DCN1) covalent adducts useful for analyzing modulation of DCN1 and / or a biological target associated with DCN1 , comprising:(a) obtaining a solution comprising DCN1 ;(b) contacting the DCN1 of the solution of (a) with a test compound having the ability to covalently bind to DCN1 or is suspected to have the ability to bind to DCN1 to form one or more DCN1 covalent adducts,(c) analyzing modulation of DCN1 and / or a biological target associated produced in the solution of (b).

[0050] In aspects, the disclosure provides a method for making a therapeutic composition, comprising:(a) contacting a test compound having the ability to covalently bind to Defective in Cullin Neddylation 1 (DCN1) or is suspected to have the ability to bind to DCN1 with DCN1 , and quantifying modulation of DCN1 and / or a biological target associated with DCN1 by the test compound;(b) selecting the test compound that modulates DCN1 and / or a biological target associated with DCN1 ;(c) classifying the test compound of (b) as a candidate compound; and(d) formulating the candidate compound for administration to a subject, thereby making the therapeutic composition.

[0051] In aspects, the disclosure provides a method for making a therapeutic composition, comprising:(a) contacting a test compound having the ability to covalently bind to Defective in Cullin Neddylation 1 (DCN1) or is suspected to have the ability to bind to DCN1 with DCN1 , and quantifying changes in levels of fetal hemoglobin (HbF) protein and / or function by the test compound;(b) selecting the test compound that changes levels of fetal hemoglobin (HbF) protein and / or function;(c) classifying the test compound of (b) as a candidate compound; and(d) formulating the candidate compound for administration to a subject, thereby making the therapeutic composition.

[0052] The function of DCN1 (also known as DCNL1 , DCUN1D1 , or SCCRO (squamous cell carcinoma-related oncogene)) includes binding to the acetylated N-terminus of UBC12 (an E2 enzyme for the UBL NEDD8) and the “Cullin (or CUL)” family of proteins to act as a co-E3 promoting NEDD8 modification (neddylation) of the CULs. In a non-limiting example, inhibition of the DCN1-UBC12 interaction can regulate CUL activity without completely blocking neddylation and provide efficacious compounds with less severe off-target effects and toxicity relative to existing drugs that target the UPS system. It is now shown herein that compounds that bind DCN-1 can induce fetal hemoglobin. In some embodiments, the compounds that bind DCN-1 are covalent binders.

[0053] In embodiments, modulation of DCN1 and / or a biological target associated with DCN1 comprises inhibition of a DCN1-E2 enzyme interaction. In embodiments, the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0054] In embodiments, the modulation of DCN1 and / or a biological target associated with DCN1 comprises inhibition of an E2 enzyme binding site in DCN1. In embodiments, the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0055] In embodiments, the modulation of DCN1 and / or a biological target associated with DCN1 comprises detecting or quantifying the formation of one or more DCN1 covalent adducts.

[0056] In embodiments, the modulation of DCN1 and / or a biological target associated with DCN1 is determined by detecting or quantifying inhibition of the DCN1-E2 enzyme interaction.

[0057] In embodiments, modulation of DCN1 and / or a biological target associated with DCN1 is determined by detecting or quantifying inhibition of the E2 enzyme binding site in DCN1.

[0058] In embodiments, detecting and / or quantifying inhibition of the DCN1-E2 enzyme interaction and / or inhibition of the E2 enzyme binding site in DCN1 and / or the formation of one or more DCN1 covalent adducts comprises assaying competitive binding of DCN1 (e.g. using a fluorescent or radioactive assay, e.g. fluorescence energy transfer (FRET), e.g., DCN1 time-resolved fluorescence energy transfer (TR-FRET)), and / or amount of DCN1 covalent adducts formed (e.g. using mass spectrometry, e.g., mass spectrometry to determine percent of DCN covalent adducts formed).

[0059] In embodiments, the E2 enzyme binding site in DCN1 is a cysteine (e.g. Cys115). In embodiments, the E2 enzyme is selected from UBC12 and UBE2F. In embodiments, the E2 enzyme is UBC12.

[0060] In embodiments, the modulation of DCN1 and / or a biological target associated with DCN1 comprises inhibition of cullin neddylation. In embodiments, the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0061] In embodiments, the modulation of DCN1 and / or a biological target associated with DCN1 is determined by detecting or quantifying inhibition of cullin neddylation.

[0062] In embodiments, detecting and / or quantifying inhibition of cullin neddylation comprises assaying changes in the amount of cullin neddylation from a baseline measurement (e.g. an immunoassay including but not limited to enzyme-linked immunosorbent assay (ELISA), western blot, or ALPHALisa assay) and / or formation of an E2 enzyme-NEDD8 complex (e.g. high-throughput screen capable of measuring the formation of a E2 enzyme-NEDD8 enzyme complex). In embodiments, the E2 enzyme is selected from UBC12 and UBE2F. In embodiments, the E2 enzyme is UBC12. For a non-limiting example of an ALPHALisa assay, see Nat. Methods 5:an8-an9 (2008), which is incorporated by reference herein in its entirety.

[0063] In embodiments, inhibition of cullin neddylation comprises inhibition of the cullin-NEDD8 interaction. In embodiments, the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound. In embodiments, the cullin is selected from cullin-1 (CUL1), cullin-2 (CUL2), cullin-3 (CUL3),cullin-4A (CUL4A), cullin-4B (CUL4B), cullin-5 (CUL5), and cullin-7 (CUL7). In embodiments, the cullin is cullin-3 (CUL3). In embodiments, inhibition of cullin neddylation comprises formation of an unneddylated cullin and / or inactive cullin. In embodiments, inhibition of cullin neddylation comprises formation of an unneddylated cullin-3 (CUL3) and / or inactive cullin-3 (CUL3).

[0064] In embodiments, the method further comprising contacting DCN1 and / or the test compound having the ability to covalently bind to Defective in Cullin Neddylation 1 (DCN1) or is suspected to have the ability to bind to DCN1 with one or more cells.

[0065] In embodiments, the one or more cells comprise or consist of erythroid cells. In embodiments, the erythroid cells are CD34+erythroid cells. In embodiments, the erythroid cells are primary CD34+erythroid cells.

[0066] In embodiments, modulation of DCN1 and / or a biological target associated with DCN1 is determined by detecting and / or quantifying fetal hemoglobin (HbF) protein, nucleic acids, and / or function. In embodiments, the increase in HbF induction is caused at least in part by modulation of DCN1 and / or a biological target associated with DCN1.

[0067] In embodiments, detecting and / or quantifying fetal hemoglobin (HbF) comprises assaying changes in levels of HbF protein and / or function (e.g. measuring the amount of HbF induction from a baseline measurement (e.g. determining HbF transcript levels by measuring y-globin gene (HBG) mRNA using Nanostring analysis); and / or amount of HbF protein formed (e.g. using high-throughput liquid chromatography (HPLC) to determine percent of HbF protein formed in cell lysates).

[0068] In embodiments, modulation of DCN1 and / or a biological target associated with DCN1 is determined by detecting and / or quantifying fetal hemoglobin (HbF) protein, nucleic acids, and / or function, and adult hemoglobin (HbA) protein, nucleic acids, and / or function. In embodiments, the increase in HbF induction and decrease in HbA is caused at least in part by modulation of DCN1 and / or a biological target associated with DCN1.

[0069] In embodiments, detecting and / or quantifying fetal hemoglobin (HbF) and adult hemoglobin (HbA) comprises assaying changes in levels of HbF and HbA proteins and / or function (e.g. measuring the amount of HbF and HbA induction from a baseline measurement (e.g. determining HbF transcript levels by measuring y-globin gene (HBG) mRNA, and determining HbA transcript levels by measuring beta-globin gene (HBB), using Nanostring analysi ); and / or amount of HbF and HbA protein formed (e.g. using high-throughput liquid chromatography (HPLC) to determine percent of HbF and HbA protein formed in cell lysates).

[0070] In embodiments, detecting and / or quantifying fetal hemoglobin (HbF) comprises measuring reactivation of fetal hemoglobin (HbF).

[0071] In embodiments, fetal hemoglobin (HbF) can be detected and / or quantified and / or measured by assaying one or more of:• expression of HBG1 and / or HBG2; or• presence of subunits of HbF (e.g. one or more a subunits and / or one or more y subunits of HbF).

[0072] In embodiments, the modulation of DCN1 and / or a biological target associated with DCN1 comprises detecting or quantifying the formation of one or more DCN1 covalent adducts and by detecting and / or quantifying fetal hemoglobin (HbF) protein, nucleic acids, and / or function.

[0073] In embodiments, the modulation of DCN1 and / or a biological target associated with DCN1 comprises inhibition of DCN1 and induction of fetal hemoglobin (HbF) protein, nucleic acids, and / or function. In embodiments, inhibition of DCN1 is detected or quantified by the formation of one or more DCN1 covalent adducts. In embodiments, the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 in the absence of the test compound.

[0074] In embodiments, the modulation of DCN1 and / or a biological target associated with DCN1 (e.g. inhibition of DCN1) results in an increase in total induction and / or levels of fetal hemoglobin (HbF) protein, nucleic acids, and / or function compared to baseline. In embodiments, total induction and / or levels of fetal hemoglobin (HbF) is increased by about 1 -fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, 1000- fold, or greater than about 1000-fold compared to baseline (e.g. total induction and / or levels of HbF in the absence of the test compound and / or electrophile).

[0075] In embodiments, the modulation of DCN1 and / or a biological target associated with DCN1 (e.g. inhibition of DCN1) results in an increase in the levels of fetal hemoglobin (HbF) mRNA and fetal hemoglobin (HbF) protein compared to baseline. In embodiments, levels of fetal hemoglobin (HbF) mRNA and fetal hemoglobin (HbF) protein is increased by about 1-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold,500-fold, 1000-fold, or greater than about 1000-fold compared to baseline (e.g. levels of fetal hemoglobin (HbF) mRNA and fetal hemoglobin (HbF) protein in the absence of the test compound and / or electrophile).

[0076] In embodiments, the modulation of DCN1 and / or a biological target associated with DCN1 (e.g. inhibition of DCN1) comprises reversal of the globin switch (e.g. fetal-to-adult hemoglobin switch which results in a switch from gamma-globin to beta-globin). In embodiments, reversal of the globin switch comprises an increase in the amount and / or levels of gamma-globin to beta-globin compared to baseline. In embodiments, the amount and / or levels of gamma-globin is increased by about 1-fold, 5-fold, 10-fold, 20- fold, 50-fold, 100-fold, 500-fold, 1000-fold, or greater than about 1000-fold compared to baseline (e.g. amount and / or level of gamma-globin in the absence of the test compound and / or electrophile). In embodiments, the ratio of gamma-globin to beta-globin is increased compared to baseline.

[0077] In embodiments, the test compound is classified as a candidate compound if a percent of DCN1 covalent adduct formation is increased by about 1-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500- fold, 1000-fold, or greater than about 1000-fold compared to baseline (e.g. percent of DCN1 covalent adduct formation in the absence of the test compound) and fetal hemoglobin (HbF) induction is increased by about 1-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or greater than about 1000-fold compared to baseline (e.g. HbF induction in the absence of the test compound).

[0078] In embodiments, modulation of DCN1 and / or a biological target associated with DCN1 is determined by detecting and / or quantifying Nrf2 protein.

[0079] In embodiments, the test compound and / or candidate compound is capable of increasing a level of Nrf2 protein compared to the level of Nrf2 protein in the absence of the test compound and / or candidate compound.

[0080] In embodiments, the test compound and / or the candidate compound is capable of forming a covalent bond with a cysteine of DCN1 (e.g. Cys115).

[0081] In embodiments, the assaying is performed in vitro, ex vivo, or in vivo.

[0082] In embodiments, the test compound and / or candidate compound comprises one or more electrophilic groups.

[0083] In embodiments, the one or more electrophilic groups are selected from nitrile, pentafluorophenyl ester, NHS-ester, acrylamides, cycloalkenyl carboxamides, acyl azides, acyl halides, aldehydes or ketones, alkyl halides, alkyl sulfonates, anhydrides, aryl halides, aziridines, boronates,carboxylic acids, carbodiimides, diazoalkenes, epoxides, haloacetamides, halotriazines, imido esters, isocyanates, isothiocyanates, maleimides, phosphoramidites, silyl halides, sulfonate esters and sulfonyl halides.

[0084] In embodiments, the candidate compound is formulated for use in a therapy. In embodiments, the therapy is suitable for treatment or prevention of a hemoglobin-related disorder. In embodiments, the therapy is suitable for treatment or prevention of a sickle cell disorder, disease, or condition. In embodiments, the sickle cell disorder, disease, or condition is a sickle cell anemia. In embodiments, the therapy is suitable for treatment or prevention of thalassemia.

[0085] It should be appreciated that test compounds and candidate compounds include compounds that are known DCN1 binders. Accordingly, known DCN1 binders can be used according to the methods disclosed herein, including the treatment of hemoglobin-related disorders including sickle cell disorders, diseases, and conditions, and thalassemia.Preparations

[0086] In aspects, the disclosure provides a method of preparing a non-natural cell-based preparation, comprising:(a) combining Defective in Cullin Neddylation 1 (DCN1), an electrophile, and cells (e.g., CD34+cells) to form one or more DCN1 covalent adducts, and(b) measuring an increase in fetal hemoglobin (HbF) induction above baseline, the increase indicating the formation of the preparation.

[0087] In aspects, the disclosure provides a method of preparing non-natural biochemical preparation of Defective in Cullin Neddylation 1 (DCN1) covalent adducts, comprising:(a) combining DCN1 and an electrophile (e.g. via biochemical assay) to form a non-natural biochemical preparation comprising DCN1 covalent adducts;(b) measuring a percent of DCN1 covalent adducts in the preparation of (a) above a baseline;(c) comparing the measurement from (b) and with a reference measurement obtained by combining DCN1 and an electrophile (e.g. via a cell based (e.g. CD34+ cells) assay) to form a non- natural biochemical preparation comprising DCN1 covalent adducts and measuring an increase in fetal hemoglobin (HbF) induction in the preparation above a baseline; and(d) obtaining a positive correlation among a percent of DCN1 covalent adduct formation and an increase in fetal hemoglobin (HbF) induction above baseline.

[0088] In embodiments, the increase in HbF induction is caused at least in part by modulation of DCN1 and / or a biological target associated with DCN1.

[0089] In embodiments, measuring an increase in fetal hemoglobin (HbF) induction above baseline comprises detecting and / or quantifying fetal hemoglobin (HbF) protein, nucleic acids, and / or function.

[0090] In embodiments, the electrophile is capable of forming a covalent bond with a cysteine of DCN1 (e.g. Cys115).

[0091] In embodiments, the electrophile is capable of increasing a level of Nrf2 protein compared to the level of Nrf2 protein in the absence of the electrophile.

[0092] In embodiments, the electrophile is a test compound comprising one or more electrophilic groups. In embodiments, wherein the one or more electrophilic groups are selected from nitrile, pentafluorphenyl ester, NHS-ester, acrylamides, cycloalkenyl carboxamides, acyl azides, acyl halides, aldehydes or ketones, alkyl halides, alkyl sulfonates, anhydrides, aryl halides, aziridines, bonorates, carboxylic acids, carbodiimides, diazoalkenes, epoxides, haloacetamides, halotriazines, imido esters, isocyanates, isothiocyanates, maleimides, phosphoramidites, silyl halides, sulfonate esters and sulfonyl halides.

[0093] In embodiments, the method further comprises classifying the test compound as a candidate compound if fetal hemoglobin (HbF) induction is increased by about 1-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or greater than about 1000-fold compared to baseline (e.g. HbF induction in the absence of the test compound).

[0094] In embodiments, the candidate compound is formulated for use in a therapy. In some embodiments, the therapy is suitable for treatment or prevention of a hemoglobin-related disorder. Nonlimiting examples of hemoglobin-related disorders include sickle cell disorders, diseases, and conditions, and thalassemia. In some embodiments, the therapy is suitable for treatment or prevention of a sickle cell disorder, disease, or condition. In some embodiments, the sickle cell disorder, disease, or condition is a sickle cell anemia. In embodiments, the therapy is suitable for treatment or prevention of thalassemia.

[0095] In aspects, the disclosure provides a method of a non-natural biochemical preparation of Defective in Cullin Neddylation 1 (DCN1) covalent adducts, comprising:(a) combining DCN1 and an electrophile (e.g. via biochemical assay) to form a non-natural biochemical preparation of DCN1 covalent adducts;(b) measuring a percent of DCN1 covalent adducts in the preparation of (a) above baseline;(c) combining DCN1 and an electrophile (e.g. via a cell based (e.g. CD34+cells) assay) to form a non-natural cell-based preparation comprising DCN1 covalent adducts;(d) measuring an increase in fetal hemoglobin (HbF) induction in the preparation of (c) above baseline;(e) comparing the measurements from (b) and measurements from (d); and(f) obtain a positive correlation among a percent of DCN1 covalent adduct formation and an increase in fetal hemoglobin (HbF) induction above baseline.

[0096] In embodiments, the increase in HbF induction is caused at least in part by modulation of DCN1 and / or a biological target associated with DCN1.

[0097] In embodiments, measuring a percent of DCN1 covalent adducts in the preparation of (a) above baseline comprises detecting and / or quantifying the formation of one or more DCN1 covalent adducts.

[0098] In embodiments, measuring an increase in fetal hemoglobin (HbF) induction in the preparation of (c) above baseline comprises detecting and / or quantifying fetal hemoglobin (HbF) protein, nucleic acids, and / or function.

[0099] In embodiments, the modulation of DCN1 and / or a biological target associated with DCN1 comprises detecting or quantifying the formation of one or more DCN1 covalent adducts and by detecting and / or quantifying fetal hemoglobin (HbF) protein, nucleic acids, and / or function.

[0100] In embodiments, the modulation of DCN1 and / or a biological target associated with DCN1 comprises inhibition of DCN1 and induction of fetal hemoglobin (HbF) protein, nucleic acids, and / or function. In embodiments, inhibition of DCN1 is detected or quantified by the formation of one or more DCN1 covalent adducts. In embodiments, inhibition of DCN1 is detected or quantified by the formation of one or more DCN1 covalent adducts, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 in the absence of the electrophile.

[0101] In embodiments, the electrophile is capable of increasing level of Nrf2 protein compared to the level of Nrf2 protein in the absence of the electrophile.

[0102] In embodiments, the electrophile is capable of forming a covalent bond with a cysteine of DCN1 (e.g. Cys115).

[0103] In embodiments, the electrophile is a test compound comprising one or more electrophilic groups. In embodiments, the one or more electrophilic groups are selected from nitrile, pentafluorophenyl ester, NHS-ester, acrylamides, cycloalkenyl carboxamides, acyl azides, acyl halides, aldehydes or ketones, alkyl halides, alkyl sulfonates, anhydrides, aryl halides, aziridines, boronates, carboxylic acids, carbodiimides, diazoalkenes, epoxides, haloacetamides, halotriazines, imido esters, isocyanates, isothiocyanates, maleimides, phosphoramidites, silyl halides, sulfonate esters and sulfonyl halides.

[0104] In embodiments, the test compound is classified as a candidate compound if the percent of DCN1 covalent adduct formation is increased by about 1-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500- fold, 1000-fold, or greater than about 1000-fold compared to baseline (e.g. percent of DCN1 covalent adduct formation in the absence of the test compound) and fetal hemoglobin (HbF) induction is increased by about 1-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or greater than about 1000-fold compared to baseline (e.g. HbF induction in the absence of the test compound).

[0105] In embodiments, the candidate compound is formulated for use in a therapy. In some embodiments, the therapy is suitable for treatment or prevention of a hemoglobin-related disorder. Nonlimiting examples of hemoglobin-related disorders include sickle cell disorders, diseases, and conditions, and thalassemia. In some embodiments, the therapy is suitable for treatment or prevention of a sickle cell disorder, disease, or condition. In some embodiments, the sickle cell disorder, disease, or condition is a sickle cell anemia. In embodiments, the therapy is suitable for treatment or prevention of thalassemia.

[0106] In aspects, the disclosure provides a non-natural cell-based preparation, comprising:(a) increased levels of Defective in Cullin Neddylation (DCN1) covalent adducts, above baseline (e.g. baseline adduct levels), wherein the adducts derive from DCN1 and an electrophile, and(b) increased levels of induction of fetal hemoglobin (HbF) above baseline (e.g. baseline induction levels).

[0107] Thus, in one aspect, the test compounds and candidate compounds disclosed herein, which includes the compounds described in the “Exemplary test compounds and candidate compounds” below canbe used for the treatment of sickle cell disorder, disease, or condition. In some embodiments, the treatment is suitable for treatment or prevention of thalassemia.

[0108] It should be appreciated that, in addition to DCN1 , modulation, e.g., up- or down-regulation, of other members of the Neddylation pathway can alter the levels of fetal hemoglobin or otherwise treat a sickle cell disorder, disease, or condition. In some embodiments, the sickle cell disorder, disease, or condition is a sickle cell anemia. In some embodiments, the treatment is suitable for treatment or prevention of thalassemia.

[0109] In some embodiments, the member of the Neddylation pathway is selected from DCN2, DCN3, DCN4, DCN5, NEDD8, UBC12, UBE2M, UBE2F, NAE or a component thereof, NAE1 , APPBP1, UBA3, and one or more cull i n, optionally wherein the one or more cullin is selected from one or more of cu Hi n- 1 (CUL1), cullin-2 (CUL2), cullin-3 (CUL3), cullin-4A (CUL4A), cullin-4B (CUL4B), cullin-5 (CUL5), and cullin- 7 (CUL7).Compositions and methods of treatment

[0110] In aspects, the disclosure provides a pharmaceutical composition comprising a candidate compound identified using the methods of the disclosure and a pharmaceutically acceptable carrier.

[0111] In aspects, the disclosure provides a pharmaceutical composition comprising a solution prepared by the methods of the disclosure and a pharmaceutically acceptable carrier.

[0112] In aspects, the disclosure provides a method of treating a hemoglobin-related disorder. In embodiments, the method comprises administering a candidate compound identified using any of methods of the disclosure to a subject afflicted with the hemoglobin-related disorder and / or at risk of developing the hemoglobin-related disorder. In embodiments, the method comprises administering a solution prepared by any of the methods of the disclosure to a subject afflicted with the hemoglobin-related disorder and / or at risk of developing the hemoglobin-related disorder. Non-limiting examples of hemoglobin-related disorders include sickle cell disorders, diseases, and conditions, and thalassemia. In embodiments, the sickle cell disorders, diseases, and / or condition is a sickle cell anemia.

[0113] In aspects, the disclosure provides a method of treating a sickle cell disorder, disease, or condition, the method comprising administering a candidate compound identified using any of the methodsof the disclosure to a subject afflicted with the sickle cell disorder, disease, or condition and / or at risk of developing the sickle cell disorder, disease, or condition.

[0114] In aspects, the disclosure provides a method of treating a sickle cell disorder, disease, or condition, the method comprising administering a solution prepared by any of the methods of the disclosure to a subject afflicted with the sickle cell disorder, disease, or condition and / or at risk of developing the sickle cell disorder, disease, or condition.

[0115] In embodiments, the sickle cell disorder, disease, or condition is a sickle cell anemia.

[0116] In aspects, the disclosure provides a method of treating thalassemia, the method comprising administering a candidate compound identified using any of the methods of the disclosure to a subject afflicted with thalassemia and / or is at risk of developing thalassemia.

[0117] In aspects, the disclosure provides a method of treating thalassemia, the method comprising administering a solution prepared by any of the methods of the disclosure to a subject afflicted with thalassemia and / or is at risk of developing thalassemia.

[0118] As used herein, the terms “subject” and “patient” are used interchangeably and refer to organisms to be treated by the methods of the present invention. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and, most preferably, includes humans

[0119] As used herein, the term “inhibitor” is defined as a compound that binds to and / or inhibits the target with measurable affinity. In some embodiments, inhibition in the presence of the inhibitor is observed in a dose-dependent manner. In some embodiments, the measured signal (e.g., signaling activity or biological activity) is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% lower than the signal measured with a negative control under comparable conditions.

[0120] As used herein, the term “effective amount” refers to the amount of a compound sufficient to effect beneficial or desired results (e.g., a therapeutic, ameliorative, inhibitory, or preventative result). Aneffective amount can be administered in one or more administrations, applications, or dosages and is not intended to be limited to a particular formulation or administration route.

[0121] As used herein, the term “treating” includes any effect, e.g., lessening, reducing, modulating, ameliorating, or eliminating, that results in the improvement of the condition, disease, disorder, and the like, or ameliorating a symptom thereof. In some embodiments, treatment can be administered after one or more symptoms have developed. In other embodiments, treatment can be administered in the absence of symptoms. For example, treatment can be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors). Treatment can also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.

[0122] As used herein, the term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0123] As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil / water or water / oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see e.g., Martin, Remington’s Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA

[1975] .

[0124] For therapeutic use, salts of the compounds of the present invention are contemplated as being pharmaceutically acceptable. However, salts of acids and bases that are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.

[0125] In addition, when a compound of the invention contains both a basic moiety (such as, but not limited to, a pyridine or imidazole) and an acidic moiety (such as, but not limited to, a carboxylic acid) zwitterions (“inner salts”) may be formed. Such acidic and basic salts used within the scope of the invention are pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts. Such salts of the compounds of the invention may be formed, for example, by reacting a compound of the invention with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.

[0126] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.

[0127] As a general matter, compositions specifying a percentage are by weight unless otherwise specified.Methods of Use

[0128] It has now surprisingly been found that compounds and compositions that can modulate, e.g., inhibit, DCN-1 are useful in treating hemoglobinopathy such as sickle cell disorder or disease, or thalassemia disorder or disease. In some embodiments, modulating DCN-1 is inhibiting or reducing the activity of DCN-1 . Without being limited to a specific mechanism, as shown herein, inhibiting or reducing the activity of DCN-1 results in reduced neddylation and other downstream effects.

[0129] In aspects, the disclosure provides a method of inducing fetal hemoglobin (HbF) in a subject, the method comprising administering to a subject a compound that binds Defective in Cullin Neddylation 1 (DCN1) in an amount sufficient to induce HbF in the subject.

[0130] In aspects, the disclosure provides a method of treating a hemoglobin-related disorder, the method comprising administering to a subject a compound that binds Defective in Cullin Neddylation 1 (DCN1) in an amount sufficient to treat the hemoglobin-related disorder.

[0131] In aspects, the disclosure provides a method of treating sickle cell disease, the method comprising administering to a subject a compound that binds Defective in Cullin Neddylation 1 (DCN1) in an amount sufficient to treat sickle cell disease.

[0132] In aspects, the disclosure provides a method of treating thalassemia, the method comprising administering to a subject a compound that binds Defective in Cullin Neddylation 1 (DCN1) in an amount sufficient to treat thalassemia.

[0133] In some embodiments of the aspect provided herein a compound that can bind Defective in Cullin Neddylation 1 (DCN1) covalently binds Defective in Cullin Neddylation 1 (DCN1).

[0134] In aspects, the disclosure provides a pharmaceutical composition comprising a compound that binds Defective in Cullin Neddylation 1 (DCN1) in an amount sufficient to treat a hemoglobin-related disorder, and a pharmaceutically acceptable carrier.

[0135] In aspects, the disclosure provides a pharmaceutical composition comprising a compound that binds Defective in Cullin Neddylation 1 (DCN1) in an amount sufficient to treat sickle cell disease, and a pharmaceutically acceptable carrier.

[0136] In aspects, the disclosure provides a pharmaceutical composition comprising a compound that binds Defective in Cullin Neddylation 1 (DCN1) in an amount sufficient to treat thalassemia, and a pharmaceutically acceptable carrier.

[0137] In aspects, the disclosure provides a method of inducing fetal hemoglobin (HbF) in a subject, the method comprising modifying a reversible inhibitor of Defective in Cullin Neddylation 1 (DCN1) with a warhead, thereby generating a covalent inhibitor of DCN1 , and administering the covalent inhibitor of DCN1 to a subject in an amount sufficient to induce HbF in the subject.

[0138] In one aspect, the present disclosure provides a method of modulating the activity of DCN- 1 in vivo, comprising contacting DCN-1 with a compound or composition that can modulate DCN-1 , or a pharmaceutically acceptable salt thereof, to thereby treat the hemoglobinopathy. It should be appreciated that DCN-1 and DCN-2 are similar and structure and that compounds or compositions that can modulate DCN-1 can also modulate DCN-2 to thereby treat the hemoglobinopathy.

[0139] It should be appreciated that DCN1 and DCN-1 can be used interchangeably herein and that they both refer to the Defective in Cullin Neddylation 1 protein.

[0140] In one aspect, the disclosure provides methods, compounds and compositions for the treatment of hemoglobinopathies such as sickle cell disorder or disease or thalassemia disorder or disease. In one aspect, as shown herein, the compounds and compositions that can modulate DCN-1 (e.g., reduce the activity of) induce HbF (fetal hemoglobin; expressed by the gamma globin genes HBG1 and HBG2). It should be appreciated that induction of HbF allows for the treatment of hemoglobinopathies such as sickle cell disorder or disease or thalassemia disorder or disease. Thus, in one aspect, the disclosure provides methods for the treatment of sickle cell disease including administering the compounds and compositions that can modulate DCN-1.

[0141] In one aspect, the disclosure provides compounds and compositions for the treatment of hemoglobinopathies such as sickle cell disorder or disease or thalassemia disorder or disease. In some embodiments, compounds and compositions that can modulate DCN-1 can both induce HbF (fetal hemoglobin; expressed by the gamma globin genes HBG1 and HBG2) and reduce HbA (adult hemoglobin; expressed by the beta globin gene HBB), thus inducing production of fetal hemoglobin and reducing the expression of the hemoglobin beta gene. It should be appreciated that induction of HbF and reduction of HbA allows for the treatment of hemoglobinopathies such as sickle cell disorder or disease or thalassemia disorder or disease.

[0142] In some embodiments, a compound that can modulate DCN-1 is an inhibitor of DCN-1. In some embodiments, an inhibitor of DCN-1 can be used to treat hemoglobinopathy such as sickle cell disorder or disease, or thalassemia disorder or disease.

[0143] In some embodiments, a compound that can modulate DCN-1 is an inhibitor of DCN-1. In some embodiments, the inhibitor of DCN-1 is a reversible inhibitor of DCN-1 or a covalent inhibitor of DCN- 1 . In some embodiments, the inhibitor of DCN-1 is a covalent inhibitor of DCN-1 . In some embodiments, a covalent inhibitor of DCN-1 can be used to treat hemoglobinopathy such as sickle cell disorder or disease, or thalassemia disorder or disease. In some embodiments, the covalent inhibitor of DCN-1 is an irreversible covalent inhibitor. In some embodiments, the covalent inhibitor of DCN-1 is a reversible covalent inhibitor.

[0144] It should be appreciated that a reversible inhibitor of DCN-1 can be modified with a warhead, thereby providing a covalent inhibitor of DCN-1. Thus, in some embodiments, the disclosure provides a method of inducing fetal hemoglobin (HbF) in a subject, the method comprising modifying a reversible inhibitor of Defective in Cullin Neddylation 1 (DCN1) with a warhead, thereby generating a covalent inhibitor of DCN1 , and administering the covalent inhibitor of DCN1 to a subject in an amount sufficient to induce HbF in the subject.

[0145] In some embodiment, the covalent inhibitor of DCN1 inhibits DCN1 activity by covalently modifying a cysteine. In some embodiment, the covalent inhibitor of DCN1 inhibits DCN1 activity by covalently modifying a Cys115.

[0146] In some embodiments, the disclosure provides methods for generating covalent inhibitors of DCN-1 that can be used for the treatment of hemoglobinopathy such as sickle cell disorder or disease, or thalassemia disorder or disease. In some embodiments, the methods include modifying a non-covalent inhibitor to result in a covalent inhibitor. In some embodiments, the non-covalent inhibitor is converted into acovalent inhibitor by equipping the non-covalent inhibitor with a covalent warhead. Covalent warheads are known in the art and include terminal vinyl groups.

[0147] In one aspect, the present disclosure provides a method of treating a hemoglobinopathy disorder or disease, comprising administering to a subject in need thereof a covalent inhibitor of DCN-1 , or a pharmaceutically acceptable salt thereof. In some embodiments, the hemoglobinopathy is a sickle cell disorder or disease. In some embodiments, the hemoglobinopathy is a thalassemia disorder or disease. In another aspect, the present disclosure provides a method of inducing or increasing production of fetal hemoglobin by administering a covalent inhibitor of DCN-1 . Such methods are useful, for example, in treating hemoglobin-related disorders including sickle cell disorders, diseases and conditions and thalassemia.

[0148] As also shown herein, hydroxyurea can be used to induce fetal hemoglobin. Accordingly, in one aspect, the present disclosure provides a method to increase red blood cell levels and / or hemoglobin levels in a subject in need thereof, treat or prevent an anemia in a subject in need thereof, treat sickle-cell disease or thalassemia in a subject in need thereof, or treat one or more complications of sickle-cell disease or thalassemia in a subject in need thereof, including administering to a subject in need thereof a compound that modulates DCN-1 , such as a DCN-I inhibitor, or a covalent DCN-1 inhibitor, or a pharmaceutically acceptable salt thereof, in combination with hydroxyurea or a pharmaceutically acceptable salt thereof.

[0149] In one aspect, the present disclosure provides a method to increase fetal hemoglobin levels in a subject in need thereof, treat or prevent an anemia in a subject in need thereof, treat sickle-cell disease or thalassemia in a subject in need thereof, or treat one or more complications of sickle-cell disease or thalassemia in a subject in need thereof, including administering to a subject in need thereof a compound disclosed herein, e.g., a compound that can inhibit DCN-1 , or a pharmaceutically acceptable salt thereof, in combination with hydroxyurea or a pharmaceutically acceptable salt thereof.

[0150] In some embodiments, any of the methods of treatment provided herein optionally include co-administration of a second therapeutic agent. In some embodiments, the second therapeutic agent is hydroxyurea or a pharmaceutically acceptable salt thereof.

[0151] In one aspect, the present disclosure provides a method of treating a hemoglobinopathy disorder or disease, comprising administering to a subject in need thereof a compound disclosed herein, or a pharmaceutically acceptable salt thereof, in combination with a second agent such as hydroxyurea or a pharmaceutically acceptable salt thereof. In some embodiments, the hemoglobinopathy is a sickle cell disorder or disease. In some embodiments, the hemoglobinopathy is a thalassemia disorder or disease. Insome embodiments, the compound or pharmaceutically acceptable salt thereof and the hydroxyurea or a pharmaceutically acceptable salt thereof act synergistically.

[0152] In some embodiments, the compound or pharmaceutically acceptable salt thereof is selected from one of those shown in the Section entitled “Exemplary test compounds and candidate compounds”. However, it should be appreciated that, in some embodiments, any compound or composition that can bind DCN-1 can be used according to the methods provided herein. In some embodiments, the compound or composition that can bind DCN-1 according to the methods provided herein is a covalent inhibitor of DCN-1.

[0153] In one aspect, the present disclosure provides a method of increasing efficacy and / or reducing toxicity of hydroxyurea treatment in a subject undergoing said treatment, comprising administering to the subject a compound disclosed herein, or a pharmaceutically acceptable salt thereof. In some embodiments, the hydroxyurea treatment is for a hemoglobinopathy. In some embodiments, the hydroxyurea treatment is for sickle cell disease. In some embodiments, the hydroxyurea treatment is for a thalassemia disorder. In some embodiments, the method further comprises the step of decreasing an amount of hydroxyurea being administered to the subject. In some embodiments, the amount of hydroxyurea being administered is decreased by 10-90%.

[0154] In one aspect, the present disclosure provides a method of decreasing the dose of hydroxyurea or a pharmaceutically acceptable salt thereof needed for effective treatment of a hemoglobinopathy disorder or disease, comprising administering to a subject in need thereof a compound disclosed herein, e.g., a DCN-1 inhibitor, or a pharmaceutically acceptable salt thereof, in combination with hydroxyurea or a pharmaceutically acceptable salt thereof, wherein the dose of hydroxyurea or a pharmaceutically acceptable salt thereof needed for effective treatment of the hemoglobinopathy disorder or disease is less than the dose needed for treatment in the subject using hydroxyurea or a pharmaceutically acceptable salt thereof as a monotherapy. In some embodiments, the dose of hydroxyurea or a pharmaceutically acceptable salt thereof co-administered with the compound or pharmaceutically acceptable salt thereof is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% relative to the dose needed for treatment in the subject using hydroxyurea or a pharmaceutically acceptable salt thereof as a monotherapy.

[0155] In some embodiments, the present disclosure provides a method to treat or prevent one or more complications of sickle cell disease including, for example, anemia, anemia crisis, splenomegaly, paincrisis, chest syndrome, acute chest syndrome, blood transfusion requirement, organ damage, pain medicine (management) requirement, splenic sequestration crises, hyperhemolytic crisis, vaso-occlusion, vasoocclusion crisis, acute myocardial infarction, sickle-cell chronic lung disease, thromboemboli, hepatic failure, hepatomegaly, hepatic sequestration, iron overload and complications of iron overload (e.g., congestive heart failure, cardiac arrhythmia, myocardial infarction, other forms of cardiac disease, diabetes mellitus, dyspnea, hepatic disease and adverse effects of iron chelation therapy), splenic infarction, acute and / or chronic D renal failure, pyelonephritis, aneurysm, ischemic stroke, intraparenchymal hemorrhage, subarachnoid hemorrhage, intraventricular hemorrhage, peripheral retinal ischemia, proliferative sickle retinopathy, vitreous hemorrhage, and / or priapism; comprising administering to a subject in need thereof a disclosed compound or pharmaceutically acceptable salt thereof, optionally in combination with a second therapeutic agent such as hydroxyurea or a pharmaceutically acceptable salt thereof.

[0156] In some embodiments, the compound or pharmaceutically acceptable salt thereof acts synergistically in combination with the second therapeutic agent, e.g., hydroxyurea or a pharmaceutically acceptable salt thereof.Combination Therapies

[0157] In one aspect, the compounds of the present disclosure are used advantageously in combination with a second therapeutic agent. Such a second therapeutic agent includes, in some embodiments, hydroxyurea or a pharmaceutically acceptable salt thereof.

[0158] In some embodiments, the disclosure provides methods for using a compound or combination therapy (for example, a disclosed compound or pharmaceutically acceptable salt thereof in combination with hydroxyurea or a pharmaceutically acceptable salt thereof) to treat or prevent vascular occlusion (vaso-occlusion) in a sickle-cell disease patient in need thereof as well as various complications associated with vaso-occlusion in a sickle-cell disease patient (e.g., vaso-occlusion crisis, pain crisis, etc.). In some embodiments, the disclosure provides methods for using a disclosed compound or combination therapy to treat or prevent anemia in a sickle-cell disease patient in need thereof as well as various complications associated with anemia in a sickle-cell disease patient (e.g., aplastic crisis, hyperhemolytic crisis, etc.). In such methods, a disclosed compound or combination therapy can be used to increase red blood cell levels while reducing the need for red blood cell transfusions and / or iron chelation therapy, and thereby reduce morbidity and mortality associated with iron accumulation in vulnerable tissues / organs. In such methods, a disclosed compound or combination therapy can also be used to reduce the need for othersupportive therapies for treating sickle-cell disease [e.g., treatment with hydroxyurea, treatment with an EPO or other EPO agonist, and / or pain management (e.g., treatment with one or more of opioid analgesic agents, non-steroidal anti-inflammatory drugs, and / or corticosteroids)]. In part, a disclosed compound or combination therapy can be used in combination with existing supportive therapies for sickle-cell disease including, for example, transfusion of red blood cells, iron chelation therapy, hydroxyurea therapy, EPO or EPO agonist therapy, and / or pain management therapy. Optionally, a disclosed compound or combination therapy can be used to reduce the amount, duration, etc. of an existing supportive therapy for sickle-cell disease. For example, while transfusion of red blood cells and iron chelation therapy may help treat certain complications of sickle-cell disease, they sometimes result in adverse side effects. Therefore, in certain aspects, a disclosed compound or combination therapy can be used to reduce the amount of a second supportive therapy, e.g., reduce blood cell transfusion burden or reduce the dosage of a chelation therapeutic. In certain aspects, the disclosure provides uses of a disclosed compound or combination therapy (optionally in combination with one or more supportive therapies for sickle-cell disease) for making a medicament for the treatment or prevention of sickle-cell disease, particularly one or more complications of sickle-cell disease as disclosed herein.

[0159] The present disclosure also provides compositions that comprise or deliver a compound as provided herein. In some embodiments, the present disclosure provides compositions comprising a compound provided herein with one or more other components.

[0160] In some embodiments, provided compositions comprise and / or deliver a compound described herein. In some embodiments, a provided composition is a pharmaceutical composition that comprises and / or delivers a compound provided herein and further comprises a pharmaceutically acceptable carrier.

[0161] Pharmaceutical compositions typically contain an active agent (e.g., a compound described herein) in an amount effective to achieve a desired therapeutic effect while avoiding or minimizing adverse side effects. In some embodiments, provided pharmaceutical compositions comprise a compound described herein and one or more carriers or excipients (e.g., fillers, disintegrants, lubricants, glidants, anti-adherents, and / or anti-statics, etc.) Provided pharmaceutical compositions can be in a variety of forms including oral dosage forms, topical creams, topical patches, iontophoresis forms, suppository, nasal spray and / or inhaler, eye drops, intraocular injection forms, depot forms, as well as injectable and infusible solutions.

[0162] Provided pharmaceutical compositions can be prepared with any appropriate available technologies.

[0163] In some embodiments, provided compounds are formulated in a unit dosage form for ease of administration and uniformity of dosage. The expression “unit dosage form” as used herein refers to a physically discrete unitof an active agent (e.g., a compound described herein) for administration to a subject. Typically, each such unit contains a predetermined quantity of active agent. In some embodiments, a unit dosage form contains an entire single dose of the agent. In some embodiments, more than one unit dosage form is administered to achieve a total single dose. In some embodiments, administration of multiple unit dosage forms is required, or expected to be required, in order to achieve an intended effect. A unit dosage form may be, for example, a liquid pharmaceutical composition containing a predetermined quantity of one or more active agents, a solid pharmaceutical composition (e.g., a tablet, a capsule, or the like) containing a predetermined amount of one or more active agents, a sustained release formulation containing a predetermined quantity of one or more active agents, or a drug delivery device containing a predetermined amount of one or more active agents, etc.

[0164] Provided compositions may be administered in accordance with a dosing regimen (i.e., that includes a single dose or multiple doses separated from one another in time, administered via a particular route of administration) that is (e.g., has been demonstrated to be) effective for treating (e.g., delaying onset of and / or decreasing incidence and / or intensity of) a disease or disorder, for example as described herein.

[0259] The present disclosure also provides methods of preparing pharmaceutical compositions provided herein. In some embodiments, provided methods comprise (i) providing a provided compound or a pharmaceutically acceptable salt thereof; and (ii) formulating the compound with suitable excipients to give a pharmaceutical composition.Exemplary test compounds and candidate compounds

[0165] As shown herein, DCN-1 binding compounds can be used for the induction of fetal hemoglobin and the treatment of sickle cell disease and thalassemia. In aspect, the DCN-1 binding compounds that are used for the induction of fetal hemoglobin and the treatment of sickle cell disease and thalassemia are covalent binders of DCN-1.

[0166] In some embodiments, the compound or pharmaceutically acceptable salt thereof that can be used according to the methods provided herein is selected from one of those shown in this section entitled“Exemplary test compounds and candidate compounds”. However, it should be appreciated that, in some embodiments, any compound or composition that can bind DCN-1 can be used according to the methods provided herein. In some embodiments, the compound or composition that can bind DCN-1 and can be used in the methods provided herein is an inhibitor of DCN-1 . In some embodiments, the compound or composition that can bind DCN-1 and can be used in the methods provided herein is a covalent inhibitor of DCN-1.

[0167] In one aspect, the compounds provided herein (including test compound and candidate compounds) can be used in the methods provided herein including the induction of fetal hemoglobin and the treatment of sickle cell disease. In some embodiments, test compounds and candidate compounds of the disclosure are selected from:L1 reference: WO2018191199 COVALENT SMALL MOLECULE DCN1 INHIBITORS AND THERAPEUTICMETHODS USING THE SAMEL2 = DI-1548, Zhou H, et al. Selective inhibition of cullin 3 neddylation through covalent targeting DCN1 protects mice from acetaminophen-induced liver toxicity. Nat Commun. 2021 ;12(1 ):2621L3 reference: WO2017049295 METHODS AND COMPOSITIONS OF INHIBITING DCN1-UBC12 INTERACTION

[0168] In some embodiments, test compounds and candidate compounds of the disclosure are selected from:L832SUBSTITUTE SHEET (RULE 26)or a pharmaceutically acceptable salt thereof.L4 = DI-591 , Zhou H, et al. A potent small-molecule inhibitor of the DCN1-UBC12 interaction that selectively blocks cullin 3 neddylation. Nat Commun. 2017 Oct 27;8(1 ):1150;L5 = Compound 40; Kim et al., J. Med. Chem. 2021, 64, 5850.L6 reference: Kim et al., J. Med. Chem. 2021 , 64, 5850.L7 reference: Kim et al., J. Med. Chem. 2021 , 64, 5850.L8 reference: Kim et al., J. Med. Chem. 2021 , 64, 5850.

[0169] In some embodiments, test compounds and candidate compounds of the disclosure are selected from:L11 L12L13 L14 or a pharmaceutically acceptable salt thereof.L9 reference: Hammill JT, et al. Discovery of an Orally Bioavail able Inhibitor of Defective in Cui I i n Neddylation 1 (DCNI)-Mediated Cullin Neddylation. J Med Chem. 2018 Apr 12;61 (7):2694-2706L10 reference: Zhou H, et al. High-Affinity Peptidomimetic Inhibitors of the DCN1-UBC12 Protein-Protein Interaction. J Med Chem. 2018 Mar 8;61 (5): 1934-1950L11 reference: He ZX, et al. Discovery of Potent and Selective 2-(Benzylthio)pyrimidine-based DCN1-UBC12 Inhibitors for Anticardiac Fibrotic Effects. J Med Chem. 2022;65(1 ):163-190L12 reference: Zhou H, et al. High-Affinity Peptidomimetic Inhibitors of the DCN1-UBC12 Protein-Protein Interaction. J Med Chem. 2018 Mar 8;61 (5): 1934-1950L13 reference: Wang S, et al. Development of Highly Potent, Selective, and Cellular Active Triazolo[1 ,5- a]pyrimidine-Based Inhibitors Targeting the DCN1-UBC12 Protein-Protein Interaction. J Med Chem. 2019 Mar 14;62(5):2772-2797L14 reference: Zhou H, et al. Selective inhibition of cullin 3 neddylation through covalent targeting DCN1 protects mice from acetaminophen-induced liver toxicity. Nat Commun. 2021 ;12(1 ):2621.

[0170] In some embodiments, the test compounds and candidate compounds are selected from those described in International patent publications WO2023 / 244691 , WC2020 / 257790, WO2018 / 191199 WO2018 / 13411 , WO2017 / 147386, WO2017 / 049295, US patent publication US2018 / 0289677, and patent publications CN111925336, CN109516984, CN108864067, and CN108484612; or a pharmaceutically acceptable salt thereof; the contents of which are incorporated by reference herein.

[0171] In some embodiments, the test compounds and candidate compounds are selected from those described in J. Med. Chem. 2019, 62, 8429; or a pharmaceutically acceptable salt thereof; the contents of which are incorporated by reference herein.

[0172] In some embodiments, the test compounds and candidate compounds are selected from those described in J. Med. Chem. 2021, 64, 5850; or a pharmaceutically acceptable salt thereof; the contents of which are incorporated by reference herein.

[0173] In some embodiments, the test compounds and candidate compounds are selected from those described in J. Med. Chem. 2018, 61 , 2680; or a pharmaceutically acceptable salt thereof; the contents of which are incorporated by reference herein.

[0174] In some embodiments, the test compounds and candidate compounds are selected from those described in Hammill JT, et al. Discovery of an Orally Bioavailable Inhibitor of Defective in Cullin Neddylation 1 (DCNI)-Mediated Cullin Neddylation. J Med Chem. 2018 Apr 12;61 (7):2694-2706; or a pharmaceutically acceptable salt thereof; the contents of which are incorporated by reference herein.

[0175] In some embodiments, the test compounds and candidate compounds are selected from those described in Zhou H, et al. High-Affinity Peptidomimetic Inhibitors of the DCN1-UBC12 Protein-Protein Interaction. J Med Chem. 2018 Mar 8;61 (5):1934-1950; or a pharmaceutically acceptable salt thereof; the contents of which are incorporated by reference herein.

[0176] In some embodiments, the test compounds and candidate compounds are selected from those described in He ZX, et al. Discovery of Potent and Selective 2-(Benzylthio)pyrimidine-based DCN1- UBC12 Inhibitors for Anticardiac Fibrotic Effects. J Med Chem. 2022;65(1): 163-190; or a pharmaceutically acceptable salt thereof; the contents of which are incorporated by reference herein.

[0177] In some embodiments, the test compounds and candidate compounds are selected from those described in Zhou H, et al. A potent small-molecule inhibitor of the DCN1-UBC12 interaction that selectively blocks cullin 3 neddylation. Nat Commun. 2017 Oct 27;8(1):1150; or a pharmaceutically acceptable salt thereof; the contents of which are incorporated by reference herein.

[0178] In some embodiments, the test compounds and candidate compounds are selected from those described in Wang S, et al. Development of Highly Potent, Selective, and Cellular Active Triazolo[1 , 5- a]pyrimidine-Based Inhibitors Targeting the DCN1-UBC12 Protein-Protein Interaction. J Med Chem. 2019Mar 14;62(5):2772-2797; or a pharmaceutically acceptable salt thereof; the contents of which are incorporated by reference herein.

[0179] In some embodiments, the test compounds and candidate compounds are selected from those described in Zhou H, et al. Selective inhibition of cullin 3 neddylation through covalent targeting DCN1 protects mice from acetaminophen-induced liver toxicity. Nat Commun. 2021 ;12(1):2621 ; or a pharmaceutically acceptable salt thereof; the contents of which are incorporated by reference herein.

[0180] In some embodiments, the compound or composition that can bind DCN-1 and can be used in the methods provided herein is a covalent inhibitor of DCN-1. It should be appreciated that non-covalent DCN-1 binding compounds can modified to become covalent modifiers of DCN-1, for instance, by adding a covalent warhead to the non-covalent inhibitor

[0181] In some embodiments the compound that can bind DCN-1 is not a CUL3 inhibitor. CUL3 inhibitors are described for instance in US 2022 / 0017908 and include MLN4924, suramin and DI-591.

[0182] It will also be understood that, although the terms first, second, etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. For example, a first subject could be termed a second subject, and, similarly, a second subject could be termed a first subject, without departing from the scope of the present disclosure. The first subject and the second subject are both subjects, but they are not the same subject.

[0183] The terminology used in the present disclosure is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will also be understood that the term “and / or” as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0184] As used herein, the term “if” may be construed to mean “when” or “upon” or “in response to determining” or “in response to detecting,” depending on the context. Similarly, the phrase “if it is determined” or “if [a stated condition or event] is detected” may be construed to mean “upon determining” or “in response to determining” or “upon detecting (the stated condition or event)” or “in response to detecting (the stated condition or event),” depending on the context.

[0185] The foregoing description, for purpose of explanation, has been described with reference to specific implementations. However, the illustrative discussions above are not intended to be exhaustive or to limit the implementations to the precise forms disclosed. Many modifications and variations are possible in view of the above teachings. The implementations were chosen and described in order to best explain the principles and their practical applications, to thereby enable others skilled in the art to best utilize the implementations and various implementations with various modifications as are suited to the particular use contemplated.EXAMPLESExample 1 : NBSGW Humanized mouse model for HbF induction

[0186] Animals

[0187] Female, 6-week-old NOD.Cg-KitW-41 J Tyr+ Prkdcscid H2rgtm1 Wjl / ThomJ (NBSGW) mice (Jackson Laboratory strain #02662) were used for these studies. The mice were acclimatized to laboratory conditions for 5 days prior to inoculation.

[0188] Cell Preparation and Inoculation

[0189] GCSF-mobilized human CD34+ cells were removed from liquid nitrogen storage, thawed in a 37C water bath and transferred quickly into a 50 mL conical tube. Cryovial was rinsed once with thaw buffer, 0.1 % BSA in phosphate buffered saline (PBS), and buffer was transferred combined with the original contents in the 50 mL conical tube. Next, doubling volumes of thaw buffer was added to the conical and gently swirled for ~30 seconds to one minute until the volume in the conical was 32 mL. Cells and buffer were centrifuged at 300G for 8 minutes, and the supernatants were aspirated. Cells were counted by resuspending in 1 mL of thawing buffer per million of cells to a target concentration range of 0.5 to 2M / ml) and counting with ACPI (1 :1) on a luna cell counter to confirm the concentration of cells / mL. The cell concentration was adjusted to 3x10A6 cells / ml. For each mouse, 300 thousand cells in 0.1 ml were injected into the tail vein with a 25- gauge needle.

[0190] Engraftment Checkpoint

[0191] On day 56 after human cell adoptive transfer, whole blood was collected from each mouse by submandibular bleed and a 100 pL sample of EDTA whole blood was transferred to a 2 ml tube containing 1.8 mL ACK Lysing Buffer at room temperature (RT), and then inverted to mix. Samples were incubated at RT for 15 min in the dark to lyse. After lysis, samples were centrifuged at 500xg for 5 minutes at RT to enablesupernatant decanting. Remaining cells were washed with 1 mL of PBS-0.5% BSA and centrifuged at 500xg for 5 minutes at 4C. Supernatant was decanted and cells were stained with leukocyte markers (human and mouse CD45 antibodies; BD347464, BD557659) to confirm human cell engraftment. Mice having less than one percent, or greater than ten percent, human CD45 positive cells were excluded from the subsequent study. The remaining mice were then randomized into treatment groups based on percentage of human cell engraftment. Each treatment group included 10-11 mice.

[0192] Compound Administration

[0193] Hydroxyurea was solubilized in PBS. Formulations were prepared fresh daily. Commencing on day 84 post human cell engraftment, mice were treated by oral gavage with hydroxyurea or their respective vehicles, for a period of three weeks using either once daily (QD) or twice daily (BID) dosing regimens. Mice were monitored daily for body weight and condition. Mice which lost greater than 20% body weight prior to study completion were removed from the study and humanely euthanized.

[0194] Bone Marrow Collection and Analysis

[0195] After 21 days of dosing, all mice were euthanized and prepared for bone marrow collection. Both femurs were collected from each mouse by first disinfecting the skin with 70% ethanol and then, using a pair of scissors and forceps, removing the limb and dissecting the muscles both above and below the femur and tibia, taking care not to damage the bone. Femurs were placed in PBS-0.5%BSA-2mM EDTA-containing tubes on ice during collections. Each femur was flushed to extract marrow with 1 mL of 0.5% BSA-PBS 2mM EDTA using a 27 gauge needle a total of three times. Extracted cells were counted and aliquoted to prepare for analysis. For detection of fetal (HbF) and adult (HbB) hemoglobin protein, bone marrow cells expressing human glycophorin A (GlyA) were isolated by flow cytometry and frozen. Frozen cells were analyzed by HPLC. For assessment of fetal hemoglobin mRNA (HBG1) expression by Nanostring™, whole RBC-lysed bone marrow cells were used. Resulting mRNA expression levels were normalized based on the percentage of GlyA positive cells in the bone marrow of each mouse. The data are shown in Figures 1 and 2.Example 2: AlphaLISA Assay

[0196] The AlphaLISA assay for detecting Cullin-3 (CUL3) neddylation was performed according to manufacturer instructions (Revvity, Hopkinton, MA). Briefly, TF1 cells (ATCC) were plated in Iscove’s Modified Dulbecco's Medium (IMDM) without supplements in 384-well plates. These cells were treated with11 concentrations for 3 hours before lysing them with AlphaLI SA lysis buffer, 5X (Revvity). To detect the level of CUL3 neddylation, biotinylated anti-NEDD8 antibody, was added followed by AlphaLISA Acceptor beads conjugated with anti-CU L3 antibody. After overnight incubation, Streptavidin-coated Alpha Donor beads were added and incubated for 1 hour. The AlphaLISA signal was then read on a VICTOR Nivo Multimode Microplate Reader (Revvity, Hopkinton, MA). The positive control was 1 M DI-1548, which is reported to reduce CUL3 neddylation (Zhou et al. Nature Comm. 2021). The neddylation signal was normalized such that the percent inhibition is 0% for vehicle control and 100% for positive control. The dose-response curves were fitted with the Hill equation to obtain IC50 values (Graphpad Prism). The results are shown in Table 1 below.Table 1 . AlphaLISA AssayExample 3: HBG1 Nanostring Assay

[0197] A Nanostring Assay was performed to evaluate the ability of the compounds to induce expression of the fetal hemoglobin gene HBG1 in cells. The data are shown as EC50, with stronger inducers having a lower EC50 value and higher YMax.

[0198] CD34 culture and sample preparation

[0199] Mobilized peripheral blood (mPB) derived CD34+ hematopoietic stem and progenitor cells (HSPCs) were thawed and plated at 500,000 cells / ml on day of cell thaw (day -4) in StemSpan (StemCell Tech, 09600) complete media supplemented with 1 % StemSpan CC100 (StemCell Tech,02690), and 0.2% human recombinant thrombopoietin (StemCell Tech, 02822). Forty eight hours later (day -2), the cells were passaged at a density of 200,000 cells / ml in complete expantion media. The cells were plated at 200,000 cells / ml on day 0 and day3, 400,000 cells / ml on day 5 in phase 1 erythroid differentiation media containing StemSpan with 2.5U / mL of EPO (R&D systems, 287-TC-500), 0.5 mg / mL of Holo- Transferrin (Sigma, T0665-500MG), 1x Glutamine (Gluta-Max) (Gibco, 35050-061), 5 uL / mL Lipid Mixture (Sigma, L0288-100ML), 50 ng / mL SCF (R&D Systems, 255-SV-050), 10 ng / mL IL-3 (R&D Systems, 203- IL010) and 10 ng / mL Insulin (Sigma, I9278-5ML). Cells were then passaged at 500,000 cells / ml on day 7 in phase 2 Erythroid differentiation (Phase 1 erythroid differentiation removing IL-3) until collection day (Day10).

[0200] For all cell passagings, cells were centrifuged at 300g for 8 minutes at room temperature and cell number were normalized per well. All the compounds are diluted in DMSO and added to complete media by Formulatrix FAST and mixed starting from day -2. Cells were counted by Luna cell counter with ACPI Staining Solution (Nexcelom Bioscience, CS2-0106-25mL) on day-2. Cells were counted by BD FACSCelesta Flow Cytometer with CountBright™ Plus Absolute Counting Beads (Thermal Fisher Scientific, C36995) and SYTOX AADvanced™ Ready Flow™ Reagent (Invitrogen, R37173) for other passages. Cells were cultured at 37 °C and 5% CO2.

[0201] On the collection day (Day10), 100K cells were collected and stored at -80 °C. For direct hybridization for Nanostring, 100K cell pellet was lysed in 25 pL of RLT (QIAGEN, 79216) with lx (3- Mercaptoethanol (Gibco, 21985-023) and was shaken at 300-500 RPM for 5 minutes at room temperature. Cell lysate was stored in -80°C after lysis.Hybridization and imaging

[0202] All hybridizations were done in a total volume of 15 pL (3 pL of RNA lysate added to master mix of 12 pL probe A / B, capture probe / reporter probes, proteinase K and attenuation oligos with HBG1 , HBG2, HBA1 and HBB suspended in hybridization buffer). Samples were hybridized at 67 °C for 22 hr (referring to MAN-10040-06_PlexSet_Reagents_User_Manual). Following hybridization, referring to NanoString manual MAN-C0035_nCounter„Analysis„System„MAX„FLEX for instrument operation.Quality control (QC) metrics and data analysis

[0203] The quality control (QC) metric included limit of detection QC by checking for wells with less than 100 total counts for positive controls.

[0204] Data analysis, refers to NanoString manual “MAN-C0011-04 Gene Expression Data Analysis Guidelines”.

[0205] The results of the assay are shown in Table 2 below. The Nanostring Ymax observed data is relative to a control compound, presented in % which is shown in Table 2 below.Table 2. HBG1 Nanostring AssayExample 4: HbF HPLC Cell Culture and Analysis

[0206] A Cell Culture Assay was performed to evaluate the ability of compounds to induce fetal hemoglobin in a cell culture. The amount fetal hemoglobin protein induced was measured by HPLC.

[0207] Cell culture began on Day -4 (thaw day). Thaw buffer was prepared by sterile filtering 6 mL of Human Serum Albumin and 144 mL of PBS to make sterile 1% HSA / PBS. Cells were removed from liquid nitrogen storage and mostly thawed in a 37 °C water bath. Once the ice in the cryovials was melted, cells were transferred quickly into a 50 mL conical tube. Cryovial was rinsed once with thaw buffer and buffer was transferred over to the conical tube as well. Next, doubling volumes of thaw buffer was added to the conical and gently swirled for ~30 seconds to one minute (for example: 2 mL was added and swirled, then 4 mL, then 8 mL, and so on) until the volume in the conical is 32 mL. Cells and buffer were centrifuged at 300G for 8 minutes, and the supernatant was aspirated. Another 32 mL of thaw buffer was added slowly, swirling the tube. The tube was again centrifuged at 300G for 8 minutes and the supernatant was aspirated. Cells werecounted by resuspending in 1 mL of expansion media and counting with AOPI to determine the concentration of cells / mL.

[0208] On day 2, Passage & Treatment Day, each cell culture well was counted. Next fresh CD34 Expansion Media was made which contained StemSpan SFEM, CC100 and TPO all from StemCell technologies. Appropriate volume of cells were collected, centrifuged at 300G and resuspended in fresh media. Cells were plated in treatment format at a density of 200,000 cells / mL, 25,000 cells / well. Finally, they were treated with test compounds as well as positive and negative controls.

[0209] On Days 0, 3 & 5 the same process was carried out. First, each cell culture well was counted. Next Phase 1 Erythroid Differentiation Media was freshly made, which contained StemSpan SFEM, 2.5U / mLEPO, 0.5mg / ml Holo-TF, 1x Glutamax, 5pL / mL chemically defined lipid mixture, 10ng / mL insulin, 50ng / mL SCF, 10ng / mL IL-3. Appropriate volume of cells were collected, centrifuged at 300G and resuspended in fresh media. Cells should now be in fresh wells at a density of 100,000 cells / m L. 60,000 cells, 60,000 cells and 100,000 cells for days 0, 3, 5 respectively. Finally, fresh compound were added to each well at each timepoint (Days 0, 3 & 5).

[0210] On Day 7 media and cell culture density changes. Each cell culture well was counted. Next, Phase 2 Erythroid Differentiation Media was freshly made, which contains StemSpan SFEM, 2.5U / mL EPO, 0.5mg / mL Holo-TF, 1x Glutamax, 5|jL / mL chemically defined lipid mixture, 10ng / mL insulin, 50ng / mL SCF. Appropriate volume of cells was collected, centrifuged at 300G and resuspended in fresh media. Cells were plated in fresh wells at a density of 500,000 cells / mL, 300,000 cells / well. Finally, fresh compound was added to each well at Day 7 timepoint.

[0211] On Day 10 media formulation is once again changed. Each cell culture well was counted. Fresh Phase 3 Erythroid Differentiation Media was made which contains StemSpan SFEM, 2.5U / mL EPO, 0.5mg / mL Holo-TF, 1x Glutamax, 5pl / ml chemically defined lipid mixture, 10ng / mL insulin. Appropriate volume of cells was collected, centrifuged at 300G and resuspended in fresh media. Cells were plated in fresh wells at a density of 500,000 cells / mL, 500,000 cells / well. Finally, fresh compound was added to each well at Day 10 timepoint.

[0212] On Day 12 the cell density is once again changed. Each cell culture well was counted. Fresh Phase 3 Erythroid Differentiation Media was made. Appropriate volume of cells was collected, centrifuged at 300G and resuspended in fresh media. Cells were plated in fresh wells at a density of 1 ,000,000 cells / mL, 1 ,000,000 cells / well. Finally fresh compound was added to each well.

[0213] Day 14 (18thday of experiment) refers to the terminal day of culture. Each cell culture well was counted. Next between 150K and 650K cells were placed in a uniquely labeled 1.5mL standard tube. Cells were centrifuged in media at 300G for 8 minutes, then as much of the media as possible was removed without disturbing the pellet. The pellet was washed with 500|jl dPBS and once again spun at 300g for 8 minutes. As much of the supernatant as possible was removed without disturbing the pellet and immediately frozen at -80°C. Cell pellets are now ready for HPLC lysis and analysis.

[0214] All centrifugation were run at 300x G for 8 minutes at room temperature. Cells were cultured in a standard incubator at 37 °C and 5% C). Cell culture plates were either a 96-well treated plate for Day - 2 or 24-well cell culture treated plates for day 0 through 14. Culture wells were counted using a 1 :1 mix of cells and AOPI. Counting was done on Nexcelom Cellaca.

[0215] Methods for assaying %HBF and %F+ cells are well known in the art. Non-limiting examples include high performance liquid chromatography (HPLC), flow cytometry, or ion-exchange chromatography. The HbF% is usually measured by HPLC. The flow cytometry assay, the standard clinical method, may be used for assaying %F+ cells by immunofluorescent techniques. In addition to flow cytometry, ion-exchange chromatography may be used to measure the fraction HbF relative to all other hemoglobin (HbF / HbA+HbF).

[0216] The Hemoglobin HPLC Ymax observed data is relative to a control compound, presented in % which is shown in Table 3 below.Table 3. HbF HPLC Cell Culture AssayExample 5: Assay for Transposase-Accessible Chromatin via Sequencing (ATAC-seq)

[0217] Reagents: ATAC-seq kit (Active Motif - 53150)

[0218] Method: Mobilized peripheral blood CD34+ cells (Stemcells Tech), were differentiated using an 18-day erythroid differentiation protocol in the presence of either DMSO or 3 uM of DI-1548 (n=5 for each condition). Cells were harvested on day 5 and were processed according to manufacturer’s standard protocol with minor modifications. Briefly, nuclei were isolated from 50,000 cells using the ATAC-seq lysis buffer then washed with PBS. Intact nuclei were then treated with Tn5 transposase at 37C for 40 minutes which allows DNA to be tagged with Illumina-compatible adaptors at positions of open chromatin (also known as “tagmentation”). DNA is then captured, washed and eluted using a purification column. To enrich and add a sample-level index to the tagmented fragments representative of open chromatin, a PCR was performed on the purified DNA using primers specific to the adaptors (i5 / i7) added during the tagmentation process using the suggested PCR cycling conditions. Amplified DNA was purified using the SPRI beads supplied by the kit, however, a double-sided clean-up was performed. In short, SPRI beads were added at 0.5X, incubated with sample for 5 minutes, then beads were separated on a magnet for another 5 minutes. Supernatant was then transferred to a new tube and 1 .3X SPRI beads were added and left to incubate for 5 minutes. Beads were clarified on magnet and supernatant removed followed by two ethanol washes. DNA was eluted off beads using DNA purification elution buffer. Libraries were assessed and quantified using the Bioanalyzer High Sensitivity DNA chip and Qubit, respectively. Libraries were pooled equimolar at 0.8nM and run on the NovaSeq6000 using cycling parameters Readl : 50bp, Read2: 50bp, I ndexl : 8bp, I ndex2: 8bp and sequenced at a depth of 200M reads per sample. Raw sequence reads (BCL) were converted to fastQ using bcl2fastq tool.

[0219] The data are shown in Figure 3. Profiling the chromatin tri-methyl ation mark on histone H3 lysine 4 (H3K4me3) in CD34 cells at the fetal hemoglobin locus revealed 4 sites where H3K4me3 is deposited. This result shows an upward trend in H3K4me3 at these sites, indicating increased transcriptional activity of fetal hemoglobin genes and genes associated with switching from adult hemoglobin to fetal hemoglobin. (See also Example 5)Bioinformatic analysis

[0220] Data was processed via in-house implementation of the NF-Core ATAC-seq pipeline (github.com / nf-core / atacseq, version 2.1.2) using NextFlow (nextflow.io / , version 23.10.0). Briefly, FastQ files were subjected to read quality control via FASTQC (bioinformatics.babraham.ac.uk / projects / fastqc / , version 0.11.9) to determine basic fastQ quality, sequence duplication, adapter and base content. Sequence adapters were then trimmed using cutadapt (cutadapt.readthedocs.io / , version 3.4). Paired-end sequencing reads were aligned to the hg38 reference genome (GENCODE, release 29) using the Burrows- Wheeler Aligner (BWA, bio-bwa.sourceforge.net / , version 0.7.17). Duplicates and poor quality alignments were removed and / or merged via a combination of Picard (broadinstitute.github.io / picard / , version 3.0.0), SAMtools (htslib.org / doc / samtools.html, version 1.17), BAMtools (github.com / pezmaster31 / bamtools,version 2.5.2), and Pysam (pysam.readthedocs.io / version 0.19.0). Output BAM files were converted to BED files via BEDTools (bedtools.readthedocs.io / , version 2.30.0). Peaks were called in narrowPeak mode (q-value=0.05) using MACS2 (github.com / macs3-project / MACS, version 2.2.7.1) using the following parameters: -g 2700000000. Peaks were annotated to relevant gene features using HOMER (homer.ucsd.edu / homer / , version 4.11). For differential peak analysis, consensus peaks were called across conditions and counts were created per sample replicate across consensus peaks using the merge and multicov functions from BEDTools. Next, differential peak calling was performed using limma (bioconductor.org / packages / release / bioc / html / limma.html, version 3.60.6). Genome tracks were visualized using pyGenomeTracks (pygenometracks.readthedocs.io / , version 3.9).Example 6: CUTANA CUT & RUN, Illumina sequencing, and data analysis.Reagents-. CUTANA CUT AND RUN kit

[0221] Methods: Mobilized peripheral blood CD34+ cells (Stemcells Tech), were differentiated using an 18-day erythroid differentiation protocol in the presence of either DMSO or 3 uM of DI-1548. Cells were harvested on day 5 and nuclei were isolated from cell preps. CUTANA™ CUT&RUN was performed with Primary Bone Marrow Derived CD34+nuclei samples on an automated protocol (autoCUT&RUN) derived from those previously described. In brief, for each CUT&RUN reaction 500K nuclei [5 million nuclei / mL prepared in CUTANA™ Nuclei Extraction Buffer (EpiCypher# -1026)] were dispensed to individual wells of a 96-well plate, immobilized onto Concanavalin-A beads (Con- A; EpiCypher #21 -1401), and incubated overnight (4°C) with 0.5 pg of antibody (IgG, H3K4me3) (IgG, H3K4me3 antibodies validated to histone post-translation modification (PTM)-defined SNAP-CUTANA nucleosome standards as previously). pAG-MNase (EpiCypher #15-1016) was added / activated (2 hours @ 4°C) and CUT&RUN enriched DNA was purified using Serapure beads after mixing at 2:1 (Bead:DNA) ratio. Recovered DNA was quantified using PicoGreen and reactions were normalized to 5ng DNA (or entirety of the reaction if <5ng DNA was recovered) before preparing sequencing libraries (CUTANA CUT&RUN Library Prep kit; EpiCypher# 4-1001 ). All autoCUT&RUN steps were optimized I performed on Tecan Freedom EVO robotics platforms with gentle rocking for incubation steps and magnetic capture for media exchange I washing steps. Sequencing was performed on Illumina NextSeq2000, 2x50bp paired-end reads. Paired-end fastq files were aligned to the hg38 reference genome using Bowtie2 v.2.2.5 after removing multi-aligned reads, PCR duplicates, and, ENCODE DAC exclusion list regions. Bigwigs were generated using Deeptoolsv.3.5.1 , peak calling was performed with MACS2 v.2.2.7.1 , and peaks were annotated with HOMER. Consensus peaks were called across conditions using the multicov function from BEDTools v.2.31.1. Genome tracks were visualized using pyGenomeTracks v.3.9.

[0222] The data are shown in Fig 4: Profiling chromatin accessibility of CD34 cells at the fetal hemoglobin locus revealed 7 sites where accessibility was significantly increased by treatment with DI- 1548 (adj p-value < 0.05, N=5). These sites were coordinated at the promoter and gene body regions of fetal hemoglobin genes HBG2, HBG1, and known regulatory IncRNA genes BGLT3, and HBBP1. Overall these results show a dramatic change in accessibility of the hemoglobin locus after 7 days treatment with DI- 1548 and 5 days into erythroid differentiation. (1) Marunde et al (2024) Elife 13:e78866 [PMID: 38319148]; (2) Yusufova et al (2021) Nature 589:299 [PMID: 33299181]; (3) Skene et al (2018) Nat Protoc 13:1006 [DOI: doi.org / 10.1038 / nprot.2018.015]; (4) Shah et al (2018) Molecular Cell 72:172 [PMID: 30244833]; (5) Amemiya et al (2019) Scientific Reports ^, Article number: 9354 [PMID: 31249361].Example 7: Differential Gene Expression Analysis.Methods

[0223] Mobilized peripheral blood CD34+ cells (Stemcells Tech), were differentiated using an 18- day erythroid differentiation protocol in the presence of either DMSO, 0.1 uM, or 3 uM of DM 548. Cells were harvested after 48 hours and 7 days (5 days of erythroid differentiation) and subjected to single cell RNA sequencing to determine differential gene expression compared between DMSO and compound treated cells.

[0224] Single-cell RNA-seq libraries were prepared from cells using 10X Genomics Next GEM or GEM-X Single Cell 3’ kits or according to the manufacturer’s recommended protocol (s). Briefly, up to 10,000 cells were loaded into a single channel of the appropriate Next GEM or GEM-X chip and partitioned into droplets with gel beads using a Chromium or Chromium X controller. After emulsion droplets were formed and collected, reverse transcription reactions were performed and barcoded transcripts are purified, amplified, fragmented and ligated to unique dual indexed sequencing adapters, all according to the manufacturer’s recommended protocol(s). Libraries were sequenced to a targeted depth of -40,000 reads per cell on an Illumina NextSeq2000 or NovaSeq6000 using paired end reads as follows: Readl = 28 cycles, i7 Index = 10 cycles, i5 = 10 cycles, Read2 = 89 cycles (NovaSeq) or 91 cycles (NextSeq). CellRanger (v5.1 .0 or v8.0) mkfastq was used to generate demultiplexed FASTQ files from the raw sequencing data. Cell Ranger count was used to align reads to the human GRCh38 genome reference and quantify gene and UMI counts. Quality assessments of the libraries were performed using our standard Seuratbased pipeline which provides information on a wide range of standard and fit for purpose metrics (e.g., ribosomal / mitochondrial read fraction, cell clustering and annotation, etc.). QC and cell filtering operations were performed using functions from scanpy v.1 .9.8. Differential expression analysis was performed using limma v.3.60.6. Mobilized peripheral blood CD34 cells from healthy volunteers were exposed to either 100 nM or 3 uM of CY-4219 and probed for differential gene expression by scRNAseq on days 2 and 7 (N=2)

[0225] The data are shown in Figure 5: On day 2 none of the genes in the 100 nM treatment were differentially regulated that had a fold change of >1.5 and an adjusted p-value < 0.05; At 3 uM DI-1548, 18 genes were upregulated and 8 were downregulated. Among the genes differentially upregulated are NQO1 and FTL (markers of NRF2 activation) and HBBP1 (marker of HbF switching from adult to fetal hemoglobin). Analysis of erythroid subset after 7 days treatment with DI-1548 and 5 days into erythroid differentiation revealed 8 genes significantly upregulated in the 100 nM treated cells and 18 genes up and 7 genes downregulated in the 3 uM treated cells. Among the upregulated genes are HBG1 , HBG2, and BGLT3 (marker of HbF switching from adult to fetal hemoglobin).Example 8: A TR-FRET assay shows the ability of the compounds to bind the DCN-1 protein.

[0226] The TR-FRET assay was designed following the Scott et al. protocol (Scott et al., Nat Chem Biol. 2017 August; 13(8): 850-857. Doi:10.1038 / nchembio.2386). The recombinant form of the DCN1 (DCUND1) protein PONY domain was produced using an E.coli expression system at Viva Biotech (China). The DCN1 protein was biotinylated (EZ sulfo-NHS-LC-biotin; Thermofisher) for labeling with streptavidin terbium (Tb) cryptate in the reaction. The probe was changed to a non-covalent DCN1 inhibitor labeled with carboxyfluorescein (FAM; Zhou et al., Nat Commun. 2017; 8: 1150. Doi: 10.1038 / s41467-017- 01243-7). Buffer conditions were modified to enhance protein stability by exchanging Tween20 for TritonX and increasing NaCI to 200 mM. The compounds were screened against 5 nM DCN1 and 20 nM FAM- probe or 0.31 nM DCN1 and 900 nM total probe (100 nM FAM-labeled plus 800 nM unlabeled). The TR- FRET ratio between Tb-DCN1 and the FAM-labeled probe was measured in a 384-well opti-plate (Perkin Elmer) using a plate reader (BMG) at 1, 5, and 24 hrs after treatment with compound (final DMSO concentration of 0.1 %). The ratio was normalized to the high (DCN1 and FAM-probe) and low (DCN1 andno probe) controls for a readout of % activity (= 100* (x - low) / (high - low). The % activity across concentrations is used to determine the IC50. The data are shown in Table 4 below.Table 4. TR-FRET AssayNON-LIMITING ASPECTS OF THE DISCLOSURE

[0227] The disclosure provides the following non-limiting aspects:

[0228] Aspect 1 : A method for identifying a candidate compound, comprising:(a) obtaining a test compound having the ability to covalently bind to Defective in Cullin Neddylation 1 (DCN1) or is suspected to have the ability to bind to DCN1 ;(b) contacting the test compound with DCN1;(c) assaying for modulation of DCN1 and / or a biological target associated with DCN1; and(d) classifying the test compound as a candidate compound if modulation of the DCN1 and / or a biological target associated with DCN1 is detected.

[0229] Aspect 2: A method for identifying a candidate compound, comprising:(a) obtaining a test compound having the ability to covalently bind to Defective in Cullin Neddylation 1 (DCN1) or is suspected to have the ability to bind to DCN1 ;(b) contacting the test compound with DCN1;(c) assaying for changes in levels of fetal hemoglobin (HbF) protein and / or function; and(d) classifying the test compound as a candidate compound if changes in levels of fetal hemoglobin (HbF) protein and / or function are detected.

[0230] Aspect 3: The method of aspect 1 or 2, wherein the modulation of DCN1 and / or the biological target associated with DCN1 comprises inhibition of a DCN1-E2 enzyme interaction, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0231] Aspect 4: The method of any one of aspects 1-3, wherein the modulation of DCN1 and / or a biological target associated with DCN1 comprises inhibition of an E2 enzyme binding site in DCN1, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0232] Aspect 5: The method of any one of aspects 1-4, wherein the modulation of DCN1 and / or a biological target associated with DCN1 comprises detecting or quantifying the formation of one or more DCN1 covalent adducts.

[0233] Aspect 6: The method of any one of aspects 1-5, wherein the modulation of DCN1 and / or a biological target associated with DCN1 is determined by detecting or quantifying inhibition of the DCN1-E2 enzyme interaction.

[0234] Aspect 7: The method of any one of aspects 1-6, wherein the modulation of DCN1 and / or a biological target associated with DCN1 is determined by detecting or quantifying inhibition of the E2 enzyme binding site in DCN1.

[0235] Aspect 8: The method of any one of aspects 5-7, wherein detecting and / or quantifying inhibition of the DCN1-E2 enzyme interaction and / or inhibition of the E2 enzyme binding site in DCN1 and / or the formation of one or more DCN1 covalent adducts comprises assaying competitive binding of DCN1 (e.g. using a fluorescent or radioactive assay, e.g. fluorescence energy transfer (FRET), e.g., DCN1 time-resolved fluorescence energy transfer (TR-FRET)), and / or amount of DCN1 covalent adducts formed(e.g. using mass spectrometry, e.g., mass spectrometry to determine percent of DCN covalent adducts formed).

[0236] Aspect 9: The method of any one of aspects 3-8, wherein the E2 enzyme binding site in DCN1 is a cysteine (e.g. Cys115).

[0237] Aspect 10: The method of aspect 9, wherein the E2 enzyme is selected from UBC12 and UBE2F, optionally wherein the E2 enzyme is UBC12.

[0238] Aspect 11: The method of any one of aspects 1-10, wherein the modulation of DCN1 and / or a biological target associated with DCN1 comprises inhibition of cullin neddylation, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0239] Aspect 12: The method of any one of aspects 1-11 , wherein the modulation of DCN1 and / or a biological target associated with DCN1 is determined by detecting or quantifying inhibition of cullin neddylation.

[0240] Aspect 13: The method of aspect 12, wherein detecting and / or quantifying inhibition of cullin neddylation comprises assaying changes in the amount of cullin neddylation from a baseline measurement (e.g. an immunoassay including but not limited to enzyme-linked immunosorbent assay (ELISA), western blot, or ALPHALisa assay) and / or formation of an E2 enzyme-NEDD8 complex (e.g. high- throughput screen capable of measuring the formation of a E2 enzyme-NEDD8 enzyme complex), optionally wherein the E2 enzyme is selected from UBC12 and UBE2F, optionally wherein the E2 enzyme is UBC12.

[0241] Aspect 14: The method of aspects 12 or 13, wherein inhibition of cullin neddylation comprises inhibition of the cullin-NEDD8 interaction, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0242] Aspect 15; The method of any one of aspects 12-14, wherein the cullin is selected from cullin-1 (CUL1 ), cullin-2 (CUL2), cullin-3 (CUL3), cullin-4A (CUL4A), cullin-4B (CUL4B), cullin-5 (CUL5), and cullin-7 (CUL7), optionally cullin-3 (CUL3).

[0243] Aspect 16: The method of any one of aspects 12-15, wherein inhibition of cullin neddylation comprises formation of an unneddylated cullin and / or inactive cullin, optionally wherein inhibition of cullin neddylation comprises formation of an unneddylated cullin-3 (CUL3) and / or inactive cullin-3 (CUL3).

[0244] Aspect 17: The method of any one of aspects 1-16, the method further comprising contacting DCN1 and / or the test compound having the ability to covalently bind to Defective in Cullin Neddylation 1 (DCN1) or is suspected to have the ability to bind to DCN1 with one or more cells.

[0245] Aspect 18; The method of aspect 17, wherein the one or more cells comprise or consist of erythroid cells, optionally CD34+erythroid cells, optionally primary CD34+erythroid cells.

[0246] Aspect 19: The method of any one of aspects 1-18, wherein modulation of DCN1 and / or a biological target associated with DCN1 is determined by detecting and / or quantifying fetal hemoglobin (HbF) protein, nucleic acids, and / or function.

[0247] Aspect 20; The method of aspect 19, wherein detecting and / or quantifying fetal hemoglobin (HbF) comprises assaying changes in levels of HbF protein and / or function (e.g. measuring the amount of HbF induction from a baseline measurement (e.g. determining HbF transcript levels by measuring y-globi n gene (HBG) mRNA using Nanostring analysis); and / or amount of HbF protein formed (e.g. using high-throughput liquid chromatography (HPLC) to determine percent of HbF protein formed in cell lysates).

[0248] Aspect 21 : The method of aspect 19 or 20, wherein detecting and / or quantifying fetal hemoglobin (HbF) comprises measuring reactivation of fetal hemoglobin (HbF).

[0249] Aspect 22: The method of any one of aspects 1-21 , wherein the modulation of DCN1 and / or a biological target associated with DCN1 comprises detecting or quantifying the formation of one or more DCN1 covalent adducts and by detecting and / or quantifying fetal hemoglobin (HbF) protein, nucleic acids, and / or function.

[0250] Aspect 23: The method of any one of aspects 1-22, wherein the modulation of DCN1 and / or a biological target associated with DCN1 comprises inhibition of DCN1 and induction of fetalhemoglobin (HbF) protein, nucleic acids, and / or function, optionally wherein inhibition of DCN1 is detected or quantified by the formation of one or more DCN1 covalent adducts, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 in the absence of the test compound.

[0251] Aspect 24: The method of any one of aspects 21-23, wherein fetal hemoglobin (HbF) can be detected and / or quantified and / or measured by assaying one or more of:• expression of HBG1 and / or HBG2; or• presence of subunits of HbF (e.g. one or more a subunits and / or one or more y subunits of HbF).

[0252] Aspect 25: The method of any one of aspects 1-24, wherein the test compound is classified as a candidate compound if a percent of DCN1 covalent adduct formation is increased by about 1-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or greater than about 1000-fold compared to baseline (e.g. percent of DCN1 covalent adduct formation in the absence of the test compound) and fetal hemoglobin (HbF) induction is increased by about 1-fold, 5-fold, 10-fold, 20-fold, 50- fold, 100-fold, 500-fold, 1000-fold, or greater than about 1000-fold compared to baseline (e.g. HbF induction in the absence of the test compound).

[0253] Aspect 26: The method of any one of aspects 1-25, wherein modulation of DCN1 and / or a biological target associated with DCN1 is determined by detecting and / or quantifying Nrf2 protein.

[0254] Aspect 27: The method of any one of aspects 1-26, wherein the test compound and / or candidate compound is capable of increasing a level of Nrf2 protein compared to the level of Nrf2 protein in the absence of the test compound and / or candidate compound.

[0255] Aspect 28: The method of any one of aspects 1-27, wherein the test compound and / or the candidate compound is capable of forming a covalent bond with a cysteine of DCN1 (e.g. Cys115).

[0256] Aspect 29: The method of any one of aspects 8-28, wherein the assaying is performed in vitro, ex vivo, or in vivo.

[0257] Aspect 30: The method of any one of aspects 1-29, wherein the test compound and / or candidate compound comprises one or more electrophilic groups.

[0258] Aspect 31 : The method of aspect 30, wherein the one or more electrophilic groups are selected from nitrile, pentafluorphenyl ester, NHS-ester, acrylamides, cycloalkenyl carboxamides, acyl azides, acyl halides, aldehydes or ketones, alkyl halides, alkyl sulfonates, anhydrides, aryl halides, aziridines, bonorates, carboxylic acids, carbodiimides, diazoalkenes, epoxides, haloacetamides, halotriazines, imido esters, isocyanates, isothiocyanates, maleimides, phosphoramidites, silyl halides, sulfonate esters and sulfonyl halides.

[0259] Aspect 32: The method of any one of aspects 1-31 , wherein the candidate compound is formulated for use in a therapy, optionally wherein the therapy is suitable for treatment or prevention of a hemoglobin-related disorder, optionally wherein the therapy is suitable for treatment or prevention of a sickle cell disorder, disease, or condition, optionally wherein the sickle cell disorder, disease, and / or condition is a sickle cell anemia, optionally wherein the therapy is suitable for treatment or prevention of thalassemia.

[0260] Aspect 33: A method for making a therapeutic composition, comprising:(a) identifying a candidate compound by:(i) obtaining a test compound having the ability to covalently bind to Defective in Cullin Neddylation 1 (DCN1) or is suspected to have the ability to bind to DCN1 ;(ii) contacting the test compound with DCN1 ;(iii) assaying for modulation of DCN1 and / or a biological target associated with DCN1 ; and(iv) classifying the test compound as a candidate compound if modulation of DCN1 and / or the biological target associated with DCN1 is detected; and(b) formulating the candidate compound for use in a therapy.

[0261] Aspect 34: A method for making a therapeutic composition, comprising:(a) identifying a candidate compound by:(i) obtaining a test compound having the ability to covalently bind to Defective in Cullin Neddylation 1 (DCN1) or is suspected to have the ability to bind to DCN1 ;(ii) contacting the test compound with DCN1 ;(iii) assaying for changes in levels of fetal hemoglobin (HbF) protein and / or function; and(iv) classifying the test compound as a candidate compound if the changes in levels of fetal hemoglobin (HbF) protein and / or function are detected; and(b) formulating the candidate compound for use in a therapy.

[0262] Aspect 35: The method of aspect 33 or 34, wherein the modulation of DCN1 and / or the biological target associated with DCN1 comprises inhibition of a DCN1-E2 enzyme interaction, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0263] Aspect 36: The method of any one of aspects 33-35, wherein the modulation of DCN1 and / or a biological target associated with DCN1 comprises inhibition of an E2 enzyme binding site in DCN1 , optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0264] Aspect 37: The method of any one of aspects 33-36, wherein the modulation of DCN1 and / or a biological target associated with DCN1 comprises detecting or quantifying the formation of one or more DCN1 covalent adducts.

[0265] Aspect 38: The method of any one of aspects 33-37, wherein the modulation of DCN1 and / or a biological target associated with DCN1 is determined by detecting or quantifying inhibition of the DCN1-E2 enzyme interaction.

[0266] Aspect 39: The method of any one of aspects 33-38, wherein the modulation of DCN1 and / or a biological target associated with DCN1 is determined by detecting or quantifying inhibition of the E2 enzyme binding site in DCN1.

[0267] Aspect 40: The method of any one of aspects 37-39, wherein detecting and / or quantifying inhibition of the DCN1-E2 enzyme interaction and / or inhibition of the E2 enzyme binding site in DCN1 and / or the formation of one or more DCN1 covalent adducts comprises assaying competitive binding of DCN1 (e.g. using a fluorescent or radioactive assay, e.g. fluorescence energy transfer (FRET), e.g.,DCN1 time-resolved fluorescence energy transfer (TR-FRET)), and / or amount of DCN1 covalent adducts formed (e.g. using mass spectrometry, e.g., mass spectrometry to determine percent of DCN covalent adducts formed).

[0268] Aspect 41 : The method of any one of aspects 36-40, wherein the E2 enzyme binding site in DCN1 is a cysteine (e.g. Cys115).

[0269] Aspect 42: The method of any one of aspects 35-41 , wherein the E2 enzyme is selected from UBC12 and UBE2F, optionally wherein the E2 enzyme is UBC12.

[0270] Aspect 43: The method of any one of aspects 33-42, wherein the modulation ofDCN1 and / or a biological target associated with DCN1 comprises inhibition of cullin neddylation, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0271] Aspect 44.: The method of any one of aspects 33-43, wherein the modulation of DCN1 and / or a biological target associated with DCN1 is determined by detecting or quantifying inhibition of cullin neddylation.

[0272] Aspect 45: The method of aspect 43 or 44, wherein detecting and / or quantifying inhibition of cullin neddylation comprises assaying changes in the amount of cullin neddylation from a baseline measurement (e.g. an immunoassay including but not limited to enzyme-linked immunosorbent assay (ELISA), western blot, or ALPHALisa assay) and / or formation of an E2 enzyme-NEDD8 complex (e.g. high-throughput screen capable of measuring the formation of a E2 enzyme-NEDD8 enzyme complex), optionally wherein the E2 enzyme is selected from UBC12 and UBE2F, optionally wherein the E2 enzyme is UBC12.

[0273] Aspect 46: The method of any one of aspects 43-45, wherein inhibition of cullin neddylation comprises inhibition of the cullin-NEDD8 interaction, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0274] Aspect 47: The method of any one of aspects 43-46, wherein the cullin is selected from cullin-1 (CUL1 ), cullin-2 (CUL2), cullin-3 (CUL3), cullin-4A (CUL4A), cullin-4B (CUL4B), cullin-5 (CUL5), and cullin-7 (CUL7), optionally cullin-3 (CUL3).

[0275] Aspect 48: The method of any one of aspects 43-47, wherein inhibition of cullin neddylation comprises formation of an unneddylated cullin and / or inactive cullin, optionally wherein inhibition of cullin neddylation comprises formation of an unneddylated cullin-3 (CUL3) and / or inactive cullin-3 (CUL3).

[0276] Aspect 49: The method of any one of aspects 33-48, the method further comprising contacting the DCN1 and / or the test compound having the ability to covalently bind to Defective in Cullin Neddylation 1 (DCN1) or is suspected to have the ability to bind to DCN1 with one or more cells.

[0277] Aspect 50: The method of aspect 49, wherein the one or more cells comprise or consist of erythroid cells, optionally CD34+erythroid cells, optionally primary CD34+erythroid cells.

[0278] Aspect 51: The method of any one of aspects 33-50, wherein modulation of DCN1 and / or a biological target associated with DCN1 is determined by detecting and / or quantifying fetal hemoglobin (HbF) protein, nucleic acids, and / or function.

[0279] Aspect 52: The method of aspect 51 , wherein detecting and / or quantifying fetal hemoglobin (HbF) comprises assaying changes in levels of HbF protein and / or function (e.g. measuring the amount of HbF induction from a baseline measurement (e.g. determining HbF transcript levels by measuring y-globi n gene (HBG) mRNA using Nanostring analysis); and / or amount of HbF protein formed (e.g. using high-throughput liquid chromatography (HPLC) to determine percent of HbF protein formed in cell lysates).

[0280] Aspect 53: The method of aspect 51 or 52, wherein detecting and / or quantifying fetal hemoglobin (HbF) comprises measuring reactivation of fetal hemoglobin (HbF).

[0281] Aspect 54: The method of any one of aspects 51-53, wherein fetal hemoglobin (HbF) can be detected and / or quantified and / or measured by assaying one or more of:• expression of HBG1 and / or HBG2; or• presence of subunits of HbF (e.g. one or more a subunits and / or one or more y subunits of HbF).

[0282] Aspect 55: The method of any one of aspects 33-54, wherein the modulation of DCN1 and / or a biological target associated with DCN1 comprises detecting or quantifying the formation ofone or more DCN1 covalent adducts and by detecting and / or quantifying fetal hemoglobin (HbF) protein, nucleic acids, and / or function.

[0283] Aspect 56: The method of any one of aspects 33-55, wherein the modulation of DCN1 and / or a biological target associated with DCN1 comprises inhibition of DCN1 and induction of fetal hemoglobin (HbF) protein, nucleic acids, and / or function, optionally wherein inhibition of DCN1 is detected or quantified by the formation of one or more DCN1 covalent adducts, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 in the absence of the test compound.

[0284] Aspect 57: The method of any one of aspects 33-56, wherein the test compound is classified as a candidate compound if a percent of DCN1 covalent adduct formation is increased by about 1-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or greater than about 1000-fold compared to baseline (e.g. percent of DCN1 covalent adduct formation in the absence of the test compound) and fetal hemoglobin (HbF) induction is increased by about 1-fold, 5-fold, 10-fold, 20-fold, 50- fold, 100-fold, 500-fold, 1000-fold, or greater than about 1000-fold compared to baseline (e.g. HbF induction in the absence of the test compound).

[0285] Aspect 58: The method of any one of aspects 33-57, wherein modulation of DCN1 and / or a biological target associated with DCN1 is determined by detecting and / or quantifying Nrf2 protein.

[0286] Aspect 59: The method of any one of aspects 40-58, wherein the assaying is performed in vitro, ex vivo, or in vivo.

[0287] Aspect 60: The method of any one of aspects 33-59, wherein the test compound and / or candidate compound is capable of increasing a level of Nrf2 protein compared to the level of Nrf2 protein in the absence of the test compound and / or candidate compound.

[0288] Aspect 61 : The method of any one of aspects 33-60, wherein the test compound and / or the candidate compound is capable of forming a covalent bond with a cysteine of DCN1 (e.g. Cys115).

[0289] Aspect 62: The method of any one of aspects 33-61 , wherein the test compound and / or candidate compound comprises one or more electrophilic groups.

[0290] Aspect 63: The method of aspect 62, wherein the one or more electrophilic groups are selected from nitrile, pentafluorphenyl ester, NHS-ester, acrylamides, cycloalkenyl carboxamides, acyl azides, acyl halides, aldehydes or ketones, alkyl halides, alkyl sulfonates, anhydrides, aryl halides, aziridines, bonorates, carboxylic acids, carbodiimides, diazoalkenes, epoxides, haloacetamides, halotriazines, imido esters, isocyanates, isothiocyanates, maleimides, phosphoramidites, silyl halides, sulfonate esters and sulfonyl halides.

[0291] Aspect 64: The method of any one of aspects 33-63, wherein the therapy is suitable for treatment or prevention of a hemoglobin-related disorder, optionally wherein the therapy is suitable for treatment or prevention of a sickle cell disorder, disease, or condition, optionally wherein the sickle cell disorder, disease, and / or condition is a sickle cell anemia, optionally wherein the therapy is suitable for treatment or prevention of thalassemia.

[0292] Aspect 65: A method of preparing a solution comprising one or more Defective in Cullin Neddylation 1 (DCN1) covalent adducts useful for analyzing modulation of DCN1 and / or a biological target associated with DCN1 , comprising:(a) obtaining a solution comprising DCN1 ;(b) contacting the DCN1 of the solution of (a) with a test compound having the ability to covalently bind to DCN1 or is suspected to have the ability to bind to DCN1 to form one or more DCN1 covalent adducts,(c) analyzing modulation of DCN1 and / or a biological target associated produced in the solution of (b).

[0293] Aspect 66: The method of aspect 65, wherein the modulation of DCN1 and / or the biological target associated with DCN1 comprises inhibition of a DCN1-E2 enzyme interaction, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0294] Aspect 67: The method of aspect 65 or 66, wherein the modulation of DCN1 and / or a biological target associated with DCN1 comprises inhibition of an E2 enzyme binding site in DCN1, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0295] Aspect 68: The method of any one of aspects 65-67, wherein the modulation of DCN1 and / or a biological target associated with DCN1 comprises detecting or quantifying the formation of one or more DCN1 covalent adducts.

[0296] Aspect 69: The method of any one of aspects 65-68, wherein the modulation of DCN1 and / or a biological target associated with DCN1 is determined by detecting or quantifying inhibition of the DCN1-E2 enzyme interaction.

[0297] Aspect 70: The method of any one of aspects 65-69, wherein the modulation of DCN1 and / or a biological target associated with DCN1 is determined by detecting or quantifying inhibition of the E2 enzyme binding site in DCN1.

[0298] Aspect 71: The method of any one of aspects 68-70, wherein detecting and / or quantifying inhibition of the DCN1-E2 enzyme interaction and / or inhibition of the E2 enzyme binding site in DCN1 and / or the formation of one or more DCN1 covalent adducts comprises assaying competitive binding of DCN1 (e.g. using a fluorescent or radioactive assay, e.g. fluorescence energy transfer (FRET), e.g. , DCN1 time-resolved fluorescence energy transfer (TR-FRET)), and / or amount of DCN1 covalent adducts formed (e.g. using mass spectrometry, e.g., mass spectrometry to determine percent of DCN covalent adducts formed).

[0299] Aspect 72: The method of any one of aspects 66-71 , wherein the E2 enzyme binding site in DCN1 is a cysteine (e.g. Cys115).

[0300] Aspect 73: The method of aspect72, wherein the E2 enzyme is selected from UBC12 and UBE2F, optionally wherein the E2 enzyme is UBC12.

[0301] Aspect 74: The method of any one of aspects 65-73, wherein the modulation ofDCN1 and / or a biological target associated with DCN1 comprises inhibition of cullin neddylation, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0302] Aspect 75: The method of aspect 74, wherein the modulation of DCN1 and / or a biological target associated with DCN1 is determined by detecting or quantifying inhibition of cullin neddylation.

[0303] Aspect 76: The method of aspect 75, wherein detecting and / or quantifying inhibition of cullin neddylation comprises assaying changes in the amount of cullin neddylation from a baseline measurement (e.g. an immunoassay including but not limited to enzyme-linked immunosorbent assay (ELISA), western blot, or ALPHALisa assay) and / or formation of an E2 enzyme-NEDD8 complex (e.g. high- throughput screen capable of measuring the formation of a E2 enzyme-NEDD8 enzyme complex), optionally wherein the E2 enzyme is selected from UBC12 and UBE2F, optionally wherein the E2 enzyme is UBC12.

[0304] Aspect 77: The method of any one of aspects 74-76, wherein inhibition of cullin neddylation comprises inhibition of the cullin-NEDD8 interaction, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0305] Aspect 78: The method of any one of aspects 74-77, wherein the cullin is selected from cullin-1 (CUL1), cullin-2 (CUL2), cullin-3 (CUL3), cullin-4A (CUL4A), cullin-4B (CUL4B), cullin-5 (CUL5), and cullin-7 (CUL7), optionally cullin-3 (CUL3).

[0306] Aspect 79: The method of any one of aspects 74-78, wherein inhibition of cullin neddylation comprises formation of an unneddylated cullin and / or inactive cullin, optionally wherein inhibition of cullin neddylation comprises formation of an unneddylated cullin-3 (CUL3) and / or inactive cullin-3 (CUL3).

[0307] Aspect 80: The method of any one of aspects 65-79, the method further comprising contacting the DCN1 and / or the test compound having the ability to covalently bind to Defective in Cullin Neddylation 1 (DCN1) or is suspected to have the ability to bind to DCN1 with one or more cells.

[0308] Aspect 81 : The method of aspect 80, wherein the one or more cells comprise or consist of erythroid cells, optionally CD34+erythroid cells, optionally primary CD34+erythroid cells.

[0309] Aspect 82: The method of any one of aspects 65-81 , wherein modulation of DCN1 and / or a biological target associated with DCN1 is determined by detecting and / or quantifying fetal hemoglobin (HbF) protein, nucleic acids, and / or function.

[0310] Aspect 83: The method of aspect 82, wherein detecting and / or quantifying fetal hemoglobin (HbF) comprises assaying changes in levels of HbF protein and / or function (e.g. measuring the amount of HbF induction from a baseline measurement (e.g. determining HbF transcript levels by measuring y-globi n gene (HBG) mRNA using Nanostring analysis); and / or amount of HbF protein formed (e.g. using high-throughput liquid chromatography (HPLC) to determine percent of HbF protein formed in cell lysates).

[0311] Aspect 84: The method of aspect 82 or 83, wherein detecting and / or quantifying fetal hemoglobin (HbF) comprises measuring reactivation of fetal hemoglobin (HbF).

[0312] Aspect 85: The method of any one of aspects 82-84, wherein fetal hemoglobin (HbF) can be detected and / or quantified and / or measured by assaying one or more of:• expression of HBG1 and / or HBG2; or• presence of subunits of HbF (e.g. one or more a subunits and / or one or more y subunits of HbF).

[0313] Aspect 86: The method of any one of aspects 65-85, wherein modulation of DCN1 and / or a biological target associated with DCN1 is determined by detecting and / or quantifying Nrf2 protein.

[0314] Aspect 87: The method of any one of aspects 71-86, wherein the assaying is performed in vitro, ex vivo, or in vivo.

[0315] Aspect 88: The method of any one of aspects 65-88, wherein the method further comprises classifying the test compound as a candidate compound if modulation of DCN1 and / or the biological target associated with DCN1 is detected.

[0316] Aspect 89: The method of any one of aspects 65-88, wherein the modulation of DCN1 and / or a biological target associated with DCN1 comprises detecting or quantifying the formation of one or more DCN1 covalent adducts and by detecting and / or quantifying fetal hemoglobin (HbF) protein, nucleic acids, and / or function.

[0317] Aspect 90: The method of any one of aspects 65-89, wherein the modulation of DCN1 and / or a biological target associated with DCN1 comprises inhibition of DCN1 and induction of fetal hemoglobin (HbF) protein, nucleic acids, and / or function, optionally wherein inhibition of DCN1 is detectedor quantified by the formation of one or more DCN1 covalent adducts, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 in the absence of the test compound.

[0318] Aspect 91 : The method of any one of aspects 65-90, wherein the test compound is classified as a candidate compound if a percent of DCN1 covalent adduct formation is increased by about 1-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or greater than about 1000-fold compared to baseline (e.g. percent of DCN1 covalent adduct formation in the absence of the test compound) and fetal hemoglobin (HbF) induction is increased by about 1-fold, 5-fold, 10-fold, 20-fold, 50- fold, 100-fold, 500-fold, 1000-fold, or greater than about 1000-fold compared to baseline (e.g. HbF induction in the absence of the test compound).

[0319] Aspect 92: The method of any one of aspects65-91 , wherein the test compound and / or candidate compound is capable of increasing a level of Nrf2 protein compared to the level of Nrf2 protein in the absence of the test compound and / or candidate compound.

[0320] Aspect 93: The method of aspect65-92, wherein the test compound and / or the candidate compound is capable of forming a covalent bond with a cysteine of DCN1 (e.g. Cys115).

[0321] Aspect 94: The method of any one of aspects 65-93, wherein the test compound and / or candidate compound comprises one or more electrophilic groups.

[0322] Aspect 95: The method of aspect 94, wherein the one or more electrophilic groups are selected from nitrile, pentafluorphenyl ester, NHS-ester, acrylamides, cycloalkenyl carboxamides, acyl azides, acyl halides, aldehydes or ketones, alkyl halides, alkyl sulfonates, anhydrides, aryl halides, aziridines, bonorates, carboxylic acids, carbodiimides, diazoalkenes, epoxides, haloacetamides, halotriazines, imido esters, isocyanates, isothiocyanates, maleimides, phosphoramidites, silyl halides, sulfonate esters and sulfonyl halides.

[0323] Aspect 96: The method of any one of aspects 88-95, wherein the candidate compound is formulated for use in a therapy, optionally wherein the therapy is suitable for treatment or prevention of a hemoglobin-related disorder, optionally wherein the therapy is suitable for treatment or prevention of a sickle cell disorder, disease, or condition, optionally wherein the sickle cell disorder,disease, and / or condition is a sickle cell anemia, optionally wherein the therapy is suitable for treatment or prevention of thalassemia.

[0324] Aspect 97: A method for making a therapeutic composition, comprising:(a) contacting a test compound having the ability to covalently bind to Defective in Cullin Neddylation 1 (DCN1) or is suspected to have the ability to bind to DCN1 with DCN1 , and quantifying modulation of DCN1 and / or a biological target associated with DCN1 by the test compound;](b) selecting the test compound that modulates DCN1 and / or a biological target associated with DCN1 ;(c) classifying the test compound of (b) as a candidate compound; and(d) formulating the candidate compound for administration to a subject, thereby making the therapeutic composition.

[0325] Aspect 98: A method for making a therapeutic composition, comprising:(a) contacting a test compound having the ability to covalently bind to Defective in Cullin Neddylation 1 (DCN1) or is suspected to have the ability to bind to DCN1 with DCN1 , and quantifying changes in levels of fetal hemoglobin (HbF) protein and / or function by the test compound;(b) selecting the test compound that changes levels of fetal hemoglobin (HbF) protein and / or function;(c) classifying the test compound of (b) as a candidate compound; and(d) formulating the candidate compound for administration to a subject, thereby making the therapeutic composition.

[0326] Aspect 99: The method of aspect 97 or 98, wherein the modulation of DCN1 and / or the biological target associated with DCN1 comprises inhibition of a DCN1-E2 enzyme interaction, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0327] Aspect 100: The method of any one of aspects 97-99, wherein the modulation of DCN1 and / or a biological target associated with DCN1 comprises inhibition of an E2 enzyme binding site in DCN1 , optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or atleast partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0328] Aspect 101 : The method of any one of aspects 97-100, wherein the modulation of DCN1 and / or a biological target associated with DCN1 comprises detecting or quantifying the formation of one or more DCN1 covalent adducts.

[0329] Aspect 102: The method of any one of aspects 97-101 , wherein the modulation of DCN1 and / or a biological target associated with DCN1 is determined by detecting or quantifying inhibition of the DCN1-E2 enzyme interaction.

[0330] Aspect 103: The method of any one of aspects 97-102, wherein the modulation of DCN1 and / or a biological target associated with DCN1 is determined by detecting or quantifying inhibition of the E2 enzyme binding site in DCN1.

[0331] Aspect 104: The method of any one of aspects 101-103, wherein detecting and / or quantifying inhibition of the DCN1-E2 enzyme interaction and / or inhibition of the E2 enzyme binding site in DCN1 and / or the formation of one or more DCN1 covalent adducts comprises assaying competitive binding of DCN1 (e.g. using a fluorescent or radioactive assay, e.g. fluorescence energy transfer (FRET), e.g., DCN1 time-resolved fluorescence energy transfer (TR-FRET)), and / or amount of DCN1 covalent adducts formed (e.g. using mass spectrometry, e.g., mass spectrometry to determine percent of DCN covalent adducts formed).

[0332] Aspect 105: The method of any one of aspects 100-104, wherein the E2 enzyme binding site in DCN1 is a cysteine (e.g. Cys115).

[0333] Aspect 106: The method of any one of aspects 99-105, wherein the E2 enzyme is selected from UBC12 and UBE2F, optionally wherein the E2 enzyme is UBC12.

[0334] Aspect 107: The method of any one of aspects 97-106, wherein the modulation ofDCN1 and / or a biological target associated with DCN1 comprises inhibition of cullin neddylation, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0335] Aspect 108: The method of any one of aspects 97-107, wherein the modulation of DCN1 and / or a biological target associated with DCN1 is determined by detecting or quantifying inhibition of cullin neddylation.

[0336] Aspect 109: The method of aspect 108, wherein detecting and / or quantifying inhibition of cullin neddylation comprises assaying changes in the amount of cullin neddylation from a baseline measurement (e.g. an immunoassay including but not limited to enzyme-linked immunosorbent assay (ELISA), western blot, or ALPHALisa assay) and / or formation of an E2 enzyme-NEDD8 complex (e.g. high- throughput screen capable of measuring the formation of a E2 enzyme-NEDD8 enzyme complex), optionally wherein the E2 enzyme is selected from UBC12 and UBE2F, optionally wherein the E2 enzyme is UBC12.

[0337] Aspect 110: The method of any one of aspects 107-109, wherein inhibition of cullin neddylation comprises inhibition of the cullin-NEDD8 interaction, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0338] Aspect 111 : The method of any one of aspects 107-110, wherein the cullin is selected from cullin-1 (CUL1), cullin-2 (CUL2), cullin-3 (CUL3), cullin-4A (CUL4A), cullin-4B (CUL4B), cullin-5 (CUL5), and cullin-7 (CUL7), optionally cullin-3 (CUL3).

[0339] Aspect 112: The method of any one of aspects 107-111, wherein inhibition of cullin neddylation comprises formation of an unneddylated cullin and / or inactive cullin, optionally wherein inhibition of cullin neddylation comprises formation of an unneddylated cullin-3 (CUL3) and / or inactive cullin-3 (CUL3).

[0340] Aspect 113: The method of any one of aspects 97-112, the method further comprising contacting the DCN1 and / or the test compound having the ability to covalently bind to Defective in Cullin Neddylation 1 (DCN1) or is suspected to have the ability to bind to DCN1 with one or more cells.

[0341] Aspect 114: The method of aspect 113, wherein the one or more cells comprise or consist of erythroid cells, optionally CD34+erythroid cells, optionally primary CD34+erythroid cells.

[0342] Aspect 115: The method of any one of aspects 97-114, wherein modulation of DCN1 and / or a biological target associated with DCN1 is determined by detecting and / or quantifying fetal hemoglobin (HbF) protein, nucleic acids, and / or function.

[0343] Aspect 116: The method of aspect 115, wherein detecting and / or quantifying fetal hemoglobin (HbF) comprises assaying changes in levels of HbF protein and / or function (e.g. measuring the amount of HbF induction from a baseline measurement (e.g. determining HbF transcript levels by measuring y-globi n gene (HBG) mRNA using Nanostring analysis); and / or amount of HbF protein formed (e.g. using high-throughput liquid chromatography (HPLC) to determine percent of HbF protein formed in cell lysates).

[0344] Aspect 117: The method of aspect 115 or 116, wherein detecting and / or quantifying fetal hemoglobin (HbF) comprises measuring reactivation of fetal hemoglobin (HbF).Aspect 118: The method of any one of aspects 115-117, wherein fetal hemoglobin (HbF) can be detected and / or quantified and / or measured by assaying one or more of:• expression of HBG1 and / or HBG2; or• presence of subunits of HbF (e.g. one or more a subunits and / or one or more y subunits of HbF).

[0345] Aspect 119: The method of any one of aspects 97-118, wherein the modulation of DCN1 and / or a biological target associated with DCN1 comprises detecting or quantifying the formation of one or more DCN1 covalent adducts and by detecting and / or quantifying fetal hemoglobin (HbF) protein, nucleic acids, and / or function.

[0346] Aspect 120: The method of any one of aspects 97-119, wherein the modulation of DCN1 and / or a biological target associated with DCN1 comprises inhibition of DCN1 and induction of fetal hemoglobin (HbF) protein, nucleic acids, and / or function, optionally wherein inhibition of DCN1 is detected or quantified by the formation of one or more DCN1 covalent adducts, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 in the absence of the test compound.

[0347] Aspect 121 : The method of any one of aspects 97-120, wherein the test compound is classified as a candidate compound if a percent of DCN1 covalent adduct formation is increased by about 1-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or greater than about 1000-fold compared to baseline (e.g. percent of DCN1 covalent adduct formation in the absence of the test compound) and fetal hemoglobin (HbF) induction is increased by about 1-fold, 5-fold, 10-fold, 20-fold, 50- fold, 100-fold, 500-fold, 1000-fold, or greater than about 1000-fold compared to baseline (e.g. HbF induction in the absence of the test compound).

[0348] Aspect 122: The method of any one of aspects 97-121, wherein modulation of DCN1 and / or a biological target associated with DCN1 is determined by detecting and / or quantifying Nrf2 protein.

[0349] Aspect 123: The method of any one of aspects 104-122, wherein the assaying is performed in vitro, ex vivo, or in vivo.

[0350] Aspect 124: The method of any one of aspects 97-123, wherein the method further comprises classifying the test compound as a candidate compound if modulation of DCN1 and / or the biological target associated with DCN1 is detected.

[0351] Aspect 125: The method of any one of aspects 97-124, wherein the test compound and / or candidate compound is capable of increasing level of Nrf2 protein compared to the level of Nrf2 protein in the absence of the test compound and / or candidate compound.

[0352] Aspect 126: The method of any one of aspects 97-125, wherein the test compound and / or the candidate compound is capable of forming a covalent bond with a cysteine of DCN1 (e.g. Cys115).

[0353] Aspect 127: The method of any one of aspects 97-126, wherein the test compound and / or candidate compound comprises one or more electrophilic groups.

[0354] Aspect 128: The method of aspect 127, wherein the one or more electrophilic groups are selected from nitrile, pentafluorphenyl ester, NHS-ester, acrylamides, cycloalkenyl carboxamides, acyl azides, acyl halides, aldehydes or ketones, alkyl halides, alkyl sulfonates, anhydrides, aryl halides, aziridines, bonorates, carboxylic acids, carbodiimides, diazoalkenes, epoxides, haloacetamides, halotriazines, imido esters, isocyanates, isothiocyanates, maleimides, phosphoramidites, silyl halides, sulfonate esters and sulfonyl halides.

[0355] Aspect 129: The method of any one of aspects 97-128, wherein the candidate compound is formulated for use in a therapy, optionally wherein the therapy is suitable for treatment or prevention of a hemoglobin-related disorder, optionally wherein the therapy is suitable for treatment or prevention of a sickle cell disorder, disease, or condition, optionally wherein the sickle cell disorder, disease, and / or condition is a sickle cell anemia, optionally wherein the therapy is suitable for treatment or prevention of thalassemia.

[0356] Aspect 130: A method of preparing a non-natural cell-based preparation, comprising:(a) combining Defective in Cullin Neddylation 1 (DCN1), an electrophile, and cells (e.g., CD34+cells) to form one or more DCN1 covalent adducts, and(b) measuring an increase in fetal hemoglobin (HbF) induction above baseline, the increase indicating the formation of the preparation.

[0357] Aspect 131 : The method of aspect 130, wherein the increase in HbF induction is caused at least in part by modulation of DCN1 and / or a biological target associated with DCN1 .

[0358] Aspect 132: The method of aspect 131 , wherein the modulation of DCN1 and / or the biological target associated with DCN1 comprises inhibition of a DCN1-E2 enzyme interaction, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0359] Aspect 133: The method of aspectl 31 or 132, wherein the modulation of DCN1 and / or a biological target associated with DCN1 comprises inhibition of an E2 enzyme binding site in DCN1 , optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0360] Aspect 134: The method of aspect 133, wherein the E2 enzyme binding site in DCN1 is a cysteine (e.g. Cys115).

[0361] Aspect 135: The method of any one of aspects 132-134, wherein the E2 enzyme is selected from UBC12 and UBE2F, optionally wherein the E2 enzyme is UBC12.

[0362] Aspect 136: The method of any one of aspects 131-135, wherein the modulation of DCN1 and / or a biological target associated with DCN1 comprises inhibition of cullin neddylation, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0363] Aspect 137: The method of any one of aspects 131-136, wherein inhibition of cullin neddylation comprises inhibition of the cullin-NEDD8 interaction, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0364] Aspect 138: The method of aspect 136 or 137, wherein the cullin is selected from cullin- 1 (CUL1), cullin-2 (CUL2), cullin-3 (CUL3), cullin-4A (CUL4A), cullin-4B (CUL4B), cullin-5 (CUL5), and cullin-7 (CUL7), optionally cullin-3 (CUL3).

[0365] Aspect 139: The method of any one of aspects 136-138, wherein inhibition of cullin neddylation comprises formation of an unneddylated cullin and / or inactive cullin, optionally wherein inhibition of cullin neddylation comprises formation of an unneddylated cullin-3 (CUL3) and / or inactive cullin-3 (CUL3).

[0366] Aspect 140: The method of aspect 139, wherein the one or more cells comprise or consist of erythroid cells, optionally CD34+ erythroid cells, optionally primary CD34+ erythroid cells.

[0367] Aspect 141 : The method of any one of aspects 130-140, wherein the combining of (a) is in a cellular assay, optionally wherein the cellular assay comprises or consists of erythroid cells, optionally CD34+ erythroid cells, optionally primary CD34+ erythroid cells.

[0368] Aspect 142: The method of any one of aspects 130-141 , wherein measuring an increase in fetal hemoglobin (HbF) induction above baseline comprises detecting and / or quantifying fetal hemoglobin (HbF) protein, nucleic acids, and / or function.

[0369] Aspect 143: The method of aspect 142, wherein detecting and / or quantifying fetal hemoglobin (HbF) comprises assaying changes in levels of HbF protein and / or function (e.g. measuring theamount of HbF induction from a baseline measurement (e.g. determining HbF transcript levels by measuring y-globi n gene (HBG) mRNA using Nanostring analysis); and / or amount of HbF protein formed (e.g. using high-throughput liquid chromatography (HPLC) to determine percent of HbF protein formed in cell lysates).

[0370] Aspect 144: The method of aspect 142 or 143, wherein detecting and / or quantifying fetal hemoglobin (HbF) comprises measuring reactivation of fetal hemoglobin (HbF).

[0371] Aspect 145: The method of any one of aspects 130-144, wherein fetal hemoglobin(HbF) can be detected and / or quantified and / or measured by assaying one or more of:• expression of HBG1 and / or HBG2; or• presence of subunits of HbF (e.g. one or more a subunits and / or one or more y subunits of HbF).

[0372] Aspect 146: The method of any one of aspects 131-145, wherein the modulation of DCN1 and / or a biological target associated with DCN1 comprises detecting or quantifying the formation of one or more DCN1 covalent adducts and by detecting and / or quantifying fetal hemoglobin (HbF) protein, nucleic acids, and / or function.

[0373] Aspect 147: The method of any one of aspects 131-146, wherein the modulation of DCN1 and / or a biological target associated with DCN1 comprises inhibition of DCN1 and induction of fetal hemoglobin (HbF) protein, nucleic acids, and / or function, optionally wherein inhibition of DCN1 is detected or quantified by the formation of one or more DCN1 covalent adducts, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 in the absence of the test compound.

[0374] Aspect 148: The method of any one of aspects 130-147, wherein the measuring is performed in vitro, ex vivo, or in vivo.

[0375] Aspect 149: The method of any one of aspects 130-148, wherein the electrophile is capable of forming a covalent bond with a cysteine of DCN1 (e.g. Cys115).

[0376] Aspect 150: The method of any one of aspects 130-149, wherein the electrophile is capable of increasing a level of Nrf2 protein compared to the level of Nrf2 protein in the absence of the electrophile.

[0377] Aspect 151 : The method of any one of aspects 130-150, wherein the electrophile is a test compound comprising one or more electrophilic groups.Aspect 152: The method of aspect 151 , wherein the one or more electrophilic groups are selected from nitrile, pentafluorphenyl ester, NHS-ester, acrylamides, cycloalkenyl carboxamides, acyl azides, acyl halides, aldehydes or ketones, alkyl halides, alkyl sulfonates, anhydrides, aryl halides, aziridines, bonorates, carboxylic acids, carbodiimides, diazoalkenes, epoxides, haloacetamides, halotriazines, imido esters, isocyanates, isothiocyanates, maleimides, phosphoramidites, silyl halides, sulfonate esters and sulfonyl halides.

[0378] Aspect 153: The method of aspect 151 or 152, wherein the method further comprises classifying the test compound as a candidate compound if fetal hemoglobin (HbF) induction is increased by about 1-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or greater than about 1000-fold compared to baseline (e.g. HbF induction in the absence of the test compound).

[0379] Aspect 154: The method of aspect 153, wherein the candidate compound is formulated for use in a therapy, optionally wherein the therapy is suitable for treatment or prevention of a hemoglobin- related disorder, optionally wherein the therapy is suitable for treatment or prevention of a sickle cell disorder, disease, or condition, optionally wherein the sickle cell disorder, disease, and / or condition is a sickle cell anemia, optionally wherein the therapy is suitable for treatment or prevention of thalassemia.

[0380] Aspect 155: A method of preparing non-natural biochemical preparation of Defective in Cullin Neddylation 1 (DCN1) covalent adducts, comprising:(a) combining DCN1 and an electrophile (e.g. via biochemical assay) to form a non-natural biochemical preparation comprising DCN1 covalent adducts;(b) measuring a percent of DCN1 covalent adducts in the preparation of (a) above a baseline;(c) comparing the measurement from (b) and with a reference measurement obtained by combining DCN1 and an electrophile (e.g. via a cell based (e.g. CD34+ cells) assay) to form a non-natural biochemical preparation comprising DCN1 covalent adducts and measuring an increase in fetal hemoglobin (HbF) induction in the preparation above a baseline; and(d) obtaining a positive correlation among a percent of DCN1 covalent adduct formation and an increase in fetal hemoglobin (HbF) induction above baseline.

[0381] Aspect 156: The method of aspect 155, wherein the increase in HbF induction is caused at least in part by modulation of DCN1 and / or a biological target associated with DCN1 .

[0382] Aspect 157: The method of aspect 156, wherein the modulation of DCN1 and / or the biological target associated with DCN1 comprises inhibition of a DCN1-E2 enzyme interaction, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0383] Aspect 158: The method of aspect 156 or 157, wherein the modulation of DCN1 and / or a biological target associated with DCN1 comprises inhibition of an E2 enzyme binding site in DCN1 , optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0384] Aspect 159: The method of any one of aspects 155-158, wherein measuring a percent of DCN1 covalent adducts in the preparation of (a) above baseline comprises detecting and / or quantifying the formation of one or more DCN1 covalent adducts.

[0385] Aspect 160: The method of aspect 159, wherein the detecting and / or quantifying the formation of one or more DCN1 covalent adducts comprises assaying competitive binding of DCN1 (e.g. using a fluorescent or radioactive assay, e.g. fluorescence energy transfer (FRET), e.g., DCN1 time- resolved fluorescence energy transfer (TR-FRET)), and / or amount of DCN1 covalent adducts formed (e.g. using mass spectrometry, e.g., mass spectrometry to determine percent of DON covalent adducts formed).

[0386] Aspect 161 : The method of any one of aspects 158-160, wherein the E2 enzyme binding site in DCN1 is a cysteine (e.g. Cys115).

[0387] Aspect 162: The method of any one of aspects 157-161 , wherein the E2 enzyme is selected from UBC12 and UBE2F, optionally wherein the E2 enzyme is UBC12.

[0388] Aspect 163: The method of any one of aspects 156-162, wherein the modulation ofDCN1 and / or a biological target associated with DCN1 comprises inhibition of cullin neddylation, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition(e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0389] Aspect 164: The method of aspect 163, wherein inhibition of cullin neddylation comprises inhibition of the cullin-NEDD8 interaction, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0390] Aspect 165: The method of aspect 163 or 164, wherein the cullin is selected from cullin- 1 (CUL1), cullin-2 (CUL2), cullin-3 (CUL3), cullin-4A (CUL4A), cullin-4B (CUL4B), cullin-5 (CUL5), and cullin-7 (CUL7), optionally cullin-3 (CUL3).

[0391] Aspect 166: The method of any one of aspects 163-165, wherein inhibition of cullin neddylation comprises formation of an unneddylated cullin and / or inactive cullin, optionally wherein inhibition of cullin neddylation comprises formation of an unneddylated cullin-3 (CUL3) and / or inactive cullin-3 (CUL3).

[0392] Aspect 167: The method of aspect 166, wherein the cells comprise or consist of erythroid cells, optionally CD34+ erythroid cells, optionally primary CD34+ erythroid cells.

[0393] Aspect 168: The method of any one of aspects 156-167, wherein measuring an increase in fetal hemoglobin (HbF) induction in the preparation of (c) above baseline comprises detecting and / or quantifying fetal hemoglobin (HbF) protein, nucleic acids, and / or function.

[0394] Aspect 169: The method of aspect 168, wherein detecting and / or quantifying fetal hemoglobin (HbF) comprises assaying changes in levels of HbF protein and / or function (e.g. measuring the amount of HbF induction from a baseline measurement (e.g. determining HbF transcript levels by measuring y-globin gene (HBG) mRNA using Nanostring analysis); and / or amount of HbF protein formed (e.g. using high-throughput liquid chromatography (HPLC) to determine percent of HbF protein formed in cell lysates).

[0395] Aspect 170: The method of aspect 168 or 169, wherein detecting and / or quantifying fetal hemoglobin (HbF) comprises measuring reactivation of fetal hemoglobin (HbF).1

[0396] Aspect 171 : The method of any one of aspects 150-170, wherein fetal hemoglobin (HbF) can be detected and / or quantified and / or measured by assaying one or more of:• expression of HBG1 and / or HBG2; or• presence of subunits of HbF (e.g. one or more a subunits and / or one or more y subunits of HbF).

[0397] Aspect 172: The method of any one of aspects 156-171, wherein the modulation of DCN1 and / or a biological target associated with DCN1 comprises detecting or quantifying the formation of one or more DCN1 covalent adducts and by detecting and / or quantifying fetal hemoglobin (HbF) protein, nucleic acids, and / or function.

[0398] Aspect 173: The method of any one of aspects 156-172, wherein the modulation of DCN1 and / or a biological target associated with DCN1 comprises inhibition of DCN1 and induction of fetal hemoglobin (HbF) protein, nucleic acids, and / or function, optionally wherein inhibition of DCN1 is detected or quantified by the formation of one or more DCN1 covalent adducts, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 in the absence of the electrophile.

[0399] Aspect 174: The method of any one of aspects 155-173, wherein the measuring is performed in vitro, ex vivo, or in vivo.

[0400] Aspect 175: The method of any one of aspects 155-174, wherein the electrophile is capable of increasing level of Nrf2 protein compared to the level of Nrf2 protein in the absence of the electrophile.

[0401] Aspect 176: The method of any one of aspects 155-175, wherein the electrophile is capable of forming a covalent bond with a cysteine of DCN1 (e.g. Cys115).

[0402] Aspect 177: The method of any one of aspects 155-176, wherein the electrophile is a test compound comprising one or more electrophilic groups.

[0403] Aspect 178: The method of aspect 177, wherein the one or more electrophilic groups are selected from nitrile, pentafluorphenyl ester, NHS-ester, acrylamides, cycloalkenyl carboxamides, acyl azides, acyl halides, aldehydes or ketones, alkyl halides, alkyl sulfonates, anhydrides, aryl halides, aziridines, bonorates, carboxylic acids, carbodiimides, diazoalkenes, epoxides, haloacetamides,halotriazines, imido esters, isocyanates, isothiocyanates, maleimides, phosphoramidites, silyl halides, sulfonate esters and sulfonyl halides.

[0404] Aspect 179: The method of aspect 177 or 178, wherein the method further comprises classifying the test compound as a candidate compound if the percent of DCN1 covalent adduct formation is increased by about 1-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or greater than about 1000-fold compared to baseline (e.g. percent of DCN1 covalent adduct formation in the absence of the test compound) and fetal hemoglobin (HbF) induction is increased by about 1-fold, 5-fold, 10-fold, 20- fold, 50-fold, 100-fold, 500-fold, 1000-fold, or greater than about 1000-fold compared to baseline (e.g. HbF induction in the absence of the test compound).

[0405] Aspect 180: The method of aspect 179, wherein the candidate compound is formulated for use in a therapy, optionally wherein the therapy is suitable for treatment or prevention of a hemoglobin- related disorder, optionally wherein the therapy is suitable for treatment or prevention of a sickle cell disorder, disease, or condition, optionally wherein the sickle cell disorder, disease, and / or condition is a sickle cell anemia, optionally wherein the therapy is suitable for treatment or prevention of thalassemia.

[0406] Aspect 181 : A non-natural cell-based preparation, comprising:(a) increased levels of Defective in Cullin Neddylation (DCN1) covalent adducts, above baseline adduct levels, wherein the adducts derive from DCN1 and an electrophile, and(b) increased levels of induction of fetal hemoglobin (HbF) above baseline induction levels.

[0407] Aspect 182: The preparation of aspect 181, wherein the increased levels of HbF induction is caused at least in part by modulation of DCN1 and / or a biological target associated with DCN1 .

[0408] Aspect 183: The preparation of aspect 181, wherein the modulation of DCN1 and / or the biological target associated with DCN1 comprises inhibition of a DCN1-E2 enzyme interaction, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0409] Aspect 184: The preparation of aspect 182 or 183, wherein the modulation of DCN1 and / or a biological target associated with DCN1 comprises inhibition of an E2 enzyme binding site in DCN1 , optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or atleast partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0410] Aspect 185: The preparation of any one of aspects 182-184, wherein a percent of DCN1 covalent adducts in the preparation of (a) are measured above baseline, and measuring comprises detecting and / or quantifying the formation of one or more DCN1 covalent adducts.

[0411] Aspect 186: The preparation of aspect 185, wherein the detecting and / or quantifying the formation of one or more DCN1 covalent adducts comprises assaying competitive binding of DCN1 (e.g. using a fluorescent or radioactive assay, e.g. fluorescence energy transfer (FRET), e.g., DCN1 time- resolved fluorescence energy transfer (TR-FRET)), and / or amount of DCN1 covalent adducts formed (e.g. using mass spectrometry, e.g., mass spectrometry to determine percent of DON covalent adducts formed).

[0412] Aspect 187: The preparation of any one of aspects 184-186 wherein the E2 enzyme binding site in DCN1 is a cysteine (e.g. Cys115).

[0413] Aspect 188: The preparation of any one of aspects 183-187, wherein the E2 enzyme is selected from UBC12 and UBE2F, optionally wherein the E2 enzyme is UBC12.

[0414] Aspect 189: The preparation of any one of aspects 182-188, wherein the modulation ofDCN1 and / or a biological target associated with DCN1 comprises inhibition of cullin neddylation, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0415] Aspect 190: The preparation of aspect 189, wherein inhibition of cullin neddylation comprises inhibition of the cullin-NEDD8 interaction, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 and / or the biological target associated with DCN1 in the absence of the test compound.

[0416] Aspect 191 : The preparation of aspect 189 or 190, wherein the cullin is selected from cullin- 1 (CUL1 ), cullin-2 (CUL2), cullin-3 (CUL3), cullin-4A (CUL4A), cullin-4B (CUL4B), cullin-5 (CUL5), and cullin-7 (CUL7), optionally cullin-3 (CUL3).

[0417] Aspect 192: The preparation of any one of aspects 189-191 , wherein inhibition of cullin neddylation comprises formation of an unneddylated cullin and / or inactive cullin, optionally wherein inhibition of cullin neddylation comprises formation of an unneddylated cullin-3 (CUL3) and / or inactive cullin-3 (CUL3).

[0418] Aspect 193: The preparation of aspect 192, wherein the cells comprise or consist of erythroid cells, optionally CD34+ erythroid cells, optionally primary CD34+ erythroid cells.

[0419] Aspect 194: The preparation of any one of aspects 181-193, wherein measuring an increase in fetal hemoglobin (HbF) induction in the preparation of (c) above baseline comprises detecting and / or quantifying fetal hemoglobin (HbF) protein, nucleic acids, and / or function.

[0420] Aspect 195: The preparation of aspect 194, wherein detecting and / or quantifying fetal hemoglobin (HbF) comprises assaying changes in levels of HbF protein and / or function (e.g. measuring the amount of HbF induction from a baseline measurement (e.g. determining HbF transcript levels by measuring y-globin gene (HBG) mRNA using Nanostring analysis); and / or amount of HbF protein formed (e.g. using high-throughput liquid chromatography (HPLC) to determine percent of HbF protein formed in cell lysates).

[0421] Aspect 196: The preparation of aspect 194 or 195, wherein detecting and / or quantifying fetal hemoglobin (HbF) comprises measuring reactivation of fetal hemoglobin (HbF). Aspect 197: The preparation of any one of aspects 181-196, wherein fetal hemoglobin (HbF) is detected and / or quantified and / or measured by assaying one or more of:• expression of HBG1 and / or HBG2; or• presence of subunits of HbF (e.g. one or more a subunits and / or one or more y subunits of HbF).

[0422] Aspect 198: The preparation of any one of aspects 182-197, wherein the modulation of DCN1 and / or a biological target associated with DCN1 comprises detecting or quantifying the formation of one or more DCN1 covalent adducts and by detecting and / or quantifying fetal hemoglobin (HbF) protein, nucleic acids, and / or function.

[0423] Aspect 199: The method of any one of aspects 182-198, wherein the modulation of DCN1 and / or a biological target associated with DCN1 comprises inhibition of DCN1 and induction of fetal hemoglobin (HbF) protein, nucleic acids, and / or function, optionally wherein inhibition of DCN1 is detected or quantified by the formation of one or more DCN1 covalent adducts, optionally wherein the inhibition comprises complete inhibition (e.g. about 100% inhibition) or at least partial inhibition (e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% inhibition) compared to the activity of DCN1 in the absence of the electrophile.

[0424] Aspect 200: The preparation of any one of aspects 181-199, wherein the measuring is performed in vitro, ex vivo, or in vivo.

[0425] Aspect 201 : The preparation of any one of aspects 181 -200, wherein the electrophile is capable of increasing level of Nrf2 protein compared to the level of Nrf2 protein in the absence of the electrophile.

[0426] Aspect 202: The preparation of any one of aspects 181 -201 , wherein the electrophile is capable of forming a covalent bond with a cysteine of DCN1 (e.g. Cys115).

[0427] Aspect 203: The preparation of any one of aspects 181 -202, wherein the electrophile is a test compound comprising one or more electrophilic groups.

[0428] Aspect 204: The preparation of aspect 203, wherein the one or more electrophilic groups are selected from nitrile, pentafluorphenyl ester, NHS-ester, acrylamides, cycloalkenyl carboxamides, acyl azides, acyl halides, aldehydes or ketones, alkyl halides, alkyl sulfonates, anhydrides, aryl halides, aziridines, bonorates, carboxylic acids, carbodiimides, diazoalkenes, epoxides, haloacetamides, halotriazines, imido esters, isocyanates, isothiocyanates, maleimides, phosphoramidites, silyl halides, sulfonate esters and sulfonyl halides.

[0429] Aspect 205: The preparation of aspect 203 or 204, wherein the test compound is classified as a candidate compound if the level (e.g. percent) of DCN1 covalent adduct formation is increased by about 1-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or greater than about 1000-fold compared to baseline (e.g. level (e.g. percent) of DCN1 covalent adduct formation in the absence of the test compound) and fetal hemoglobin (HbF) induction is increased by about 1 -fold, 5-fold,10-fold, 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or greater than about 1000-fold compared to baseline (e.g. baseline induction levels, e.g. HbF induction in the absence of the test compound).

[0430] Aspect 206: The preparation of aspect 205, wherein the candidate compound is formulated for use in a therapy, optionally wherein the therapy is suitable for treatment or prevention of a hemoglobin-related disorder, optionally wherein the therapy is suitable for treatment or prevention of a sickle cell disorder, disease, or condition, optionally wherein the sickle cell disorder, disease, or condition is a sickle cell anemia, optionally wherein the therapy is suitable for treatment or prevention of thalassemia.

[0431] Aspect 207: A pharmaceutical composition comprising a candidate compound identified using the methods of any one of aspects 1-180 and a pharmaceutically acceptable carrier.

[0432] Aspect 208: A pharmaceutical composition comprising a solution prepared by the methods of any one of aspects 181-206 and a pharmaceutically acceptable carrier.

[0433] Aspect 209: A method of treating a hemoglobin-related disorder, the method comprising administering a candidate compound identified using the methods of any one of aspectsl -180 to a subject afflicted with the hemoglobin-related disorder and / or is at risk of developing the hemoglobin- related disorder.

[0434] Aspect 210: A method of treating a hemoglobin-related disorder, the method comprising administering a solution prepared by the methods of any one of aspects 181-206 to a subject afflicted with the hemoglobin-related disorder and / or is at risk of developing the hemoglobin-related disorder.

[0435] Aspect 211 : A method of treating a hemoglobin-related disorder, the method comprising administering a pharmaceutical composition of aspect 207 or 208 to a subject afflicted with the hemoglobin-related disorder and / or is at risk of developing the hemoglobin-related disorder.

[0436] Aspect 212: A method of treating a sickle cell disorder, disease, or condition, the method comprising administering a candidate compound identified using the methods of any one of aspects 1-180 to a subject afflicted with the sickle cell disorder, disease, or condition and / or is at risk of developing the sickle cell disorder, disease, or condition.

[0437] Aspect 213: A method of treating a sickle cell disorder, disease, or condition, the method comprising administering a solution prepared by the methods of any one of aspects 181-206 to asubject afflicted with the sickle cell disorder, disease, or condition and / or is at risk of developing the sickle cell disorder, disease, or condition.

[0438] Aspect 214: A method of treating a sickle cell disorder, disease, or condition, the method comprising administering a pharmaceutical composition of aspect 207 or 208 to a subject afflicted with the sickle cell disorder, disease, or condition and / or is at risk of developing the sickle cell disorder, disease, or condition.

[0439] Aspect 215: The method of any one of aspects 212-215, wherein the sickle cell disorder, disease, or condition is a sickle cell anemia.

[0440] Aspect 216: A method of treating thalassemia, the method comprising administering a candidate compound identified using the methods of any one of aspects 1-180 to a subject afflicted with thalassemia and / or is at risk of developing thalassemia.

[0441] Aspect 217: A method of treating thalassemia, the method comprising administering a solution prepared by the methods of any one of aspects 181-206 to a subject afflicted with thalassemia and / or is at risk of developing thalassemia.

[0442] Aspect 218: A method of treating thalassemia, the method comprising administering a pharmaceutical composition of aspect 207 or 208 to a subject afflicted with the thalassemia and / or is at risk of developing thalassemia.INCORPORATION BY REFERENCE

[0443] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entireties to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0444] All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

Claims

CLAIMSWhat is claimed is:1 . A method for identifying a candidate compound, comprising:(a) obtaining a test compound having the ability to covalently bind to Defective in Cullin Neddylation 1 (DCN1) or is suspected to have the ability to bind to DCN1 ;(b) contacting the test compound with DCN1;(c) assaying for modulation of DCN1 and / or a biological target associated with DCN1 ; and(d) classifying the test compound as a candidate compound if modulation of the DCN1 and / or a biological target associated with DCN1 is detected.

2. A method for identifying a candidate compound, comprising:(a) obtaining a test compound having the ability to covalently bind to Defective in Cullin Neddylation 1 (DCN1) or is suspected to have the ability to bind to DCN1 ;(b) contacting the test compound with DCN1;(c) assaying for changes in levels of fetal hemoglobin (HbF) protein and / or function; and(d) classifying the test compound as a candidate compound if changes in levels of fetal hemoglobin (HbF) protein and / or function are detected.

3. A method for making a therapeutic composition, comprising:(a) identifying a candidate compound by:(i) obtaining a test compound having the ability to covalently bind to Defective in Cullin Neddylation 1 (DCN1) or is suspected to have the ability to bind to DCN1 ;(ii) contacting the test compound with DCN1 ;(iii) assaying for modulation of DCN1 and / or a biological target associated with DCN1 ; and(iv) classifying the test compound as a candidate compound if modulation of DCN1 and / or the biological target associated with DCN1 is detected; and(b) formulating the candidate compound for use in a therapy.

4. A method for making a therapeutic composition, comprising:(a) identifying a candidate compound by:(i) obtaining a test compound having the ability to covalently bind to Defective in Cullin Neddylation 1 (DCN1) or is suspected to have the ability to bind to DCN1 ;(ii) contacting the test compound with DCN1 ;(iii) assaying for changes in levels of fetal hemoglobin (HbF) protein and / or function; and(iv) classifying the test compound as a candidate compound if the changes in levels of fetal hemoglobin (HbF) protein and / or function are detected; and(b) formulating the candidate compound for use in a therapy.

5. A method for making a therapeutic composition, comprising:(a) contacting a test compound having the ability to covalently bind to Defective in Cullin Neddylation 1 (DCN1) or is suspected to have the ability to bind to DCN1 with DCN1 , and quantifying modulation of DCN1 and / or a biological target associated with DCN1 by the test compound;(b) selecting the test compound that modulates DCN1 and / or a biological target associated with DCN1 ;(c) classifying the test compound of (b) as a candidate compound; and(d) formulating the candidate compound for administration to a subject, thereby making the therapeutic composition.

6. A method for making a therapeutic composition, comprising:(a) contacting a test compound having the ability to covalently bind to Defective in Cullin Neddylation 1 (DCN1) or is suspected to have the ability to bind to DCN1 with DCN1 , and quantifying changes in levels of fetal hemoglobin (HbF) protein and / or function by the test compound;(b) selecting the test compound that changes levels of fetal hemoglobin (HbF) protein and / or function;(c) classifying the test compound of (b) as a candidate compound; and(d) formulating the candidate compound for administration to a subject, thereby making the therapeutic composition.

7. A pharmaceutical composition comprising a candidate compound identified using the methods of any one of claims 1-6 and a pharmaceutically acceptable carrier.

8. A method of treating a hemoglobin-related disorder, the method comprising administering a pharmaceutical composition of claim 7 to a subject afflicted with the hemoglobin-related disorder and / or is at risk of developing the hemoglobin-related disorder.

9. A method of treating a sickle cell disorder, disease, or condition, the method comprising administering a pharmaceutical composition of claim 7 to a subject afflicted with the sickle cell disorder, disease, or condition and / or is at risk of developing the sickle cell disorder, disease, or condition.

10. A method of inducing fetal hemoglobin (HbF) in a subject, the method comprising administering to a subject a compound that binds Defective in Cullin Neddylation 1 (DCN1) in an amount sufficient to induce HbF in the subject.

11. A method of treating a hemoglobin-related disorder, the method comprising administering to a subject a compound that binds Defective in Cullin Neddylation 1 (DCN1) in an amount sufficient to treat the hemoglobin-related disorder.

12. A method of treating sickle cell disease, the method comprising administering to a subject a compound that binds Defective in Cullin Neddylation 1 (DCN1) in an amount sufficient to treat sickle cell disease.

13. A method of treating thalassemia, the method comprising administering to a subject a compound that binds Defective in Cullin Neddylation 1 (DCN1) in an amount sufficient to treat thalassemia.

14. The method of any one of claims 8-13, wherein the compound covalently binds Defective in Cullin Neddylation 1 (DCN1).

15. A pharmaceutical composition comprising a compound that binds Defective in Cullin Neddylation 1 (DCN1) in an amount sufficient to treat a hemoglobin-related disorder, and a pharmaceutically acceptable carrier.

16. A pharmaceutical composition comprising a compound that binds Defective in Cullin Neddylation 1 (DCN1) in an amount sufficient to treat sickle cell disease, and a pharmaceutically acceptable carrier.

17. A pharmaceutical composition comprising a compound that binds Defective in Cullin Neddylation 1 (DCN1) in an amount sufficient to treat thalassemia, and a pharmaceutically acceptable carrier.

18. A method inducing fetal hemoglobin (HbF) in a subject, the method comprising modifying a reversible inhibitor of Defective in Cullin Neddylation 1 (DCN1) with a warhead, thereby generating a covalent inhibitor of DCN1 , and administering the covalent inhibitor of DCN1 to a subject in an amount sufficient to induce HbF in the subject.

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