Chondrocytic and pharmacokinetic properties of Phlpp inhibitors

Taylor, Earnest L.; Weaver, Samantha R.; Zars, Elizabeth L.; Turner, Cortney A.; Buhrow, Sarah A.; Reid, Joel M.; Bradley, Elizabeth W.

doi:10.1016/j.ocarto.2021.100190

Cited by 5 publications

(8 citation statements)

References 32 publications

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“…The efficacy of NSC117079 and NSC45586 in promoting matrix metabolism and reducing catabolism has previously been demonstrated in articular cartilage 36 . Yet, the role of PHLPP and its inhibitors is known to be highly tissue‐specific, and our data suggest that NSC45586, but not NSC117079, promoted NP cell survival and metabolism, maintained the notochordal morphology, enhanced the expression of NP phenotypic marker KRT19, and counteracted cell apoptosis in mouse organ cultures.…”

Section: Discussionsupporting

confidence: 59%

Differential efficacy of two small molecule PHLPP inhibitors to promote nucleus Pulposus cell health

Zhang,

Gordon,

Joseph

et al. 2023

JOR Spine

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BackgroundIntervertebral disc (IVD) degeneration is associated with chronic back pain. We previously demonstrated that the phosphatase pleckstrin homology domain and leucine‐rich repeat protein phosphatase (PHLPP) 1 was positively correlated with IVD degeneration and its deficiency decelerated IVD degeneration in both mouse IVDs and human nucleus pulposus (NP) cells. Small molecule PHLPP inhibitors may offer a translatable method to alleviate IVD degeneration. In this study, we tested the effectiveness of the two PHLPP inhibitors NSC117079 and NSC45586 in promoting a healthy NP phenotype.MethodsTail IVDs of 5‐month‐old wildtype mice were collected and treated with NSC117079 or NSC45586 under low serum conditions ex vivo. Hematoxylin & eosin staining was performed to examine IVD structure and NP cell morphology. The expression of KRT19 was analyzed through immunohistochemistry. Cell apoptosis was assessed by TUNEL assay. Human NP cells were obtained from patients with IVD degeneration. The gene expression of KRT19, ACAN, SOX9, and MMP13 was analyzed via real time qPCR, and AKT phosphorylation and the protein expression of FOXO1 was analyzed via immunoblot.ResultsIn a mouse IVD organ culture model, NSC45586, but not NSC117079, preserved vacuolated notochordal cell morphology and KRT19 expression while suppressing cell apoptosis, counteracting the degenerative changes induced by serum deprivation, especially in males. Likewise, in degenerated human NP cells, NSC45586 increased cell viability and the expression of KRT19, ACAN, and SOX9 and reducing the expression of MMP13, while NSC117079 treatment only increased KRT19 expression. Mechanistically, NSC45586 treatment increased FOXO1 protein expression in NP cells, and inhibiting FOXO1 offset NSC45586‐induced regenerative potential, especially in males.ConclusionsOur study indicates that NSC45586 was effective in promoting NP cell health, especially in males, suggesting that PHLPP plays a key role in NP cell homeostasis and that NSC45586 might be a potential drug candidate in treating IVD degeneration.

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Section: Discussionsupporting

confidence: 59%

Differential efficacy of two small molecule PHLPP inhibitors to promote nucleus Pulposus cell health

Zhang,

Gordon,

Joseph

et al. 2023

JOR Spine

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“…( Figure 1 C). Notably, the two most prolifically investigated PHLPP inhibitors have a BBB score < 2, and the frontrunner for potential translation (NSC177079 [ 6 , 7 , 19 , 20 ]) had the lowest value of 0.86. The 22 unexplored inhibitors were then grouped and ordered by IC50 to block the PP2C domain of PHLPPs [ 6 ].…”

Section: Resultsmentioning

confidence: 99%

“…These compounds are non-selective for PHLPP1 and PHLPP2. Two compounds (NSC117079 and NSC45586) were subsequently investigated by others but are unlikely candidates for CNS therapeutics [ 7 , 19 , 20 ]. Specifically, their chemical properties are not ideal for targeting the brain (e.g., their BBB score < 2) and some bind albumin.…”

Section: Discussionmentioning

confidence: 99%

“…We used a 1 mg/kg (low) and 3 mg/kg (high) dose for in vivo studies. Dosing was based on a similar dose range reported with NSC117079 and NSC45586 to induce a biological effect in animals [ 20 ]. To further strengthen our in vivo studies, we used two different brain injury models across two different species, to assess the generalizability and robustness of neuroprotection.…”

Section: Discussionmentioning

confidence: 99%

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PHLPP Inhibitor NSC74429 Is Neuroprotective in Rodent Models of Cardiac Arrest and Traumatic Brain Injury

et al. 2022

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Pleckstrin homology domain and leucine rich repeat protein phosphatase (PHLPP) knockout mice have improved outcomes after a stroke, traumatic brain injury (TBI), and decreased maladaptive vascular remodeling following vascular injury. Thus, small-molecule PHLPP inhibitors have the potential to improve neurological outcomes in a variety of conditions. There is a paucity of data on the efficacy of the known experimental PHLPP inhibitors, and not all may be suited for targeting acute brain injury. Here, we assessed several PHLPP inhibitors not previously explored for neuroprotection (NSC13378, NSC25247, and NSC74429) that had favorable predicted chemistries for targeting the central nervous system (CNS). Neuronal culture studies in staurosporine (apoptosis), glutamate (excitotoxicity), and hydrogen peroxide (necrosis/oxidative stress) revealed that NSC74429 at micromolar concentrations was the most neuroprotective. Subsequent testing in a rat model of asphyxial cardiac arrest, and in a mouse model of severe TBI, showed that serial dosing of 1 mg/kg of NSC74429 over 3 days improved hippocampal survival in both models. Taken together, NSC74429 is neuroprotective across multiple insult mechanisms. Future pharmacokinetic and pharmacodynamic (PK/PD) studies are warranted to optimize dosing, and mechanistic studies are needed to determine the percentage of neuroprotection mediated by PHLPP1/2 inhibition, or potentially from the modulation of PHLPP-independent targets.

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“…(16) Traditional phosphatase inhibitors do not target the activity of PHLPP1/2, but several small molecule inhibitors (eg, NSC 117079, NSC 45586) limit their phosphatase activity and alter levels of PHLPP1/2 in vitro. (17) Our prior work demonstrates a role of Phlpp1 within the musculoskeletal system. Published work demonstrates that Phlpp1 governs skeletal development and plays a role during musculoskeletal degeneration.…”

Section: Introductionmentioning

confidence: 87%

Phlpp1 Expression in Osteoblasts Plays a Modest Role in Bone Homeostasis

Karkache,

Molstad,

et al. 2023

JBMR Plus

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Prior work demonstrated that Phlpp1 deficiency alters limb length and bone mass, but the cell types involved and requirement of Phlpp1 for this effect were unclear. To understand the function of Phlpp1 within bone‐forming osteoblasts, we crossed Phlpp1 floxed mice with mice harboring type 1 collagen (Col1a12.3kb)‐Cre. Mineralization of bone marrow stromal cell cultures derived from Phlpp1 cKOCol1a1 was unchanged, but levels of inflammatory genes (eg, Ifng, Il6, Ccl8) and receptor activator of NF‐κB ligand/osteoprotegerin (RANKL/OPG) ratios were enhanced by either Phlpp1 ablation or chemical inhibition. Micro‐computed tomography of the distal femur and L5 vertebral body of 12‐week‐old mice revealed no alteration in bone volume per total volume, but compromised femoral bone microarchitecture within Phlpp1 cKOCol1a1 conditional knockout females. Bone histomorphometry of the proximal tibia documented no changes in osteoblast or osteoclast number per bone surface but slight reductions in osteoclast surface per bone surface. Overall, our data show that deletion of Phlpp1 in type 1 collagen–expressing cells does not significantly alter attainment of peak bone mass of either males or females, but may enhance inflammatory gene expression and the ratio of RANKL/OPG. Future studies examining the role of Phlpp1 within models of advanced age, inflammation, or osteocytes, as well as functional redundancy with the related Phlpp2 isoform are warranted. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

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Chondrocytic and pharmacokinetic properties of Phlpp inhibitors

Cited by 5 publications

References 32 publications

Differential efficacy of two small molecule PHLPP inhibitors to promote nucleus Pulposus cell health

Differential efficacy of two small molecule PHLPP inhibitors to promote nucleus Pulposus cell health

PHLPP Inhibitor NSC74429 Is Neuroprotective in Rodent Models of Cardiac Arrest and Traumatic Brain Injury

Phlpp1 Expression in Osteoblasts Plays a Modest Role in Bone Homeostasis

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