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Principal Investigator: YA HA
Organization: YALE UNIVERSITY
Fiscal Year: 2024
Award: $334,149
Funding agency: National Institute of General Medical Sciences
Project Summary / Abstract
The objective of the proposed research is to elucidate the biochemical mechanisms underlying the exquisite
substrate binding and catalytic specificity of two phosphatidylinositol phosphate 5-kinases (PIP5K, PIKfyve).
The PIPK family of lipid kinases include PIP5K (type 1), PIP4K (type 2) and PIKfyve (type 3), and is primarily
responsible for converting phosphatidylinositol monophosphate lipids into PI(4,5)P2 and PI(3,5)P2. Despite
sequence homology, these kinases are highly selective in substrate binding [PIP5K binds PI(4)P, PIP4K binds
PI(5)P, and PIKfyve binds PI(3)P] and in catalytic activity [PIP5K and PIKfyve phosphorylate the C5 hydroxyl of
the lipid's inositol head group, whereas PIP4K phosphorylates the C4 hydroxyl]. We and others have
previously identified two structural elements within the kinase domain, the specificity loop and a conserved
PIP-binding motif, that contribute to substrate selectivity, but how these two elements cooperate to confer
kinase specificity remains undefined at the structural level. In aim 1, we plan crosslinking strategies to stabilize
the specificity loop to facilitate co-crystallization with lipid substrates. We also plan to generate and crystallize a
minimalistic catalytic core domain of PIKfyve. In aim 2, we propose genetic and chemical biological
experiments to examine the role of PIKfyve in the life cycle of SARS-CoV-2. Several large-scale drug
repurposing programs have identified apilimod, a PIKfyve inhibitor, as a top lead in suppressing SARS-CoV-2
replication in cell culture (a phase II clinical trial of apilimod in treating COVID-19 is ongoing at the Yale Center
for Clinical Investigation). This discovery, together with earlier observations that apilimod also reduces infection
by Ebola and Marburg viruses, has generated great interest in pharmacologically targeting PIKfyve. Drawing
on structural and biochemical knowledge about the lipid kinase family, as well as chemical tools previously
developed to target PIP4K, we have discovered a new class of potent PIKfyve inhibitors and plan to use them
together with apilimod to interrogate how PIKfyve inhibition disrupts SARS-CoV-2 infection. All previously
known PIKfyve inhibitors are structurally related to apilimod, and their binding mode to the lipid kinase is
unknown. The new inhibitor class is significant because it not only adds confidence to the proposed
involvement of PIKfyve in SARS-CoV-2 infection, but also has a known binding mode to PIPK, which should
facilitate future optimization by medicinal chemistry.
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><Binding><Biochemical><Biological><Biology><CCR><COVID-19 infection><COVID-19 therapy><COVID-19 treatment><COVID-19 virus><COVID-19 virus infection><COVID19 infection><COVID19 virus><Catalytic Core><Catalytic Domain><Catalytic Region><Catalytic Site><Catalytic Subunit><Cell Body><Cell Culture Techniques><Cell Line><CellLine><Cells><Chemicals><Chiro-Inositol><CoV-2><CoV2><Complex><Crystallization><Crystallographies><Crystallography><Development><Disease><Disorder><EBOV><Ebola virus><Ebola-like Viruses><Elements><Endosomes><Epithelial Cells><Family><Frankfurt-Marburg Syndrome Virus><Future><Genetic><Goals><Head><Host Factor><Host Factor Protein><Human><Hydroxyl><Hydroxyl Radical><In Vitro><Infection><Inositide Phospholipids><Inositol><Inositol Phosphoglycerides><Inositol Phospholipids><Integration Host Factors><Kinases><Knock-in><Knock-out><Knockout><Knowledge><Lead><Life Cycle><Life Cycle Stages><Lipids><Lung><Lung Respiratory System><Marburg><Marburg virus><Marburg-like Viruses><Marburgvirus><Medicinal Chemistry><Membrane><Mesoinositol><Modern Man><Molecular Configuration><Molecular Conformation><Molecular Interaction><Molecular Stereochemistry><PTDINS5P><Pattern><Pb element><Pharmaceutic Chemistry><Pharmaceutical Chemistry><Pharmacological Study><Pharmacology Study><Phase 2 Clinical Trials><Phase II Clinical Trials><Phosphatidyl Inositol><Phosphatidyl Inositol Phosphates><Phosphatidylinositol Phosphates><Phosphatidylinositols><Phosphoinositides><Phosphorylation><Phosphotransferase Gene><Phosphotransferases><Polyphosphoinositides><Position><Positioning Attribute><Protein Phosphorylation><PtdIns><PtdIns-5-P><Receptosomes><Refractory><Research><Resolution><Role><SARS corona virus 2><SARS-CO-V2><SARS-COVID-2><SARS-CoV-2><SARS-CoV-2 infection><SARS-CoV-2 inhibitor><SARS-CoV-2 therapy><SARS-CoV-2 treatment><SARS-CoV2><SARS-CoV2 infection><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><Sequence Homology><Severe Acute Respiratory Coronavirus 2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome coronavirus 2 infection><Severe acute respiratory syndrome coronavirus 2 inhibitor><Severe acute respiratory syndrome related corona virus 2><Specificity><Strains Cell Lines><Structure><Substrate Specificity><Testing><Transphosphorylases><Viral><Viral Activity><Viral Function><Viral Physiology><Work><Wuhan coronavirus><analog><biologic><block SARS-CoV-2><block severe acute respiratory syndrome coronavirus 2><cell culture><cell cultures><clinical center><clinical investigation><conformation><conformational><conformational state><conformationally><conformations><coronavirus disease 2019 infection><coronavirus disease 2019 therapy><coronavirus disease 2019 treatment><coronavirus disease 2019 virus><coronavirus disease-19 virus><crosslink><cultured cell line><developmental><drug repositioning><drug repurposing><experiment><experimental research><experimental study><experiments><hCoV19><heavy metal Pb><heavy metal lead><infected with COVID-19><infected with COVID19><infected with SARS-CoV-2><infected with SARS-CoV2><infected with coronavirus disease 2019><infected with severe acute respiratory syndrome coronavirus 2><inhibit SARS-CoV-2><inhibit severe acute respiratory syndrome coronavirus 2><inhibitor><interest><knockin><life course><member><membrane structure><mutant><nCoV2><pharmacologic><phase II protocol><phosphatidylinositol 5-phosphate><programs><pulmonary><repurposing agent><repurposing medication><resolutions><severe acute respiratory syndrome coronavirus 2 therapy><severe acute respiratory syndrome coronavirus 2 treatment><small molecular inhibitor><small molecule inhibitor><social role><tool><trafficking><treat COVID-19><treat SARS-CoV-2><treat coronavirus disease 2019><treat severe acute respiratory syndrome coronavirus 2>