26 research outputs found
Development of Cell Permeable NanoBRET Probes for the Measurement of PLK1 Target Engagement in Live Cells
PLK1 is a protein kinase that regulates mitosis and is both an important oncology drug target and a potential antitarget of drugs for the DNA damage response pathway or anti-infective host kinases. To expand the range of live cell NanoBRET target engagement assays to include PLK1, we developed an energy transfer probe based on the anilino-tetrahydropteridine chemotype found in several selective PLK inhibitors. Probe 11 was used to configure NanoBRET target engagement assays for PLK1, PLK2, and PLK3 and measure the potency of several known PLK inhibitors. In-cell target engagement for PLK1 was in good agreement with the reported cellular potency for the inhibition of cell proliferation. Probe 11 enabled the investigation of the promiscuity of adavosertib, which had been described as a dual PLK1/WEE1 inhibitor in biochemical assays. Live cell target engagement analysis of adavosertib via NanoBRET demonstrated PLK activity at micromolar concentrations but only selective engagement of WEE1 at clinically relevant doses
Panorganismal metabolic response modeling of an experimental Echinostoma caproni infection in the mouse.
Published versio
Association of Variants in the SPTLC1 Gene With Juvenile Amyotrophic Lateral Sclerosis
Importance: Juvenile amyotrophic lateral sclerosis (ALS) is a rare form of ALS characterized by age of symptom onset less than 25 years and a variable presentation.Objective: To identify the genetic variants associated with juvenile ALS.Design, Setting, and Participants: In this multicenter family-based genetic study, trio whole-exome sequencing was performed to identify the disease-associated gene in a case series of unrelated patients diagnosed with juvenile ALS and severe growth retardation. The patients and their family members were enrolled at academic hospitals and a government research facility between March 1, 2016, and March 13, 2020, and were observed until October 1, 2020. Whole-exome sequencing was also performed in a series of patients with juvenile ALS. A total of 66 patients with juvenile ALS and 6258 adult patients with ALS participated in the study. Patients were selected for the study based on their diagnosis, and all eligible participants were enrolled in the study. None of the participants had a family history of neurological disorders, suggesting de novo variants as the underlying genetic mechanism.Main Outcomes and Measures: De novo variants present only in the index case and not in unaffected family members.Results: Trio whole-exome sequencing was performed in 3 patients diagnosed with juvenile ALS and their parents. An additional 63 patients with juvenile ALS and 6258 adult patients with ALS were subsequently screened for variants in the SPTLC1 gene. De novo variants in SPTLC1 (p.Ala20Ser in 2 patients and p.Ser331Tyr in 1 patient) were identified in 3 unrelated patients diagnosed with juvenile ALS and failure to thrive. A fourth variant (p.Leu39del) was identified in a patient with juvenile ALS where parental DNA was unavailable. Variants in this gene have been previously shown to be associated with autosomal-dominant hereditary sensory autonomic neuropathy, type 1A, by disrupting an essential enzyme complex in the sphingolipid synthesis pathway.Conclusions and Relevance: These data broaden the phenotype associated with SPTLC1 and suggest that patients presenting with juvenile ALS should be screened for variants in this gene.</p
Collagen Prolyl 4-Hydroxylase as a Therapeutic Target
Collagen is the dominant protein of the extracellular matrix. Its distinguishing feature is a three-stranded helix of great tensile strength. (2S,4R)-4-Hydroxyproline residues are essential for the stability of this triple helix. These residues arise from the post-translational modification of (2S)-proline residues by collagen prolyl 4-hydroxylases (CP4Hs), which are members of the Fe(II)- and α-ketoglutarate (AKG)-dependent dioxygenase family. Here, we provide a framework for the inhibition of CP4Hs as the basis for treating fibrotic diseases and cancer metastasis. We begin with a summary of the structure and enzymatic reaction mechanism of CP4Hs. Then, we review the metal ions, metal chelators, mimetics of AKG and collagen strands, and natural products that are known to inhibit CP4Hs. Our focus is on inhibitors with potential utility in the clinic. We conclude with a prospectus for more effective inhibitors.National Institutes of Health (U.S.) (Grant R01 AR044276
Human Collagen Prolyl 4‑Hydroxylase Is Activated by Ligands for Its Iron Center
Collagen is the most abundant protein
in animals. The posttranslational
hydroxylation of proline residues in collagen contributes greatly
to its conformational stability. Deficient hydroxylation is associated
with a variety of disease states, including scurvy. The hydroxylation
of proline residues in collagen is catalyzed by an Fe(II)- and α-ketoglutarate-dependent
dioxygenase, collagen prolyl 4-hydroxylase (CP4H). CP4H has long been
known to suffer oxidative inactivation during catalysis, and the cofactor
ascorbate (vitamin C) is required to reactivate the enzyme by reducing
its iron center from Fe(III) to Fe(II). Herein, we report on the discovery
of the first synthetic activators of CP4H. Specifically, we find that
2,2′-bipyridine-4-carboxylate and 2,2′-bipyridine-5-carboxylate
serve as ligands for the iron center in human CP4H that enhance the
rate of ascorbate-dependent reactivation. This new mode of CP4H activation
is available to other biheteroaryl compounds but does not necessarily
extend to other prolyl 4-hydroxylases. As collagen is weakened in
many indications, analogous activators of CP4H could have therapeutic
benefits
Prolyl 4Hydroxylase: Substrate Isosteres in Which an (E)- or (Z)Alkene Replaces the Prolyl Peptide Bond
Development of Cell Permeable NanoBRET Probes for the Measurement of PLK1 Target Engagement in Live Cells
PLK1 is a protein kinase that regulates mitosis and is both an important oncology drug target and a potential antitarget of drugs for the DNA damage response pathway or anti-infective host kinases. To expand the range of live cell NanoBRET target engagement assays to include PLK1, we developed an energy transfer probe based on the anilino-tetrahydropteridine chemotype found in several selective PLK inhibitors. Probe 11 was used to configure NanoBRET target engagement assays for PLK1, PLK2, and PLK3 and measure the potency of several known PLK inhibitors. In-cell target engagement for PLK1 was in good agreement with the reported cellular potency for the inhibition of cell proliferation. Probe 11 enabled the investigation of the promiscuity of adavosertib, which had been described as a dual PLK1/WEE1 inhibitor in biochemical assays. Live cell target engagement analysis of adavosertib via NanoBRET demonstrated PLK activity at micromolar concentrations but only selective engagement of WEE1 at clinically relevant doses
Translating PROTAC chemical series optimization into functional outcomes underlying BRD7 and BRD9 protein degradation
Selective Inhibition of Collagen Prolyl 4‑Hydroxylase in Human Cells
Collagen is the most abundant protein
in animals. Its overproduction
is associated with fibrosis and cancer metastasis. The stability of
collagen relies on post-translational modifications, the most prevalent
being the hydroxylation of collagen strands by collagen prolyl 4-hydroxylases
(CP4Hs). Catalysis by CP4Hs enlists an iron cofactor to convert proline
residues to 4-hydroxyproline residues, which are essential for the
conformational stability of mature collagen. Ethyl 3,4-dihydroxybenzoate
(EDHB) is commonly used as a “P4H” inhibitor in cells,
but suffers from low potency, poor selectivity, and off-target effects
that cause iron deficiency. Dicarboxylates of 2,2′-bipyridine
are among the most potent known CP4H inhibitors but suffer from a
high affinity for free iron. A screen of biheteroaryl compounds revealed
that replacing one pyridyl group with a thiazole moiety retains potency
and enhances selectivity. A diester of 2-(5-carboxythiazol-2-yl)pyridine-5-carboxylic
acid is bioavailable to human cells and inhibits collagen biosynthesis
at concentrations that neither cause general toxicity nor disrupt
iron homeostasis. These data anoint a potent and selective probe for
CP4H and a potential lead for the development of a new class of antifibrotic
and antimetastatic agents