Pikfyve assay

Abstract

The invention provides for a screening assay-using a selective proteolysis of recombinant full-length PIKfyve at an endogenous caspase 3 cleavage site to liberate the 34 kDa C -terminal kinase domain. Despite instability of the kinase domain to isolation, the liberated kinase domain is stable in the presence of the N-terminal PIKfyve protein fragment and can thus be treated with compounds of interest and analyzed by mass spectrometry, such as RapidFire-MS, with each injection taking under one minute. The assay was established to triage small molecules for covalent modification of PIKfyve. The assay was also established using cysteine-mutant proteins, where the reactive cysteine was replaced with an inert amino acid which when run through the same experimental protocol, was used to confirm adduct formation at the target cysteine.

Description

[0001] Docket No. 4390.3003 WO

[0002] PIKfyve Assay

[0003] RELATED APPLICATION

[0004] This application claims the benefit of U.S. Provisional Application No. 63 / 734,427 filed December 16, 2024. The entire contents of the above-referenced application are incorporated by reference herein.

[0005] SEQUENCE LISTING

[0006] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing file, entitled 4390.3003WO_SeqList.xml, was created on December 12, 2025 and is 3,863 bytes in size. The information in electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

[0007] BACKGROUND OF THE INVENTION

[0008] Phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) is named after its function and domain structure (phosphoinositide kinase for five position containing a FYVE finger) and is a lipid kinase that phosphorylates phosphatidylinositol-3-phosphate (PI(3)P), producing PI(3,5)P2, which is involved in cellular processes including membrane trafficking and cytoskeletal reorganization. Inhibiting PIKfyve can increase PI3P levels to enable early endosomal maturation, which may lead to the clearance of dipeptide repeat proteins. Inhibiting PIKfyve may be useful in the treatment of various diseases or conditions such as viral infections or many neurological diseases. Thus, it is desirable to screen compounds for PIKfyve inhibition. The present inventors desire an assay that can efficiently screen inhibitors that can covalently bind to PIKfyve.

[0009] Direct detection of covalent adducts to a target protein by intact mass analysis (IMA) has been used in covalent drug discovery and can efficiently screen >10,000 compound libraries against small- to medium-sized recombinant proteins (10-50 kDa). Flying large intact proteins (>100 kDa), however, requires specialized techniques that are not amenable to screening-scale mass spectrometry. Assessing covalent adducts on a protein the size of PIKfyve (230 kDa) is most commonly achieved by peptide sequence mapping - a low- throughput approach requiring large amounts of protein, which is also prone to false negatives. Overcoming these challenges is particularly important for PIKfyve as expressing the C-terminal kinase domain alone results in an aggregation-prone construct that cannot be inhibited by known reversible inhibitors, which has likely hampered previous covalent drug Docket No. 4390.3003 WO discovery efforts. Improvements to high throughput screening assays for covalent modifiers of PIKfyve are desirable.

[0010] SUMMARY OF THE INVENTION

[0011] The inventors have now discovered that screening-scale IMA was enabled through the selective proteolysis of recombinant full-length PIKfyve at an endogenous caspase 3 cleavage site to liberate the 34 kDa C-terminal kinase domain. Despite instability of the kinase domain to isolation, the liberated kinase domain is stable in the presence of the N-terminal PIKfyve protein fragment and can thus be treated with compounds of interest and analyzed by mass spectrometry, such as RapidFire-MS, with each injection taking under one minute. The assay was established to triage small molecules for covalent modification of PIKfyve. The assay was also established using cysteine-mutant proteins, where the reactive cysteine was replaced with an inert amino acid which when run through the same experimental protocol, was used to confirm adduct formation at the target cysteine.

[0012] The assay was validated with desthiobiotin-iodoacetamide, a cysteine-reactive probe. The substituted iodoacetamide reagent reacts with accessible cysteines, including those in the kinase domain of PIKfyve. Digested full-length PIKfyve protein, generated using the selective proteolysis method described, was treated with the probe and a dose- and timedependent increase in protein modification, consistent with cysteine alkylation, was observed.

[0013] The combination of selective proteolysis of PIKfyve to generate intact stabilized C- and N-terminal domain fragments through digestion by caspase 3, along with evaluation of the compound treated digests by RapidFire-MS enables rapid and robust identification of covalent binders and has greatly accelerated the discovery and optimization of covalent PIKfyve inhibitors.

[0014] BRIEF DESCRIPTION OF THE DRAINGS

[0015] The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.

[0016] FIG. lA and IB are examples of a DMSO-treated sample (no modification observed) and compound-treated samples. Docket No. 4390.3003 WO

[0017] FIG 2 A and 2B are representative spectra for the reaction of PIKfyve WT kinase domain with desthiobiotin iodoacetamide, returning single and double modification of the protein. FIG 1C: 30 min reaction with 100 mM desthiobiotin iodoacetamide. FIG ID: 120 min reaction with 100 mM desthiobiotin iodoacetamide.

[0018] FIG 3 is a representative deconvoluted spectra of enzyme (Caspase3) obtained using RapidFire-TOF-MS at 1 pM protein concentration.

[0019] DETAILED DESCRIPTION OF THE INVENTION

[0020] The invention relates to a PIKfyve assay for screening PIKfyve inhibitors. The assay uses the C-terminal kinase domain of PIKfyve comprising a number of reactive cysteine residues.

[0021] Human PIKfyve sequences are described in the literature, including:

[0022] MATDDKTSPTLDSANDLPRSPTSPSHLTHFKPLTPDQDEPPFKSAYSSFVNLFRFNKERA

[0023] EGGQGEQQPLSGSWTSPQLPSRTQSVRSPTPYKKQLNEELQRRSSALDTRRKAEPTFGGH

[0024] DPRTAVQLRSLSTVLKRLKEIMEGKSQDSDLKQYWMPDSQCKECYDCSEKFTTFRRRHHC

[0025] RLCGQIFCSRCCNQEIPGKFMGYTGDLRACTYCRKIALSYAHSTDSNSIGEDLNALSDSA

[0026] CSVSVLDPSEPRTPVGSRKASRNIFLEDDLAWQSLIHPDSSNTPLSTRLVSVQEDAGKSP

[0027] ARNRSASITNLSLDRSGSPMVPSYETSVSPQANRTYVRTETTEDERKILLDSVQLKDLWK

[0028] KICHHSSGMEFQDHRYWLRTHPNCIVGKELVNWLIRNGHIATRAQAIAIGQAMVDGRWLD

[0029] CVSHHDQLFRDEYALYRPLQSTEFSETPSPDSDSVNSVEGHSEPSWFKDIKFDDSDTEQI

[0030] AEEGDDNLANSASPSKRTSVSSFQSTVDSDSAASISLNVELDNVNFHIKKPSKYPHVPPH

[0031] PADQKEYLISDTGGQQLSISDAFIKESLFNRRVEEKSKELPFTPLGWHHNNLELLREENG

[0032] EKQAMERLLSANHNHMMALLQQLLHSDSLSSSWRDIIVSLVCQVVQTVRPDVKNQDDDMD

[0033] IRQFVHIKKIPGGKKFDSVVVNGFVCTKNIAHKKMSSCIKNPKILLLKCSIEYLYREETK

[0034] FTCIDPIVLQEREFLKNYVQRIVDVRPTLVLVEKTVSRIAQDMLLEHGITLVINVKSQVL

[0035] ERISRMTQGDLVMSMDQLLTKPHLGTCHKFYMQIFQLPNEQTKTLMFFEGCPQHLGCHK

[0036] LRGGSDYELARVKEILIFMICVAYHSQLEISFLMDEFAMPPTLMQNPSFHSLIEGRGHEG

[0037] AVQEQYGGGSIPWDPDIPPESLPCDDSSLLELRIVFEKGEQENKNLPQAVASVKHQEHST Docket No. 4390.3003 WO

[0038] TACPAGLPCAFFAPVPESLLPLPVDDQQDALGSEQPETLQQTVVLQDPKSQIRAFRDPLQ

[0039] DDTGLYVTEEVTSSEDKRKTYSLAFKQELKDVILCISPVITFREPFLLTEKGMRCSTRDY

[0040] FAEQVYWSPLLNKEFKEMENRRKKQLLRDLSGLQGMNGSIQAKSIQVLPSHELVSTRIAE

[0041] HLGDSQSLGRMLADYRARGGRIQPKNSDPFAHSKDASSTSSGQSGSKNEGDEERGLILSD

[0042] AVWSTKVDCLNPINHQRLCVLFSSSSAQSSNAPSACVSPWIVTMEFYGKNDLTLGIFLER

[0043] YCFRPSYQCPSMFCDTPMVHHIRRFVHGQGCVQIILKELDSPVPGYQHTILTYSWCRICK QVTPVVALSNESWSMSFAKYLELRFYGHQYTRRANAEPCGHSIHHDYHQYFSYNQMVASF

[0044] SYSPIRLLEVCVPLPKIFIKRQAPLKVSLLQDLKDFFQKVSQVYVAIDERLASLKTDTFS

[0045] KTREEKMEDIFAQKEMEEGEFKNWIEKMQARLMSSSVDTPQQLQSVFESLIAKKQSLCEV

[0046] LQAWNNRLQDLFQQEKGRKRPSVPPSPGRLRQGEESKISAMDASPRNISPGLQNGEKEDR

[0047] FLTTLSSQSSTSSTHLQLPTPPEVMSEQSVGGPPELDTASSSEDVFDGHLLGSTDSQVKE

[0048] KSTMKAIFANLLPGNSYNPIPFPFDPDKHYLMYEHERVPIAVCEKEPSSIIAFALSCKEY

[0049] RNALEELSKATQWNSAEEGLPTNSTSDSRPKSSSPIRLPEMSGGQTNRTTETEPQPTKKA

[0050] SGMLSFFRGTAGKSPDLSSQKRETLRGADSAYYQVGQTGKEGTENQGVEPQDEVDGGDTQ

[0051] KKQLINPHVELQFSDANAKFYCREYYAGEFHKMREVILDSSEEDFIRSLSHSSPWQARGG

[0052] KSGAAFYATEDDRFILKQMPRLEVQSFLDFAPHYFNYITNAVQQKRPTALAKILGVYRIG

[0053] YKNSQNNTEKKLDLLVMENLFYGRKMAQVFDLKGSLRNRNVKTDTGKESCDVVLLDENLL

[0054] KMVRDNPLYIRSHSKAVLRTSIHSDSHFLSSHLIIDYSLLVGRDDTSNELVVGIIDYIRT

[0055] FTWDKKLEMVVKSTGILGGQGKMPTVVSPELYRTRFCEAMDKYFLMVPDHWTGLGLNC

[0056] SEQID NO. 1.

[0057] Shisheva et al. Cloning, Characterization, and Expression of a Novel Zn2+-Binding FYVE Finger-Containing Phosphoinositide Kinase in Insulin-Sensitive Cells, Mol. Cell Biol. 1999 Jan; 19(l):623-634 doi: 10.1128 / mcb. l9.1.623 and https: / / rest.uniprot.org / uniprotkb / Q9Y2I7.fasta which are incorporated herein by reference in its entirety. A “PIKfyve protein” is defined herein as a lipid kinase that phosphorylates phosphatidylinositol-3-phosphate (PI(3)P), producing PI(3,5)P2, Preferred PIKfyve proteins are wild-type (WT) human proteins, including the protein of SEQ ID NO. 1 and isoforms Docket No. 4390.3003 WO thereof. However, PIKfyve proteins isolated from other species, such as non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). Non-natural PIKfyve proteins can also be used, including proteins having at least 90%, such as at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO 1.

[0058] The assay of the invention uses a C-terminal fragment of a PIKfyve protein comprising a number of reactive cysteine residues. The fragment is preferably less than 50 kDa, preferably between 10 and 50 kDa, such as between 20 and 40 kDa or about 35 kDa. Preferably, the fragment is a caspase 3 digest of a PIKfyve protein.

[0059] The C-terminal fragment of a PIKfyve protein is stabilized in the presence of a N- terminal fragment. The N-terminal fragment is preferably a caspase 3 digest of a PIKfyve protein or a stabilizing fragment thereof.

[0060] The assay optionally uses a PIKfyve mutant wherein a PIKfyve protein or fragment thereof comprises a substitution of cysteine with an inert amino acid, such as glycine, alanine, serine, isoleucine, leucine, valine, or phenylalanine, preferably alanine. An “inert amino acid” is defined herein as an amino acid with a side chain which will not react with a test compound or significantly impact the folding of the protein.

[0061] The protein is preferably stored in a buffer solution, such as HEPES.

[0062] The assay protocol includes sample preparation. For example, the following experimental protocol can be used:

[0063] 1- Prepare protein solution for Caspase 3 digest.

[0064] • Optimal concentration of PIKfyve protein 0.75 pM. Other concentrations can be used, such as between 0.1 to 10 pM.

[0065] • Reaction volume for each sample can be 15 pL, for example.

[0066] 2- Dilute the protein solution using the storage buffer. In some cases, PIKfyve proteins are shipped at a desired concentration. In such cases, no dilution is required.

[0067] 3- Prepare the enzyme solution by diluting Caspase 3 in digestion buffer. The optimal molar ratio of Caspase3 to PIKfyve can be 1 :50. Other ratios can be between 1 :5 to 1 : 100. The enzyme solution can be diluted to 450 nM, for example. Docket No. 4390.3003 WO

[0068] 4- Combine the enzyme solution and PIKfyve protein solution. The optimum volumetric ratio of Caspase 3 to protein solution is 1 :30. Other ratios can be between 1 :5 to 1 : 100. 33uL of Caspase3 solution (450nM) can be added to the PIKfyve solution, resulting in a final concentration of 15nM of Caspase3 for the digestion step.

[0069] 5- Incubate the digestion solution. The digestion can preferably be completed at room temperature (25 °C). Other temperatures can be between 5 and 40°C or conditions that do not result in the deactivation of the caspase. The digestion can conveniently be completed in 4 to 24 hours, such as 8 to 15, preferably about 12 hours or overnight.

[0070] The assay contemplates contacting a compound or compounds with the protein fragment under conditions where the compound can react with the protein fragment. Preferred compounds are substituted with a reactive electrophilic moiety. Compounds can be run at a fixed stoichiometry to protein, of 5: 1 or 10: 1, or run in a dose response experiment over time to determine kinetic parameters of covalent inhibitors (kinact / Ki).

[0071] Compounds for screening can be derived from or selected from a library or can be created rationally. For example, one can identify potential inhibition scaffolds and substitute electrophilic moieties on the scaffold to then screen covalent binders. For example, the compounds can possess the pharmacophores or scaffolds of the compounds described in US 20230146395 or US 20230133203.

[0072] The compound or compounds can be dissolved in a suitable solvent. Water-soluble compounds can be dissolved in an aqueous solution, such as a buffer. Water-insoluble compounds can be dissolved in an organic solvent, preferably an organic solvent miscible in water, such as dimethylsulfoxide (DMSO) or ethanol. DMSO% in the reaction mixture can be between 1 and 25%, preferably 3 to 10%, more preferably 5% by volume. A reaction volume of 15 pL and volume of compound solution is 0.75 pL has been found to be suitable.

[0073] The compound(s) can be incubated with the PIKfyve fragment at 25°C. Other temperatures can be between 5 and 40°C. The reaction can be allowed to proceed until substantially complete. Typically, the reaction is completed in 5 min to 4 h. After completion of the reaction, a quenching solution can optionally be added. An example of a quenching solution is an acid, such as formic acid. Dilution of the solution can be desirable. For example, LC-MS grade water can be used to dilute the PIKfyve protein fragment solution by 3-fold. Docket No. 4390.3003 WO

[0074] The molecular weight of the resulting PIKfyve protein fragment can then be determined. For example, the solution can be subjected to mass spectrometry. Time of flight mass spectrometry can be used to accurately measure the mass of the reaction product and, optionally, protein fragment. RapidFire® high throughput LC / MS by Agilent using company instructions has obtained good results. The molecular weight of the reaction product can then be compared to the molecular weight of the protein fragment starting material. An increase in molecular weight is indicative of the test compound covalently binding to the PIKfyve protein fragment. Compounds that are determined to be binding can then be screened for inhibitory activity.

[0075] The assay can also include a control comprising (a) incubating a compound with a C- terminal fragment of a PIKfyve protein variant characterized by an amino acid substitution of the target cysteine under conditions where the compound can react with wild type protein, thereby preparing a control product and (b) determining a molecular weight of the control product. If the molecular weight of the control product is the same as the reaction product, then it can be concluded that the test compound is not covalently bound to the native cysteine.

[0076] The figures are examples of a DMSO-treated sample (FIG lA no modification observed) and compound-treated samples (FIG IB), showing >95% single modification in IMA with a 470 Da mass shift.

[0077] Reaction of PIKfyve with desthiobiotin iodoacetamide probe

[0078] Wild-type, full-length, PIKfyve was run through the caspase 3 digestion procedure and treated with the electrophile desthiobiotin iodoacetamide. 0.75 mM PIKfyve was treated with 5, 20, and 100 mM desthiobiotin iodoacetamide for 30 and 120 min at 25 °C. The sample was then quenched with 0.2% v / v formic acid and analyzed by RapidFire TOF-MS. Single and double modification at two cysteines of the C-terminal kinase domain are reported in Table. Docket No. 4390.3003 WO

[0079] FIG 2 A and 2B are representative spectra for the reaction of PIKfyve WT kinase domain with desthiobiotin iodoacetamide, returning single and double modification of the protein. FIG 1C: 30 min reaction with 100 mM desthiobiotin iodoacetamide. FIG ID: 120 min reaction with 100 mM desthiobiotin iodoacetamide.

[0080] PIKfyve Protein Expression and Purification:

[0081] Full length wild type PIKfyve (aa 1-2098; UniProt ID: Q9Y2I7-1) construct with N- terminal 3xFLAG and C-terminal His tag was cloned into pcDNA3.1 vector and transfected into Expi293F cells (Thermo, Cat. No. A14527). Protein was expressed for 72 hours at 30°C after transfection. The cells were harvested by centrifugation and flash-frozen for storage.

[0082] Frozen cell pellets were re-suspended in ice-cold lysis buffer (50 mM HEPES, 500 mM NaCl, 10% Glycerol, 0.1% Tween 20, 1 mM TCEP, 1 / 200 (v / v) HaltTM protease inhibitor cocktail (Thermo, Cat. No. 78429), 0.1 mg Benzonase / IL culture, pH 8.0) and transferred into a Dounce homogenizer and lysed with 10 strokes. The supernatant after centrifugation was incubated with Anti-FLAG-Gl affinity resin (GenScript, Cat. No. L00432) for 1.5 hours. Then, the resin was washed 3 times with lOx bed volumes of wash buffer (50 mM HEPES, 300 mM NaCl, 10% Glycerol, 1 mM TCEP, pH 8.0) and eluted 3 times with 2x bed volume of elution buffer (50 mM HEPES, 300 mM NaCl, 10% Glycerol, 1 mM TCEP, 300 pg / mL FLAG peptide [GenScript, Cat. No. RP10586], pH 8.0). The elution fractions were pooled and further purified using a HiLoad 16 / 600 SuperoseTM 6 pg (Cytiva, Cat. No. 29323952) gel filtration column pre-equilibrated with gel filtration buffer (50 mM HEPES, 600 mM NaCl, 5% Glycerol, 0.5 mM TCEP, pH 8.0). Peak fractions of interest were collected and flash frozen in liquid nitrogen for storage at -80°C. Quality control after one freeze-thaw cycle was performed to confirm protein was a monodisperse sample with no indication of aggregation or oligomerization using a SuperoseTM 6 Increase 5 / 150 GL (Cytiva, Cat. No. 29091597) gel filtration column pre-equilibrated with gel filtration buffer.

[0083] Enzymatic Assay for inhibition of PIKfyve

[0084] The inhibition of Recombinant PIKfyve protein [hPIKfyve( 1-2098) or commercial hPIKfyve (Carna, cat no. 11-118)] was measured using ADP-Glo kinase-assay kit (Promega). Docket No. 4390.3003 WO

[0085] 300 nl of compounds (dissolved in DMSO) in serial dilution were transferred to 384-well Optiplates (Perkin Elmer) using Echo 650 series liquid handler (Beckman Coulter). 5 pl of Recombinant Plkfyve in assay buffer (25 mM HEPES pH 7.5, 10 mM MgC12, 1 pM CaC12, 2 mM DTT, 0.05% BSA, and 0.002% Triton-XlOO) is added to the compounds and preincubated for 120 minutes at room temperature. To initiate the reaction, 5 ul of ATP and PI(3)P:PS substrate are added. For hPIKfyve( 1-2098) the final concentration of recombinant protein, ATP, and Substrate are 5 nM, 20 uM, and 62.5:500 uM, respectively while for hPIKfyve (Carna), the final concentration of recombinant protein, ATP, and Substrate are 5 nM, 50 uM, and 62.5:500 uM, respectively. The plate was incubated for 90 minutes at room temperature then 10 ul of the ADP-GLOTM reagent was added and incubated for 60 minutes. 20 ul of the kinase detection reagent was added and incubated for an additional 30 minutes then luminescence (LUM) was detected using the Pherastar microplate reader (BMG Labtech). The enzyme activity was normalized using control wells containing negative control (0% inhibition) of 3% DMSO or positive control (100% inhibition) of 10 pM YM- 201636 (MedChemExpress).

[0086] % Inhibition = 100 x [l-(LUMcmpd - LUMpos) / (LUMneg - LUMpos)]

[0087] Where LUMcmpd, LUMpos, and LUMneg are the relative luminescence units of compound, YM-201636, and DMSO treated wells respectively. The %inhibition values were plotted as a function of compound concentration and the IC50 values of compounds were determined using a four-parameter logistic fit Y = Min + [(Max-Min) / (l+(X / IC50)Hill slope)] or three-parameter fit if Max or Min of curves were fixed to 100% or 0% respectively.

[0088] Enzyme quality control (QC) test:

[0089] This experiment determined the molecular weight of recombinant Caspase 3 (Casp3). This QC test involves injection of the enzyme at a high concentration (250 nM to 1 pM) to confirm protein identity.

[0090] The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference. Docket No. 4390.3003 WO

[0091] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

Docket No. 4390.3003 WOCLAIMSWhat is claimed:

1. A method of screening compounds comprising:(a) incubating a compound with a C-terminal fragment of a PIKfyve protein in the presence of an N-terminal domain of a PIKfyve protein, preferably a cleaved N-terminal domain, under conditions where the compound can react with the protein fragment, thereby preparing a reaction product that is amenable to screening-scale mass spectrometry; and(b) determining a molecular weight of the reaction product; wherein a molecular weight of the reaction product exceeding the molecular weight of the C-terminal fragment of a PIKfyve protein indicates binding of the compound to the C- terminal fragment of a PIKfyve protein.

2. The method of claim 1 wherein the C-terminal fragment of a PIKfyve protein comprises a reactive Cys residue.

3. The method of claim 2 wherein the C- terminal fragment is less than 50 kDa, preferably between 10 and 50 kDa, such as between 20 and 40 kDa or about 35 kDa.

4. The method of claim 3 wherein the C-terminal fragment is a caspase 3 digest of a PIKfyve protein.

5. The method of claim 4 wherein the PIKfyve protein is human PIKfyve.

6. The method of claim 5 wherein the PIKfyve protein comprises SEQ ID NO. 1.

7. The method of any preceding claim where the compound comprises an electrophilic moiety.

8. The method of claim 7 wherein a library of compounds is screened.

9. The method of any preceding claim wherein the molecular weight of the reaction product is determined using time of flight mass spectrometry.Docket No. 4390.3003 WO10. The method of any preceding claim further comprising the step of measuring the molecular weight of the unreacted C-terminal fragment of a PIKfyve protein.

11. The method of any preceding claim further comprising the steps of (c) incubating a compound with a C-terminal fragment of a PIKfyve protein variant characterized by an amino acid substitution of the targeted amino acid under conditions where the compound can react with wild type protein, thereby preparing a control product and (d) determining a molecular weight of the control product.

12. The method of any preceding claim wherein PIKfyve inhibitory activity of the compound is determined.

13. A PIKfyve protein variant or C-terminal fragment thereof wherein the targeted amino acid (preferably a cysteine) is substituted by an inert amino acid, such as alanine.

14. A C-terminal fragment of a PIKfyve protein comprising a kinase domain.

15. The fragment of claim 14 characterized by a molecular weight of less than 50 kDa, preferably between 10 and 50 kDa, such as between 20 and 40 kDa or about 35 kDa.

16. The fragment of claim 15 wherein the C-terminal fragment is a caspase 3 digest of a PIKfyve protein.

17. The fragment of claim 14, 15 or 16 wherein the PIKfyve protein is human PIKfyve.

18. The fragment of any one of claims 14-17 wherein the PIKfyve protein comprises SEQ ID NO. 1.

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