Off-target autophagy inhibition by SHP2 allosteric inhibitors contributes to their anti-tumor activity in RAS-driven cancers.

Aberrant activation of RAS-MAPK signaling is common in cancer, and efforts to inhibit pathway components have yielded drugs with promising clinical activities. Unfortunately, treatment-provoked adaptive resistance mechanisms inevitably develop, limiting their therapeutic potential. As a central node essential for receptor tyrosine kinase mediated RAS activation, SHP2 has emerged as an attractive cancer target. Consequently, many SHP2 allosteric inhibitors are now in clinical testing. Here we discovered a previously unrecognized off-target effect associated with SHP2 allosteric inhibitors. We found that these inhibitors accumulate in the lysosome and block autophagic flux in a SHP2-independent manner. We showed that off-target autophagy inhibition by SHP2 allosteric inhibitors contributes to their anti-tumor activity. We also demonstrated that SHP2 allosteric inhibitors harboring this off-target activity not only suppress oncogenic RAS signaling but also overcome drug resistance such as MAPK rebound and protective autophagy in response to RAS-MAPK pathway blockage. Finally, we exemplified a therapeutic framework that harnesses both the on- and off-target activities of SHP2 allosteric inhibitors for improved treatment of mutant RAS driven and drug resistant malignancies such as pancreatic and colorectal cancers. Brief Summary: SHP2 allosteric inhibitors elicit off-target autophagy blockade that can be exploited for improved treatment of RAS-driven and drug-resistant cancers.

Discovery of a SHP2 Degrader with In Vivo Anti-Tumor Activity.

Src homology 2 domain-containing phosphatase 2 (SHP2) is an attractive target for cancer therapy due to its multifaceted roles in both tumor and immune cells. Herein, we designed and synthesized a novel series of proteolysis targeting chimeras (PROTACs) using a SHP2 allosteric inhibitor as warhead, with the goal of achieving SHP2 degradation both inside the cell and in vivo. Among these molecules, compound P9 induces efficient degradation of SHP2 (DC50 = 35.2 ± 1.5 nM) in a concentration- and time-dependent manner. Mechanistic investigation illustrates that the P9-mediated SHP2 degradation requires the recruitment of the E3 ligase and is ubiquitination- and proteasome-dependent. P9 shows improved anti-tumor activity in a number of cancer cell lines over its parent allosteric inhibitor. Importantly, administration of P9 leads to a nearly complete tumor regression in a xenograft mouse model, as a result of robust SHP2 depletion and suppression of phospho-ERK1/2 in the tumor. Hence, P9 represents the first SHP2 PROTAC molecule with excellent in vivo efficacy. It is anticipated that P9 could serve not only as a new chemical tool to interrogate SHP2 biology but also as a starting point for the development of novel therapeutics targeting SHP2.

SPSiPs, a Class of Diphosphine Ligands Based on SPSiOL with a Large Dihedral Angle.

The development and the synthetic applications of a novel class of diphosphine ligands (SPSiPs) based on chiral spirosilabiindane diol (SPSiOL) are presented. Starting from SPSiOL, the diphosphine ligands could be readily prepared in three steps with high efficiency. This novel class of diphosphine ligands features rigid configuration, a large dihedral angle, a large P-M-P angle, and a long P-P distance. The potentials of SPSiPs in asymmetric catalysis have also been preliminarily disclosed.

An Integrated Proteomic Strategy to Identify SHP2 Substrates.

Protein phosphatases play an essential role in normal cell physiology and the development of diseases such as cancer. The innate challenges associated with studying protein phosphatases have limited our understanding of their substrates, molecular mechanisms, and unique functions within highly coordinated networks. Here, we introduce a novel strategy using substrate-trapping mutants coupled with quantitative proteomics methods to identify physiological substrates of Src homology 2 containing protein tyrosine phosphatase 2 (SHP2) in a high-throughput manner. The technique integrates three parallel mass spectrometry-based proteomics experiments, including affinity isolation of substrate-trapping mutant complex using wild-type and SHP2 KO cells, in vivo global quantitative phosphoproteomics, and in vitro phosphatase reaction. We confidently identified 18 direct substrates of SHP2 in the epidermal growth factor receptor signaling pathways, including both known and novel SHP2 substrates. Docking protein 1 was further validated using biochemical assays as a novel SHP2 substrate, providing a mechanism for SHP2-mediated Ras activation. This advanced workflow improves the systemic identification of direct substrates of protein phosphatases, facilitating our understanding of the equally important roles of protein phosphatases in cellular signaling.

[Measurement Properties and Value Set of EuroQol Five-Dimensional Questionnaire-Youth Version].

Objective To summarize the research findings on the measurement properties and value set of EuroQol Five-Dimensional Questionnaire-Youth Version (EQ-5D-Y). Methods PubMed,Web of Science,CNKI,and Wanfang Data were searched for the studies involving the measurement properties and value set of EQ-5D-Y. Results According to the inclusion and exclusion criteria,38 studies were included for analysis.EQ-5D-Y had good feasibility,reliability,and validity in measuring health-related quality of life (HRQOL) of youth in different countries and populations.The advantages of 5-level EQ-5D-Y (Y-5L)over 3-level EQ-5D-Y (Y-3L) might be modest.The available studies have demonstrated that the EQ-5D value set for adults should not be adopted in youth,and the international protocol for constructing the Y-3L value set has been available.Many countries have established or are developing their own Y-3L value sets according to the protocol. Conclusion The study systematically elaborated the current status and trend of the measurement properties and value set of EQ-5D-Y,which could provide reference for the future research on the EQ-5D-Y in China.

A head-to-head comparison of the EQ-5D-3L index scores derived from the two EQ-5D-3L value sets for China.

OBJECTIVE: Two EQ-5D-3L (3L) value sets (developed in 2014 and 2018) co-exist in China. The study examined the level of agreement between index scores for all the 243 health states derived from them at both absolute and relative levels and compared the responsiveness of the two indices. METHODS: Intraclass correlations coefficient (ICC) and Bland-Altman plot were adopted to assess the degree of agreement between the two indices at the absolute level. Health gains for 29,403 possible transitions between pairs of 3L health states were calculated to assess the agreement at the relative level. Their responsiveness for the transitions was assessed using Cohen effect size. RESULTS: The mean (SD) value was 0.427 (0.206) and 0.649 (0.189) for the 3L2014 and 3L2018 index scores, respectively. Although the ICC value showed good agreement (i.e., 0.896), 88.9% (216/243) of the points were beyond the minimum important difference limit according to the Bland-Altman plot. The mean health gains for the 29,403 health transitions was 0.234 (3L2014 index score) and 0.216 (3L2018 index score). The two indices predicted consistent transitions in 23,720 (80.7%) of 29,403 pairs. For the consistent pairs, Cohen effective size value was 1.05 (3L2014 index score) or 1.06 (3L2018 index score); and the 3L2014 index score only yielded 0.007 more utility gains. However, the results based on the two measures varied substantially according to the direction and magnitude of health change. CONCLUSION: The 3L2014 and 3L2018 index scores are not interchangeable. The choice between them is likely to influence QALYs estimations.

Highly Potent and Selective N-Aryl Oxamic Acid-Based Inhibitors for Mycobacterium tuberculosis Protein Tyrosine Phosphatase B.

Tuberculosis is an infectious disease caused by the bacterium Mycobacterium tuberculosis (Mtb). Mtb protein tyrosine phosphatase B (mPTPB) is a virulence factor required for Mtb survival in host macrophages. Consequently, mPTPB represents an exciting target for tuberculosis treatment. Here, we identified N-phenyl oxamic acid as a highly potent and selective monoacid-based phosphotyrosine mimetic for mPTPB inhibition. SAR studies on the initial hit, compound 4 (IC50 = 257 nM), resulted in several highly potent inhibitors with IC50 values lower than 20 nM for mPTPB. Among them, compound 4t showed a Ki of 2.7 nM for mPTPB with over 4500-fold preference over 25 mammalian PTPs. Kinetic, molecular docking, and site-directed mutagenesis analyses confirmed these compounds as active site-directed reversible inhibitors of mPTPB. These inhibitors can reverse the altered host cell immune responses induced by the bacterial phosphatase. Furthermore, the inhibitors possess molecular weights <400 Da, log D7.4 < 2.5, topological polar surface area < 75, ligand efficiency > 0.43, and good aqueous solubility and metabolic stability, thus offering excellent starting points for further therapeutic development.

How the EQ-5D utilities are derived matters in Chinese diabetes patients: a comparison based on different EQ-5D scoring functions for China.

OBJECTIVES: In China, multiple approaches to calculating EQ-5D utilities are available, including the two EQ-5D-3L (3L2014 and 3L2018) scoring functions, the EQ-5D-5L (5L) scoring function, and the crosswalk function linking the 3L utilities and 5L health states. The study compared utilities derived from them in terms of agreement and discriminative power; and assessed whether the use of different approaches may affect QALY estimation in Chinese type 2 diabetes (T2D) patients. METHODS: Cross-sectional data of 289 T2D patients who self-completed both the 5L and 3L questions were used. Agreement were examined using intraclass correlation coefficient (ICC) and Bland-Altman plots. The ability of the EQ-5D utilities in differentiating the patients with and without clinical conditions was evaluated using F-statistics. Their influence on QALY estimation was assessed adopting mean absolute difference (MAD) in utility values between the patients. RESULTS: The ICC values were 0.881 (3L2014-3L2018), 0.958 (5L-c5L2014), and 0.806 (5L-c5L2018). The two 3L utilities and the three 5L utilities had poor agreement at the lower end of utility scale according to Bland-Altman plots. The 3L2018 utilities had lower F-statistics compared to the 3L2014 utilities; the two c5L utilities had larger or similar F-statistics compared to the 5L utilities. The mean MADs were 0.138 (5L), 0.116 (3L2014), 0.115 (c5L2014), 0.055 (c5L2018), and 0.055 (3L2018). CONCLUSION: The 3L2014 utilities is more discriminative than the 3L2018 utilities; and the two c5L utilities have no worse discriminative power compared with the 5L utilities. The choice of the approach to calculating the EQ-5D utilities is likely to affect QALY estimates.

Mechanistic insights explain the transforming potential of the T507K substitution in the protein-tyrosine phosphatase SHP2.

The protein-tyrosine phosphatase SHP2 is an allosteric enzyme critical for cellular events downstream of growth factor receptors. Mutations in the SHP2 gene have been linked to many different types of human diseases, including developmental disorders, leukemia, and solid tumors. Unlike most SHP2-activating mutations, the T507K substitution in SHP2 is unique in that it exhibits oncogenic Ras-like transforming activity. However, the biochemical basis of how the SHP2/T507K variant elicits transformation remains unclear. By combining kinetic and biophysical methods, X-ray crystallography, and molecular modeling, as well as using cell biology approaches, here we uncovered that the T507K substitution alters both SHP2 substrate specificity and its allosteric regulatory mechanism. We found that although SHP2/T507K exists in the closed, autoinhibited conformation similar to the WT enzyme, the interactions between its N-SH2 and protein-tyrosine phosphatase domains are weakened such that SHP2/T507K possesses a higher affinity for the scaffolding protein Grb2-associated binding protein 1 (Gab1). We also discovered that the T507K substitution alters the structure of the SHP2 active site, resulting in a change in SHP2 substrate preference for Sprouty1, a known negative regulator of Ras signaling and a potential tumor suppressor. Our results suggest that SHP2/T507K's shift in substrate specificity coupled with its preferential association of SHP2/T507K with Gab1 enable the mutant SHP2 to more efficiently dephosphorylate Sprouty1 at pTyr-53. This dephosphorylation hyperactivates Ras signaling, which is likely responsible for SHP2/T507K's Ras-like transforming activity.

Triterpenoids from Anchusa italica and their protective effects on hypoxia/reoxygenation induced cardiomyocytes injury.

Six new triterpenoids (1-6) and 22 known analogues (7-28), were separated from the aerial parts of Anchusa italica Retz., a traditional Uygur medicine for treating cardiovascular and cerebrovascular diseases in the Xinjiang region, China. The possible effects of compounds 1-28 on hypoxia/reoxygenation (H/R) induced cardiomyocytes injury were assayed, and compounds 4, 6-17, 21-22 and 26-28 showed significant protective effects. Further, the representative new compound 6 significantly suppressed the levels of H/R-induced apoptosis and autophagy in neonatal rat cardiomyocytes, with the reversing of the downregulated expression of Bcl-2 and upregulated expression of Bax and Beclin-1 by compound 6 treatment in neonatal rat cardiomyocytes following H/R injury. In addition, compound 6 protected cardiomyocyte from H/R injury, and pretreatment with 6 could decrease CK and LDH levels. Compound 6 also alleviated H/R-induced phosphorylation of p38 MAPK in neonatal rat cardiomyocytes. Therefore, tripterpenoid 6 and its analogues may be the pharmacodyamic material of A. italica, and offer a promising therapeutic approach for treating cardiomyocyte injury induced by H/R.

A novel 15-spiro diterpenoid dimer from Andrographis paniculata with inhibitory potential against human carboxylesterase 2.

The phytochemical investigation of Andrographis paniculata resulted in the isolation of a novel 15-spiro diterpenoid dimer bisandrographolide G (1). Its structure was determined by 1D and 2D NMR, HRESIMS, electronic circular dichroism (ECD), and TD DFT calculations of ECD spectra. It showed potent inhibitory activity against human carboxylesterase 2 (CES 2) with an IC50 value of 4.61 ± 0.23 µM, and it was defined as a mixed-competitive type inhibitor with a Ki value of 8.88 µM based on the inhibition kinetics result. This finding gave us a hit to develop new generation of human CES 2 inhibitors.

Polydopamine-Based Multifunctional Antitumor Nanoagent for Phototherapy and Photodiagnosis by Regulating Redox Balance.

The development of multifunctional nanoagents for the simultaneous achievement of high diagnostic and therapeutic performances is significant for precise cancer treatment. Herein, we report on a polydopamine (PDA)-based multifunctional nanoagent, PML, in which the methylene blue (MB) photosensitizer (PS) and l-arginine (l-Arg) tumor-targeting species are equipped. After selectively accumulating in tumor sites, glutathione (GSH)-responsive PML degradation can controllably release loaded MB to produce singlet oxygen (1O2) under near-infrared (NIR) photoirradiation. This GSH-depleted PS release process can not only weaken the body's antioxidant defence ability but also synergistically increase the 1O2 concentration. Therefore, GSH depletion-enhanced photodynamic therapy (PDT) efficiency is logically achieved by regulating the intracellular redox balance. In addition, our nanoagent can guide photoacoustic/NIR thermal dual-modal imaging and convert light into heat for cooperative cancer phototherapy because of the inherent photothermal conversion nature of PDA. As a result, excellent in vivo antitumor phototherapy (PDT + PTT) is achieved under the precise guidance of dual-modal imaging. This work not only realizes the integration of cancer diagnosis and treatment through PDA-based nanocarriers but also delivers dimensions in designing the next generation of multifunctional antitumor nanoagents for enhanced phototherapy and photodiagnosis by regulating the redox balance.

Quinic Acid-Conjugated Nanoparticles Enhance Drug Delivery to Solid Tumors via Interactions with Endothelial Selectins.

Current nanoparticle (NP) drug carriers mostly depend on the enhanced permeability and retention (EPR) effect for selective drug delivery to solid tumors. However, in the absence of a persistent EPR effect, the peritumoral endothelium can function as an access barrier to tumors and negatively affect the effectiveness of NPs. In recognition of the peritumoral endothelium as a potential barrier in drug delivery to tumors, poly(lactic-co-glycolic acid) (PLGA) NPs are modified with a quinic acid (QA) derivative, synthetic mimic of selectin ligands. QA-decorated NPs (QA-NP) interact with human umbilical vein endothelial cells expressing E-/P-selectins and induce transient increase in endothelial permeability to translocate across the layer. QA-NP reach selectin-upregulated tumors, achieving greater tumor accumulation and paclitaxel (PTX) delivery than polyethylene glycol-decorated NPs (PEG-NP). PTX-loaded QA-NP show greater anticancer efficacy than Taxol or PTX-loaded PEG-NP at the equivalent PTX dose in different animal models and dosing regimens. Repeated dosing of PTX-loaded QA-NP for two weeks results in complete tumor remission in 40-60% of MDA-MB-231 tumor-bearing mice, while those receiving control treatments succumb to death. QA-NP can exploit the interaction with selectin-expressing peritumoral endothelium and deliver anticancer drugs to tumors to a greater extent than the level currently possible with the EPR effect.

Therapeutic Targeting of Oncogenic Tyrosine Phosphatases.

Protein tyrosine phosphatases (PTP) are exciting and novel targets for cancer drug discovery that work in concert with protein tyrosine kinases (PTK) in controlling cellular homeostasis. Given the activating role that some PTKs play in initiating growth factor-mediated cellular processes, PTPs are usually perceived as the negative regulators of these events and therefore tumor suppressive in nature. However, mounting evidence indicate that PTPs do not always antagonize the activity of PTKs in regulating tyrosine phosphorylation, but can also play dominant roles in the initiation and progression of signaling cascades that regulate cell functions. It follows, therefore, that PTP malfunction can actively contribute to a host of human disorders, in particular, cancer, metabolic syndromes, and autoimmune diseases. The Src homology domain containing phosphatase 2 (SHP2) and the three-membered family of phosphatases of regenerating liver (PRL) are infamously oncogenic members of the PTP superfamily. Both are established regulators of major cancer pathways such as Ras/ERK1/2, Src, JAK/STAT, JNK, NF-κB, and PTEN/PI3K/AKT. Furthermore, upregulation, mutation, or other dysregulation of these PTPs has been positively correlated with cancer initiation and progression. This review will provide topical coverage of target validation and drug discovery efforts made in targeting these oncogenic PTPs as compelling candidates for cancer therapy. Cancer Res; 77(21); 5701-5. ©2017 AACR.

SHP2 phosphatase as a novel therapeutic target for melanoma treatment.

Melanoma ranks among the most aggressive and deadly human cancers. Although a number of targeted therapies are available, they are effective only in a subset of patients and the emergence of drug resistance often reduces durable responses. Thus there is an urgent need to identify new therapeutic targets and develop more potent pharmacological agents for melanoma treatment. Herein we report that SHP2 levels are frequently elevated in melanoma, and high SHP2 expression is significantly associated with more metastatic phenotype and poorer prognosis. We show that SHP2 promotes melanoma cell viability, motility, and anchorage-independent growth, through activation of both ERK1/2 and AKT signaling pathways. We demonstrate that SHP2 inhibitor 11a-1 effectively blocks SHP2-mediated ERK1/2 and AKT activation and attenuates melanoma cell viability, migration and colony formation. Most importantly, SHP2 inhibitor 11a-1 suppresses xenografted melanoma tumor growth, as a result of reduced tumor cell proliferation and enhanced tumor cell apoptosis. Taken together, our data reveal SHP2 as a novel target for melanoma and suggest SHP2 inhibitors as potential novel therapeutic agents for melanoma treatment.

Inhibition of Low Molecular Weight Protein Tyrosine Phosphatase by an Induced-Fit Mechanism.

The low molecular weight protein tyrosine phosphatase (LMW-PTP) is a regulator of a number of signaling pathways and has been implicated as a potential target for oncology and diabetes/obesity. There is significant therapeutic interest in developing potent and selective inhibitors to control LMW-PTP activity. We report the discovery of a novel class of LMW-PTP inhibitors derived from sulfophenyl acetic amide (SPAA), some of which exhibit greater than 50-fold preference for LMW-PTP over a large panel of PTPs. X-ray crystallography reveals that binding of SPAA-based inhibitors induces a striking conformational change in the LMW-PTP active site, leading to the formation of a previously undisclosed hydrophobic pocket to accommodate the α-phenyl ring in the ligand. This induced-fit mechanism is likely a major contributor responsible for the exquisite inhibitor selectivity.

Novel Anticancer Agents Based on Targeting the Trimer Interface of the PRL Phosphatase.

Phosphatase of regenerating liver (PRL) oncoproteins are phosphatases overexpressed in numerous types of human cancer. Elevated levels of PRL associate with metastasis and poor clinical outcomes. In principle, PRL phosphatases offer appealing therapeutic targets, but they remain underexplored due to the lack of specific chemical probes. In this study, we address this issue by exploiting a unique property of PRL phosphatases, namely, that they may function as homotrimers. Starting from a sequential structure-based virtual screening and medicinal chemistry strategy, we identified Cmpd-43 and several analogs that disrupt PRL1 trimerization. Biochemical and structural analyses demonstrate that Cmpd-43 and its close analogs directly bind the PRL1 trimer interface and obstruct PRL1 trimerization. Cmpd-43 also specifically blocks the PRL1-induced cell proliferation and migration through attenuation of both ERK1/2 and Akt activity. Importantly, Cmpd-43 exerted potent anticancer activity both in vitro and in vivo in a murine xenograft model of melanoma. Our results validate a trimerization-dependent signaling mechanism for PRL and offer proof of concept for trimerization inhibitors as candidate therapeutics to treat PRL-driven cancers. Cancer Res; 76(16); 4805-15. ©2016 AACR.

Cefsulodin Inspired Potent and Selective Inhibitors of mPTPB, a Virulent Phosphatase from Mycobacterium tuberculosis.

mPTPB is a virulent phosphatase from Mycobacterium tuberculosis and a promising therapeutic target for tuberculosis. To facilitate mPTPB-based drug discovery, we identified α-sulfophenylacetic amide (SPAA) from cefsulodin, a third generation ß-lactam cephalosporin antibiotic, as a novel pTyr pharmacophore for mPTPB. Structure-guided and fragment-based optimization of SPAA led to the most potent and selective mPTPB inhibitor 9, with a K i of 7.9 nM and more than 10,000-fold preference for mPTPB over a large panel of 25 phosphatases. Compound 9 also exhibited excellent cellular activity and specificity in blocking mPTPB function in macrophage. Given its novel structure, modest molecular mass, and extremely high ligand efficiency (0.46), compound 9 represents an outstanding lead compound for anti-TB drug discovery targeting mPTPB.

Exploring the Existing Drug Space for Novel pTyr Mimetic and SHP2 Inhibitors.

Protein tyrosine phosphatases (PTPs) are potential therapeutic targets for many diseases. Unfortunately, despite considerable drug discovery efforts devoted to PTPs, obtaining selective and cell permeable PTP inhibitors remains highly challenging. We describe a strategy to explore the existing drug space for previously unknown PTP inhibitory activities. This led to the discovery of cefsulodin as an inhibitor of SHP2, an oncogenic phosphatase in the PTP family. Crystal structure analysis of SHP2 interaction with cefsulodin identified sulfophenyl acetic amide (SPAA) as a novel phosphotyrosine (pTyr) mimetic. A structure-guided and SPAA fragment-based focused library approach produced several potent and selective SHP2 inhibitors. Notably, these inhibitors blocked SHP2-mediated signaling events and proliferation in several cancer cell lines. Thus, SPAA may serve as a new platform for developing chemical probes for other PTPs.