👤 Tyler J McNeill

Despite the growing burden of knee osteoarthritis on aging populations, our mechanistic understanding of this disease remains lacking. Though knee osteoarthritis is a whole joint disease, the impact of intra-articular structures such as the infrapatellar fat pad (IFP) on cartilage health is unclear. This study investigated the effect of age on paracrine communication between the IFP and chondrocytes. To isolate the effects of the IFP secretome on chondrocytes, aged chondrocytes from male and female mice were incubated with conditioned media from sex-matched young IFPs, aged IFPs, or control media. Extracellular matrix protein expression increased in both male and female chondrocytes exposed to young, but not aged, conditioned media relative to control media. The effect of the young IFP was not concomitant with changes in extracellular matrix degradation proteins, ADAMTS4 or MMP13. To identify factors mediating the effects of the IFP on chondrocytes that are altered with aging, we performed mass spectrometry of young and aged conditioned media and transcriptomics of aged chondrocytes treated with young or aged conditioned media. We then integrated the 2 datasets using network analyses. From the conditioned media, 2 secreted proteins, Mfge8 and Apoa4, were significantly changed with aging. In silico perturbation of the corresponding receptors of these IFP-secreted factors identified multiple enriched pathways in chondrocytes, including negative regulation of nitric oxide synthase activity. Overall, the data suggest that young IFPs release paracrine factors that promote extracellular matrix production in chondrocytes, potentially via regulation of nitric oxide levels, but that this effect is diminished with aging. Show less

SHP2 is a cytoplasmic protein tyrosine phosphatase encoded by the PTPN11 gene and is involved in cell proliferation, differentiation, and survival. Recently, we reported an allosteric mechanism of inhibition that stabilizes the auto-inhibited conformation of SHP2. SHP099 (1) was identified and characterized as a moderately potent, orally bioavailable, allosteric small molecule inhibitor, which binds to a tunnel-like pocket formed by the confluence of three domains of SHP2. In this report, we describe further screening strategies that enabled the identification of a second, distinct small molecule allosteric site. SHP244 (2) was identified as a weak inhibitor of SHP2 with modest thermal stabilization of the enzyme. X-ray crystallography revealed that 2 binds and stabilizes the inactive, closed conformation of SHP2, at a distinct, previously unexplored binding site-a cleft formed at the interface of the N-terminal SH2 and PTP domains. Derivatization of 2 using structure-based design resulted in an increase in SHP2 thermal stabilization, biochemical inhibition, and subsequent MAPK pathway modulation. Downregulation of DUSP6 mRNA, a downstream MAPK pathway marker, was observed in KYSE-520 cancer cells. Remarkably, simultaneous occupation of both allosteric sites by 1 and 2 was possible, as characterized by cooperative biochemical inhibition experiments and X-ray crystallography. Combining an allosteric site 1 inhibitor with an allosteric site 2 inhibitor led to enhanced pharmacological pathway inhibition in cells. This work illustrates a rare example of dual allosteric targeted protein inhibition, demonstrates screening methodology and tactics to identify allosteric inhibitors, and enables further interrogation of SHP2 in cancer and related pathologies. Show less