SGC-PIKFYVE-1 Chemical probe for PIKfyve

Click here to obtain this probe.

For any inquiries please contact proberequests@thesgc.org.

group new

Overview

Chemical structures of SGC-PIKFYVE-1 and the negative control SGC-PIKFYVE-1N.

From a library of indolyl pyrimidinamines, we identified a highly potent and cell-active chemical probe (SGC-PIKFYVE-1) that inhibits phosphatidylinositol-3-phosphate 5-kinase (PIKfyve). Comprehensive evaluation of kinome-wide selectivity confirmed that this PIKfyve probe demonstrates excellent selectivity. A structurally similar indolyl pyrimidinamine (SGC-PIKFYVE-1N) was characterized as a negative control that does not inhibit PIKfyve and exhibits exceptional selectivity when profiled broadly. Our PIKfyve chemical probe disrupts multiple phases of the β-coronavirus lifecycle: viral replication and viral entry. Versus published PIKfyve inhibitors, our scaffold is a distinct chemotype that lacks the canonical morpholine hinge-binder of classical lipid kinase inhibitors and has non-overlapping kinase off-targets. Our chemical probe set can be used by the community to further characterize the role of PIKfyve in virology and explore its other roles as well.

Figure 1: Kinome tree with PIKfyve highlighted as a red circle. Illustration is reproduced courtesy of Eurofins DiscoverX (http://treespot.discoverx.com)

Biological activity summary:
• Enzymatic assay (SignalChem): PIKfyve IC₅₀ = 6.9 nM
• Cellular data (NanoBRET): PIKfyve IC₅₀ = 4.0 nM
• Only 8/403 kinases with PoC <10 when screened at 1 μM

Properties

Physical and chemical properties for SGC-PIKFYVE-1
Molecular weight	331.4230
Molecular formula	C20H21N5
IUPAC name	11-(3-(dimethylamino)prop-1-yn-1-yl)-5,6,7,8-tetrahydropyrimido[4',5':3,4]cyclohepta[1,2-b]indol-2-amine
MollogP	1.52
PSA	66.01
No. of chiral centers	0
No. of rotatable bonds	1
No. of hydrogen bond acceptors	3
No. of hydrogen bond donors	2
Storage	Stable as a solid at room temperature. DMSO stock solutions (up to 10 mM) are stable at -20oC
Dissolution	Soluble in DMSO up to 10 mM

Physical and chemical properties for SGC-PIKFYVE-1N
Molecular weight	304.3970
Molecular formula	C19H20N4
IUPAC name	12-cyclopropyl-6,7,8,9-tetrahydro-5H-pyrimido[4',5':3,4]cycloocta[1,2-b]indol-2-amine
MollogP	2.47
PSA	62.77
No. of chiral centers	0
No. of rotatable bonds	1
No. of hydrogen bond acceptors	2
No. of hydrogen bond donors	2
Storage	Stable as a solid at room temperature. DMSO stock solutions (up to 10 mM) are stable at -20oC
Dissolution	Soluble in DMSO up to 10 mM

SMILES:

SGC-PIKFYVE-1: CN(CC#CC1=CC2=C(NC3=C2C4=C(C=NC(N)=N4)CCC3)C=C1)C

SGC-PIKFYVE-1N: NC1=NC2=C(CCCCC3=C2C4=C(C=CC(C5CC5)=C4)N3)C=N1

InChI:

SGC-PIKFYVE-1: InChI=1S/C20H21N5/c1-25(2)10-4-5-13-8-9-16-15(11-13)18-17(23-16)7-3-6-14-12-22-20(21)24-19(14)18/h8-9,11-12,23H,3,6-7,10H2,1-2H3,(H2,21,22,24)

SGC-PIKFYVE-1N: InChI=1S/C19H20N4/c20-19-21-10-13-3-1-2-4-16-17(18(13)23-19)14-9-12(11-5-6-11)7-8-15(14)22-16/h7-11,22H,1-6H2,(H2,20,21,23)

InChIKey:

SGC-PIKFYVE-1: DORZPJWJOBMQKC-UHFFFAOYSA-N

SGC-PIKFYVE-1N: XIVFOAKTTGQHLJ-UHFFFAOYSA-N

Selectivity Profile

SGC-PIKFYVE-1 was profiled in the KINOMEscan assay against 403 wild-type kinases at 1 μM. Only 8 kinases showed PoC <10 giving an S₁₀(1 μM) = 0.02. When the PoC <35 fraction was examined, 20 kinases were included (S₃₅(1 μM) = 0.05). Potential off-targets within the S₃₅(1 μM) fraction as well as PIKfyve were tested using a NanoBRET target engagement assay and via biochemical enzymatic assays where an assay was available. Data corresponding with off-target kinase activity is shown in the table below.

Kinase	DiscoverX PoC value	Assay format	IC50 or Kd value (nM)
PIKfyve	0.1	Enzymatic and NB	6.9 and 4.0
MYLK4	1.8	Enzymatic and NB	66 and 270
MEK1	4.5	Enzymatic	>10000
RIPK5	5.2	Enzymatic	>10000
IRAK3	9.2	NT	-
MEK2	9.6	Enzymatic	>10000
DYRK1A	9.9	Enzymatic	2040
YSK4	9.9	Enzymatic	4020
ULK3	11	Enzymatic	>10000
MEK4	12	Enzymatic	>10000
HASPIN	13	Enzymatic	1400
STK16	14	Enzymatic	560
CLK2	20	Enzymatic	290
CDK7	20	Enzymatic	>10000
IRAK4	21	Enzymatic	4500
AURKB	23	Enzymatic	1400
DYRK1B	26	Enzymatic	380
CLK1	30	Enzymatic	420
RIOK2	32	NT	-
CLK4	34	Enzymatic	440
PIP4K2C	53	NB and binding	>10000 and 1900
MAP4K5	96	Enzymatic and NB	89 and >10000

Figure 2: SGC-PIKFYVE-1 was profiled in the KINOMEscan assay against 403 wild-type kinases at 1 μM and off-target kinases inhibited PoC <35 were tested in an orthogonal assay. Rows colored green are PIKfyve and the two kinases with enzymatic IC50 values within 30-fold of the PIKfyve enzymatic IC₅₀ value: MYLK4 and MAP4K5.

SGC-PIKFYVE-1N was also tested in the DiscoverX panel and 1 kinase had a PoC <35 (S₃₅(1 μM) = 0.002). The negative control was sent to SignalChem for testing in enzyme assay for PIKfyve. All results are in the table below.

Kinase	DiscoverX PoC value	Assay format	IC50 value (nM)
MAST1	32	NT	NT
PIKfyve	88	Enzymatic and NB	715 and >10000

Figure 3: SGC-PIKFYVE-1N was profiled in the KINOMEscan assay against 403 wild-type kinases at 1 μM and a follow-up PIKfyve enzymatic assay was done to confirm no activity.

Cell-based Assay Data

A NanoBRET assay was utilized to assess the binding affinity of SGC-PIKFYVE-1 to PIKfyve. The negative control shows no binding affinity for PIKfyve.

Figure 4: SGC-PIKFYVE-1 and SGC-PIKFYVE-1N were profiled in the PIKfyve NanoBRET assay.

References

Drewry, D. H.; Potjewyd, F. M.; Smith, J. L.; Dickmander, R. J.; Bayati, A.; Howell, S.; Taft-Benz, S.; Min, S. M.; Hossain, M. A.; Heise, M.; McPherson, P. S.; Moorman, N. J.; Axtman, A. D. Identification and utilization of a chemical probe to interrogate the roles of PIKfyve in the lifecycle of β-coronaviruses. J Med Chem 2022, ASAP; doi: 10.1021/acs.jmedchem.2c00697.

Drewry, D. H.; Potjewyd, F. M.; Smith, J. L.; Dickmander, R. J.; Bayati, A.; Howell, S.; Taft-Benz, S. A.; Hossain, M. A.; Heise, M. T.; McPherson, P. S.; Moorman, N. J.; Axtman, A. D. Identification and utilization of a chemical probe to interrogate the roles of PIKfyve in the lifecycle of β-coronaviruses. ChemRxiv 2022. doi: 10.26434/chemrxiv-2022-bj274.