Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases.

The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS­ERK signalling pathway. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy. Here we report the discovery of a highly potent (IC50 = 0.071 µM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS­ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.

A Novel Evidence Conflict Measurement for Multi-Sensor Data Fusion Based on the Evidence Distance and Evidence Angle.

As an important method for uncertainty modeling, Dempster-Shafer (DS) evidence theory has been widely used in practical applications. However, the results turned out to be almost counter-intuitive when fusing the different sources of highly conflicting evidence with Dempster's combination rule. In previous researches, most of them were mainly dependent on the conflict measurement method between the evidence represented by the evidence distance. However, it is inaccurate to characterize the evidence conflict only through the evidence distance. To address this issue, we comprehensively consider the impacts of the evidence distance and evidence angle on conflicts in this paper, and propose a new method based on the mutual support degree between the evidence to characterize the evidence conflict. First, the Hellinger distance measurement method is proposed to measure the distance between the evidence, and the sine value of the Pignistic vector angle is used to characterize the angle between the evidence. The evidence distance indicates the dissimilarity between the evidence, and the evidence angle represents the inconsistency between the evidence. Next, two methods are combined to get a new method for measuring the mutual support degree between the evidence. Afterward, the weight of each evidence is determined by using the mutual support degree between the evidence. Then, the weights of each evidence are utilized to modify the original evidence to achieve the weighted average evidence. Finally, Dempster's combination rule is used for fusion. Some numerical examples are given to illustrate the effectiveness and reasonability for the proposed method.

[Clinical research of modified taohe chengqi granule combined mannitol for treating complicated edema in affected limbs of patients after tibiofibulas double fracture operation].

OBJECTIVE: To observe the therapeutic efficacy of Modified Taohe Chengqi Granule (MTCG) combined mannitol for treating complicated edema in affected limbs of patients after tibiofibulas double fracture operation (TDFO). METHODS: Totally 64 TDFO patients complicated edema were randomly assigned to the treated group and the control group, 32 in each group. Those in the treated group took MTCG combined intravenous dripping of mannitol, while those in the control group received intravenous dripping of mannitol alone. The treatment course was 1 week. The clinical efficacy, the onset time, the swelling degree, and the pain index were observed and compared between the two groups. RESULTS: One week after operation, the effective rate was 98.0% and the markedly effective rate was 87.5% in the treated group, while they were 78.0% and 56.9% respectively in the control group. Better results were obtained in the treated group, showing statistical difference when compared with the control group (P < 0.05). As for the onset time for swelling subsiding, it was (2.4 +/- 1.3) days in the treated group and (3.8 +/- 2.9) days in the control group. There was statistical difference between the two groups (P < 0.05). Better effects on the swelling subsiding degree and the pain index were obtained in the treated group, showing statistical difference when compared with the control group (P < 0.05). CONCLUSIONS: MTCG combined mannitol could obviously abate the edema in affected limbs of patients after TDFO. It was a better treatment method for managing edema in the peri-operative period of orthopedics.

Systematic Chemogenetic Library Assembly.

Chemogenetic libraries, collections of well-defined chemical probes, provide tremendous value to biomedical research but require substantial effort to ensure diversity as well as quality of the contents. We have assembled a chemogenetic library by data mining and crowdsourcing institutional expertise. We are sharing our approach, lessons learned, and disclosing our current collection of 4,185 compounds with their primary annotated gene targets (https://github.com/Novartis/MoaBox). This physical collection is regularly updated and used broadly both within Novartis and in collaboration with external partners.

Identification of TNO155, an Allosteric SHP2 Inhibitor for the Treatment of Cancer.

SHP2 is a nonreceptor protein tyrosine phosphatase encoded by the PTPN11 gene and is involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also plays an important role in the programed cell death pathway (PD-1/PD-L1). As an oncoprotein as well as a potential immunomodulator, controlling SHP2 activity is of high therapeutic interest. As part of our comprehensive program targeting SHP2, we identified multiple allosteric binding modes of inhibition and optimized numerous chemical scaffolds in parallel. In this drug annotation report, we detail the identification and optimization of the pyrazine class of allosteric SHP2 inhibitors. Structure and property based drug design enabled the identification of protein-ligand interactions, potent cellular inhibition, control of physicochemical, pharmaceutical and selectivity properties, and potent in vivo antitumor activity. These studies culminated in the discovery of TNO155, (3S,4S)-8-(6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)pyrazin-2-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine (1), a highly potent, selective, orally efficacious, and first-in-class SHP2 inhibitor currently in clinical trials for cancer.

Maize productivity and soil properties in the Loess Plateau in response to ridge-furrow cultivation with polyethylene and straw mulch.

Ridge-furrow with full film mulching (RFFM) is widely used in the Loess Plateau (LP) to increase maize yield. However, continuous RFFM application may cause excessive depletion of soil organic carbon (SOC) and soil water storage (SWS). The present study tested four production systems, namely, (1) RFFM; (2) ridge-furrow with polyethylene film and straw mulching (RFFSM); (3) non-contoured seedbed with film mulching (FFM); and (4) non-contoured seedbed without mulching (CK) in 2013 and 2014 to identify an optimal technique to increase maize yield yet minimizing the negative effects. SWS under RFFSM was significantly higher by 5.4% and 13.4% compared to RFFM and CK, respectively. The changes in SOC were -0.2, -0.2, and -0.4 g·kg-1 for RFFM, FFM, and CK, respectively, and 0.3 g·kg-1 for RFFSM. Increased root residue and extra external carbon input to soil under RFFSM directly contributed to SOC recovery. RFFSM had a comparable grain yield but higher water use efficiency compared to RFFM. The combination of RFFSM is promising for improving SOC stocks, water storage, and maize productivity.

Which aspects of HTS are empirically correlated with downstream success?

High-throughput screening (HTS) is a well-established hit-finding approach used in the pharmaceutical industry. In this article, recent experience at Novartis with respect to factors influencing the success of HTS campaigns is discussed. An inherent measure of HTS quality could be defined by the assay Z and Z' factors, the number of hits and their biological potencies; however, such measures of quality do not always correlate with the advancement of hits to the later stages of drug discovery. Also, for many target classes, such as kinases, it is easy to identify hits, but, as a result of selectivity, intellectual property and other issues, the projects do not result in lead declarations. In this article, HTS success is defined as the fraction of HTS campaigns that advance into the later stages of drug discovery, and the major influencing factors are examined. Interestingly, screening compounds in individual wells or in mixtures did not have a major impact on the HTS success and, equally interesting, there was no difference in the progression rates of biochemical and cell-based assays. Particular target types, assay technologies, structure-activity relationships and powder availability had a much greater impact on success as defined above. In addition, significant mutual dependencies can be observed - while one assay format works well with one target type, this situation might be completely reversed for a combination of the same readout technology with a different target type. The results and opinions presented here should be regarded as groundwork, and a plethora of factors that influence the fate of a project, such as biophysical measurements, chemical attractiveness of the hits, strategic reasons and safety pharmacology, are not covered here. Nonetheless, it is hoped that this information will be used industry-wide to improve success rates in terms of hits progressing into exploratory chemistry and beyond. The support that can be obtained from new in silico approaches to phase transitions are also described, along with the gaps they are designed to fill.

"Plate cherry picking": a novel semi-sequential screening paradigm for cheaper, faster, information-rich compound selection.

This work describes a novel semi-sequential technique for in silico enhancement of high-throughput screening (HTS) experiments now employed at Novartis. It is used in situations in which the size of the screen is limited by the readout (e.g., high-content screens) or the amount of reagents or tools (proteins or cells) available. By performing computational chemical diversity selection on a per plate basis (instead of a per compound basis), 25% of the 1,000,000-compound screening was optimized for general initial HTS. Statistical models are then generated from target-specific primary results (percentage inhibition data) to drive the cherry picking and testing from the entire collection. Using retrospective analysis of 11 HTS campaigns, the authors show that this method would have captured on average two thirds of the active compounds (IC(50) < 10 microM) and three fourths of the active Murcko scaffolds while decreasing screening expenditure by nearly 75%. This result is true for a wide variety of targets, including G-protein-coupled receptors, chemokine receptors, kinases, metalloproteinases, pathway screens, and protein-protein interactions. Unlike time-consuming "classic" sequential approaches that require multiple iterations of cherry picking, testing, and building statistical models, here individual compounds are cherry picked just once, based directly on primary screening data. Strikingly, the authors demonstrate that models built from primary data are as robust as models built from IC(50) data. This is true for all HTS campaigns analyzed, which represent a wide variety of target classes and assay types.

Flexible 3D pharmacophores as descriptors of dynamic biological space.

Development of a pharmacophore hypothesis related to small-molecule activity is pivotal to chemical optimization of a series, since it defines features beneficial or detrimental to activity. Although crystal structures may provide detailed 3D interaction information for one molecule with its receptor, docking a different ligand to that model often leads to unreliable results due to protein flexibility. Graham Richards' lab was one of the first groups to utilize "fuzzy" pattern recognition algorithms taken from the field of image processing to solve problems in protein modeling. Thus, descriptor "fuzziness" was partly able to emulate conformational flexibility of the target while simultaneously enhancing the speed of the search. In this work, we extend these developments to a ligand-based method for describing and aligning molecules in flexible chemical space termed FEature POint PharmacophoreS (FEPOPS), which allows exploration of dynamic biological space. We develop a novel, combinatorial algorithm for molecular comparisons and evaluate it using the WOMBAT dataset. The new approach shows superior retrospective virtual screening performance than earlier shape-based or charge-based algorithms. Additionally, we use target prediction to evaluate how FEPOPS alignments match the molecules biological activity by identifying the atoms and features that make the key contributions to overall chemical similarity. Overall, we find that FEPOPS are sufficiently fuzzy and flexible to find not only new ligand scaffolds, but also challenging molecules that occupy different conformational states of dynamic biological space as from induced fits.

[Infection of intervertebral space and the interventional therapy].

OBJECTIVE: To investigate the effect of interventional therapy in the treatment of intervertebral space infection. METHODS: The needle was punctured into the infected intervertebral space from the post-lateral side of the spine monitored by X-rays. The pus was drained, the degenerative disc tissues and necrosis tissues were excised and taken out, and at the end a drainaging catheter was put into the space through the needle. The patient should lie in bed absolutely. The antibiotics was injected into the space through the silicon catheter every day. Three to four weeks later, the catheter was removed. RESULTS: All the 8 patients got good results after the therapy. The low back pain was dramatically alleviated instantly at the day of operation. Erythrocyte sedimental rate gradually descended. After 3 approximately 4 weeks of treatment,the catheter was removed. CONCLUSION: Interventional therapy of the intervertebral space infection has notable advantage over the open operation.

Knowledge-based design of target-focused libraries using protein-ligand interaction constraints.

Here we present a new strategy for designing and filtering potentially massive combinatorial libraries using structural information of a binding site. We have developed a variation of the structural interaction fingerprint (SIFt) named r-SIFt, which incorporates the binding interactions of variable fragments in a combinatorial library. This method takes into account the 3D structure of the active site of the target molecule and translates desirable ligand-target binding interactions into library filtering constraints. We show using the MAP kinase p38 as a test case that we can efficiently analyze and classify compounds on the basis of their abilities to interact with the target in the desired binding mode. On the basis of these classifications, decision tree models were generated using the molecular descriptors of the compounds as predictor variables. Our results suggest that r-SIFt coupled with the classification models should be a valuable tool for structure-based focusing of combinatorial chemical libraries.

Bridging chemical and biological space: "target fishing" using 2D and 3D molecular descriptors.

Bridging chemical and biological space is the key to drug discovery and development. Typically, cheminformatics methods operate under the assumption that similar chemicals have similar biological activity. Ideally then, one could predict a drug's biological function(s) given only its chemical structure by similarity searching in libraries of compounds with known activities. In practice, effectively choosing a similarity metric is case dependent. This work compares both 2D and 3D chemical descriptors as tools for predicting the biological targets of ligand probes, on the basis of their similarity to reference molecules in a 46,000 compound, biologically annotated chemical database. Overall, we found that the 2D methods employed here outperform the 3D (88% vs 67% success) in correct target prediction. However, the 3D descriptors proved superior in cases of probes with low structural similarity to other compounds in the database (singletons). Additionally, the 3D method (FEPOPS) shows promise for providing pharmacophoric alignment of the small molecules' chemical features consistent with those seen in experimental ligand/ receptor complexes. These results suggest that querying annotated chemical databases with a systematic combination of both 2D and 3D descriptors will prove more effective than employing single methods.

[Clinic and pathology of the knee menisci injury with sclerosis denaturalization].

OBJECTIVE: To summarize the characteristics of clinic and pathology of the knee menisci injury with sclerosis denaturalization and to discuss the mechanism of the sclerosis and its clinic significance. METHODS: We retrospectively analyzed the diagnosis and treatment and healing of the knee menisci with the sclerosis denatured injury from 1989 to 2003. The changes of the partial meniscus with sclerosis denaturalization were observed in tissue section. RESULTS: Menisci with sclerosis denaturalization occupied 24.1%, and 77.6% of them subordinated to simple sclerosis denaturalization without tear. Their characteristics were that the manifestations were not except typical knee pain, tenderness at joint line,integrity shape with dull or less lubricity and tiny grand on the super face of menisci under the arthroscopy,and trembles could be touched by a probe. Pathology showed the formation of local sclerosis with the histological changes of fibro-hyperplasia, hyaline degeneration and mucous degeneration in the menisci. No operation obtained curative effects. Symptoms can be eliminated by the excision of the menisci with sclerosis denaturalization. CONCLUSION: The clinic of simple meniscus injury with sclerosis denaturalization are non-typical and arthroscopic check-up is valuable for the diagnosis. The menisci can be removed from the patients suffered from heavy symptoms for a long time.

[Preliminary study on treating patellar instability guided by arthroscopy].

OBJECTIVE: To explore the operation method in treating patellar instability guided by arthroscopy and to evaluate its therapeutic effects. METHODS: Thirty-two patients (34 knees) with patellar instability including 2 patellar tilt (2 knees), 26 (27 knees) patellar subluxation, and 4 (5 knees) patellar dislocation, diagnosed by clinical and arthroscopical standard, were treated with anterior transfer of the gracilis or medial transfer of the tibial tuberosity based on the lateral retinacular release and medial tightening. RESULTS: Twenty-eight patients were followed up for 5-87 (mean 36.6) months. The excellence rates was 92.8%. CONCLUSION: The patellofemoral alignment can be seen directly and dynamically under the arthroscopy. The combinational treatment including anterior transfer of the gracilis in repairing patellar instability can avoid the recurrence effectively and get satisfactory results.

Discovery and Characterization of Allosteric WNK Kinase Inhibitors.

Protein kinases are known for their highly conserved adenosine triphosphate (ATP)-binding site, rendering the discovery of selective inhibitors a major challenge. In theory, allosteric inhibitors can achieve high selectivity by targeting less conserved regions of the kinases, often with an added benefit of retaining efficacy under high physiological ATP concentration. Although often overlooked in favor of ATP-site directed approaches, performing a screen at high ATP concentration or stringent hit triaging with high ATP concentration offers conceptually simple methods of identifying inhibitors that bind outside the ATP pocket. Here, we applied the latter approach to the With-No-Lysine (K) (WNK) kinases to discover lead molecules for a next-generation antihypertensive that requires a stringent safety profile. This strategy yielded several ATP noncompetitive WNK1-4 kinase inhibitors, the optimization of which enabled cocrystallization with WNK1, revealing an allosteric binding mode consistent with the observed exquisite specificity for WNK1-4 kinases. The optimized compound inhibited rubidium uptake by sodium chloride cotransporter 1 (NKCC1) in HT29 cells, consistent with the reported physiology of WNK kinases in renal electrolyte handling.

Allosteric Inhibition of SHP2: Identification of a Potent, Selective, and Orally Efficacious Phosphatase Inhibitor.

SHP2 is a nonreceptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also purportedly plays an important role in the programmed cell death pathway (PD-1/PD-L1). Because it is an oncoprotein associated with multiple cancer-related diseases, as well as a potential immunomodulator, controlling SHP2 activity is of significant therapeutic interest. Recently in our laboratories, a small molecule inhibitor of SHP2 was identified as an allosteric modulator that stabilizes the autoinhibited conformation of SHP2. A high throughput screen was performed to identify progressable chemical matter, and X-ray crystallography revealed the location of binding in a previously undisclosed allosteric binding pocket. Structure-based drug design was employed to optimize for SHP2 inhibition, and several new protein-ligand interactions were characterized. These studies culminated in the discovery of 6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine (SHP099, 1), a potent, selective, orally bioavailable, and efficacious SHP2 inhibitor.

Interaction profiles of protein kinase-inhibitor complexes and their application to virtual screening.

A major challenge facing structure-based drug discovery efforts is how to leverage the massive amount of experimental (X-ray and NMR) and virtual structural information generated from drug discovery projects. Many important drug targets have large numbers of protein-inhibitor complexes, necessitating tools to compare and contrast their similarities and differences. This information would be valuable for understanding potency and selectivity of inhibitors and could be used to define target constraints to assist virtual screening. We describe a profile-based approach that enables us to capture the conservation of interactions between a set of protein-ligand receptor complexes. The use of profiles provides a sensitive means to compare multiple inhibitors binding to a drug target. We demonstrate the utility of profile-based analysis of small molecule complexes from the protein-kinase family to identify similarities and differences in binding of ATP, p38, and CDK2 compounds to kinases and how these profiles can be applied to differentiate the selectivity of these inhibitors. Importantly, our virtual screening results demonstrate superior enrichment of kinase inhibitors using profile-based methods relative to traditional scoring functions. Interaction-based analysis should provide a valuable tool for understanding inhibitor binding to other important drug targets.

Structural interaction fingerprint (SIFt): a novel method for analyzing three-dimensional protein-ligand binding interactions.

Representing and understanding the three-dimensional (3D) structural information of protein-ligand complexes is a critical step in the rational drug discovery process. Traditional analysis methods are proving inadequate and inefficient in dealing with the massive amount of structural information being generated from X-ray crystallography, NMR, and in silico approaches such as structure-based docking experiments. Here, we present SIFt (structural interaction fingerprint), a novel method for representing and analyzing 3D protein-ligand binding interactions. Key to this approach is the generation of an interaction fingerprint that translates 3D structural binding information from a protein-ligand complex into a one-dimensional binary string. Each fingerprint represents the "structural interaction profile" of the complex that can be used to organize, analyze, and visualize the rich amount of information encoded in ligand-receptor complexes and also to assist database mining. We have applied SIFt to tackle three common tasks in structure-based drug design. The first involved the analysis and organization of a typical set of results generated from a docking study. Using SIFt, docking poses with similar binding modes were identified, clustered, and subsequently compared with conventional scoring function information. A second application of SIFt was to analyze approximately 90 known X-ray crystal structures of protein kinase-inhibitor complexes obtained from the Protein Databank. Using SIFt, we were able to organize the structures and reveal striking similarities and diversity between their small molecule binding interactions. Finally, we have shown how SIFt can be used as an effective molecular filter during the virtual chemical library screening process to select molecules with desirable binding mode(s) and/or desirable interaction patterns with the protein target. In summary, SIFt shows promise to fully leverage the wealth of information being generated in rational drug design.

Clinical significance of microRNA-93 downregulation in human colon cancer.

AIM: MicroRNA-93 (miR-93) has been shown to suppress proliferation and colony formation of colon cancer stem cells. The aim of this study was to examine the expression pattern and prognostic value of miR-93 in patients with colon cancer. MATERIALS AND METHODS: A quantitative real-time PCR analysis was carried out to detect the expression levels of miR-93 in 138 paired samples of tumoral and nontumoral colon tissues diagnosed with colon cancer. Associations of miR-93 expression with clinicopathological parameters and survival were also examined. RESULTS: miR-93 expression was significantly decreased in tumoral compared with nontumoral colon tissues (P<0.001). Low miR-93 expression was significantly correlated with advanced tumor stage (P=0.02), positive nodal metastasis (P=0.006), and positive distant metastases (P=0.01). In addition, Kaplan-Meier survival analysis by Cox regression showed that low miR-93 expression [hazard ratio (HR), 10.2; 95% confidence interval (CI), 1.9-42.8, P=0.003] was associated closely with poor overall survival in patients with colon cancer. Moreover, multivariate analysis showed that miR-93 decreased expression (HR, 4.3; 95% CI, 0.8-17.2, P=0.02), advanced tumor stage (HR, 3.1; 95% CI, 0.2-13.9, P=0.04), positive nodal metastasis (HR, 4.1; 95% CI, 0.7-16.8, P=0.02), and positive distant metastases (HR, 3.7; 95% CI, 0.5-14.1, P=0.03) were independent risk factors for overall survival in patients with colon cancer. CONCLUSION: Our data show for the first time that the downregulation of miR-93 was significantly correlated with unfavorable clinicopathologic features and short overall survival in patients with colon cancer, suggesting that decreased expression of miR-93 be used as a novel prognostic factor for this disease.

Tumor associated glycoprotein-72 is a novel marker for poor survival in hepatocellular carcinoma.

To investigate the relationship of tumor associated glycoprotein-72 (TAG-72) expression with clinicopathological features in hepatocellular carcinoma (HCC) patients. Sixty pairs of HCC and paracarcinomatous (PCLT) tissues, and 10 normal liver (NL) tissues were collected for Western blot analysis, and 244 pairs of HCC and PCLT tissues were collected for immunohistochemistry analysis. TAG-72 protein expression was elevated significantly in HCC tissues compared with PCLT and NL tissues. Its increased expression was correlated with TNM stage, Edmondson-Steiner grade, vein invasion and multiple tumor nodes. It is noteworthy that the HCC patients with high TAG-72 expression had shorter overall survival and disease-free survival than the patients with low expression. Multivariate Cox regression analysis revealed that TAG-72 expression was an independent prognostic factor for HCC patients. The current study demonstrated for the first time that the increased expression of TAG-72 was correlated with poor survival in patients with HCC, indicating that TAG-72 is a novel prognostic marker for HCC.