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Aim To explore the current status of Chinese nurses’ willingness to work during the COVID‐19 pandemic and the factors that influence them. Background The demand for front‐line nurses continues to grow during the COVID‐19 pandemic, but their willingness varies significantly. Therefore, it is crucial to explore nurses’ willingness to report for front‐line work. Methods A cross‐sectional study of 1,310 nurses from six tertiary hospitals was conducted. The participants completed self‐administered online questionnaires. Results A total of 90.5% of nurses reported that they would like to voluntarily participate in front‐line work. Those with previous training, higher self‐efficacy scores, and lower perceived risk and self‐worth scores were more likely to participate in front‐line work, while nurses, who had 11–15 years of work experience and were worried about their family and the lack of family support, were less likely to be involved in front‐line work. Conclusion This study found that the vast majority of nurses were willing to participate in front‐line work and affirmed the positive effects of previous infection prevention training, self‐efficacy and self‐worth. Implications for Nursing Management This research emphasizes the necessity of infection prevention training and provides evidence for further emergency workforce deployment and incentives.

Resistance to second-generation androgen receptor (AR) antagonists such as enzalutamide is an inevitable consequence in patients with castration-resistant prostate cancer (CRPC). There are no effective therapeutic options for this recurrent disease. The expression of truncated AR variant 7 (AR-V7) has been suggested to be one mechanism of resistance; however, its low frequency in patients with CRPC does not explain the almost universal acquisition of resistance. We noted that the ability of AR to translocate to nucleus in an enzalutamide-rich environment opens up the possibility of a posttranslational modification in AR that is refractory to enzalutamide binding. Chemical proteomics in enzalutamide-resistant CRPC cells revealed acetylation at Lys 609 in the zinc finger DNA binding domain of AR (acK609-AR) that not only allowed AR translocation but also galvanized a distinct global transcription program, conferring enzalutamide insensitivity. Mechanistically, acK609-AR was recruited to the AR and ACK1/TNK2 enhancers, up-regulating their transcription. ACK1 kinase–mediated AR Y267 phosphorylation was a prerequisite for AR K609 acetylation, which spawned positive feedback loops at both the transcriptional and posttranslational level that regenerated and sustained high AR and ACK1 expression. Consistent with these findings, oral and subcutaneous treatment with ACK1 small-molecule inhibitor, ( R )-9b, not only curbed AR Y267 phosphorylation and subsequent K609 acetylation but also compromised enzalutamide-resistant CRPC xenograft tumor growth in mice. Overall, these data uncover chronological modification events in AR that allows prostate cancer to evolve through progressive stages to reach the resilient recurrent CRPC stage, opening up a therapeutic vulnerability.

IntroductionStudies suggest that continuous glucose monitors (CGMs) play an important role in the management of diabetes. Although general acceptance has been reported by patients with type 2 diabetes towards the use of CGMs, potential barriers exist like pain due to sensor insertion, accidental removal of the device or adhesive strip, impacts of daily activities, skin reactions to sensor adhesive, etc. This systematic review of qualitative studies aims to explore the perspectives, experiences and narratives of patients and caregivers about CGM use, and its barriers and facilitators.Methods and analysisThis review will include qualitative studies and cross-sectional and longitudinal cohort studies using open-ended questions, published in English by 30 October 2021. The following electronic databases will be searched: Cochrane Library, PubMed, EMBASE, CINAHL, PsycINFO and Scopus. A search of grey literature will be conducted via an online search of Google Scholar, WorldCat, ClinicalTrials.gov and OpenGrey A combined search strategy using medical subject headings (MeSH), controlled vocabulary and ‘free-text’ terms will be appropriately revised to suit each database. Primary outcomes will include patient and caregiver perspectives on diabetes management regarding glucose control; living with CGM (quality of life, experience of wearing a CGM); psychological aspects (anxiety, depression, emotional burden); barriers (technical issues, financial issues) to use of CGM and thoughts (interpretation, understanding) on the CGM report. A qualitative meta-synthesis will be conducted employing a systematic literature search of existing literature, quality assessment using study-specific tools and an aggregative thematic synthesis by a multidisciplinary team.Ethics and disseminationEthical approval is not required since this is a systematic review. The results will help improve clinical implementation of CGMs on part of both patients and caregivers.PROSPERO registration numberCRD42020152211.

PARP inhibitors (PARPis) display single-agent anticancer activity in small cell lung cancer (SCLC) and other neuroendocrine tumors independent of BRCA1/2 mutations. Here, we determine the differential efficacy of multiple clinical PARPis in SCLC cells. Compared with the other PARPis rucaparib, olaparib, and niraparib, talazoparib displays the highest potency across SCLC, including SLFN11-negative cells. Chemical proteomics identifies PARP16 as a unique talazoparib target in addition to PARP1. Silencing PARP16 significantly reduces cell survival, particularly in combination with PARP1 inhibition. Drug combination screening reveals talazoparib synergy with the WEE1/PLK1 inhibitor adavosertib. Global phosphoproteomics identifies disparate effects on cell-cycle and DNA damage signaling thereby illustrating underlying mechanisms of synergy, which is more pronounced for talazoparib than olaparib. Notably, silencing PARP16 further reduces cell survival in combination with olaparib and adavosertib. Together, these data suggest that PARP16 contributes to talazoparib's overall mechanism of action and constitutes an actionable target in SCLC.

Although the BRAF inhibitors dabrafenib and vemurafenib have both proven successful against BRAF-mutant melanoma, there seem to be differences in their mechanisms of action. Here, we show that dabrafenib is more effective at inhibiting the growth of NRAS-mutant and KRAS-mutant cancer cell lines than vemurafenib. Using mass spectrometry-based chemical proteomics, we identified NEK9 and CDK16 as unique targets of dabrafenib. Both NEK9 and CDK16 were highly expressed in specimens of advanced melanoma, with high expression of both proteins correlating with a worse overall survival. A role for NEK9 in the growth of NRAS- and KRAS-mutant cell lines was suggested by siRNA studies in which silencing was associated with decreased proliferation, cell cycle arrest associated with increased p21 expression, inhibition of phospho-CHK1, decreased CDK4 expression, and the initiation of a senescence response. Inhibition of CDK4 but not CHK1 recapitulated the effects of NEK9 silencing, indicating this to be the likely mechanism of growth inhibition. We next turned our attention to CDK16 and found that its knockdown inhibited the phosphorylation of the Rb protein at S780 and increased expression of p27. Both of these effects were phenocopied in NRAS- and KRAS-mutant cancer cells by dabrafenib, but not vemurafenib. Combined silencing of NEK9 and CDK16 was associated with enhanced inhibition of melanoma cell proliferation. In summary, we have identified dabrafenib as a potent inhibitor of NEK9 and CDK16, and our studies suggest that inhibition of these kinases may have activity against cancers that do not harbor BRAF mutations.

Background Continuous glucose monitorings (CGMs) have been used to manage diabetes with reasonable glucose control amongst patients with type 2 diabetes (T2D) in recent decades. CGMs measure interstitial fluid glucose levels to provide information about glucose levels, which identify fluctuation that would not have been identified with conventional self-monitoring. Self-monitoring of blood glucose (SMBG) is a classical tool to measure glycaemic changes. However, the effectiveness of glucose control, hypoglycemia, weight change, quality of life and user satisfaction, are needed to evaluate and compare CGMs and SMBG amongst adults with T2D. Methods The review will compare the various forms of CGM systems (i.e flash CGM, real-time CGM, retrospective CGM) versus SMBG or usual intervention regarding diabetes management amongst adults with T2D. The following databases will be searched: Cochrane Library, PubMed, EMBASE, CINAHL, PsycINFO, Scopus and grey literature (ClinicalTrials.gov, PsycEXTRA, ProQuest Dissertations, Google Scholar and Theses Global) for the identification of studies. The studies involving adults (aged ≥ 18 years old) will be included. We will only include and summarise randomised clinical trials (RCTs) with respect to authors, publication type, year, status and type of devices. Studies published in English between February 2010 and March 2020, will be included as the field of CGMs amongst T2D patients has emerged over the last decade. Primary outcomes will be HbA1c (glycosylated haemoglobin level) (mmol/L), body weight (kg), time spent with hypoglycaemia (< 70 mg/dl) or hyperglycaemia (≥ 180 mg/dl), blood pressure (< 140/90 mmHg is considered as good management) and quality of life (understanding and feeling of living situation based on culture and value system). Secondary outcome measures will be user satisfaction (patient or treatment/intervention satisfaction or satisfaction scale) and barriers (physical and mental difficulties or issues). Study selection, data extraction and risk of bias assessment will be conducted independently by at least two reviewers. A third reviewer will determine and resolve discrepancies. Moreover, the quality of the evidence of the review will be assessed according to the Grading of Recommendations Assessment, Development and Evaluation tool (GRADE). Discussion The review will synthesise evidence on the comparison between using CGMs and SMBG. The results will support researchers and health professionals to determine the most effective methods/technologies in the overall diabetes management. Systematic review registration PROSPERO CRD42020149212

Additional file 3: Logic grid.

Additional file 1: Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) and checklist.

Additional file 6: Classification of randomised trials of biases.

Additional file 2: Preliminary search strategy for PubMed.

Additional file 4: PRISMA-2009 flow diagram.

Additional file 5: Data extraction form.

Inhibition of the WEE1 tyrosine kinase enhances anticancer chemotherapy efficacy. Accordingly, the WEE1 inhibitor AZD1775 (previously MK-1775) is currently under evaluation in clinical trials for cancer in combination with chemotherapy. AZD1775 has been reported to display high selectivity and is therefore used in many studies as a probe to interrogate WEE1 biology. However, AZD1775 also exhibits anticancer activity as a single agent although the underlying mechanism is not fully understood. Using a chemical proteomics approach, we here describe a proteome-wide survey of AZD1775 targets in lung cancer cells and identify several previously unknown targets in addition to WEE1. In particular, we observed polo-like kinase 1 (PLK1) as a new target of AZD1775. Importantly, in vitro kinase assays showed PLK1 and WEE1 to be inhibited by AZD1775 with similar potency. Subsequent loss-of-function experiments using RNAi for WEE1 and PLK1 suggested that targeting PLK1 enhances the pro-apoptotic and antiproliferative effects observed with WEE1 knockdown. Combination of RNAi with AZD1775 treatment suggested WEE1 and PLK1 to be the most relevant targets for mediating AZD1775's anticancer effects. Furthermore, disruption of WEE1 by CRISPR-Cas9 sensitized H322 lung cancer cells to AZD1775 to a similar extent as the potent PLK1 inhibitor BI-2536 suggesting a complex crosstalk between PLK1 and WEE1. In summary, we show that AZD1775 is a potent dual WEE1 and PLK1 inhibitor, which limits its use as a specific molecular probe for WEE1. However, PLK1 inhibition makes important contributions to the single agent mechanism of action of AZD1775 and enhances its anticancer effects.

Background: Small cell lung cancer (SCLC) is a highly aggressive type of neuroendocrine tumor with a low survival rate, signifying an unmet medical need. Although PARP inhibitors (PARPis) display single-agent anticancer activity in SCLC, the underlying biology is poorly understood. In the present study, we determined the differential efficacy of multiple clinically relevant PARPis and identified a novel non-canonical and actionable PARPi target in SCLC. Methods: A panel of SCLC cell lines was analyzed for sensitivity to PARPis using the Cell Titer Glow (CTG) viability assay. Chemical proteomics and western blotting were performed to investigate the targets of PARPis. The effect of PARP16 on cell viability was analyzed using RNA interference. Drug combination screening for inhibition of viability was performed, network-wide signaling changes were determined using phosphoproteomics, and efficacy of drug combinations was determined in ex vivo PDX clonogenic assays. Results: Compared to other clinically relevant PARPis (rucaparib, olaparib, niraparib) talazoparib displayed the highest potency across a panel of SCLC cell lines, including cells negative for SLFN11, which has been suggested to indicate PARPi sensitivity. Subsequent chemical proteomics with SLFN11-negative SCLC cells identified the non-canonical PARP family member PARP16 as a unique talazoparib target, along with the eminent PARP1. Transient knockdown of PARP16 significantly reduced cell survival in certain SCLC cell lines, particularly in combination with olaparib. Furthermore, drug combination screens revealed synergistic activity of talazoparib when combined with the WEE1 inhibitor adavosertib in SCLC cells, which was more pronounced than the combination of adavosertib with olaparib, which has no activity against PARP16. Silencing of PARP16, in combination with olaparib and adavosertib, with enhanced cell killing, and global phosphoproteomics identified network-wide signaling crosstalk inhibition by the talazoparib/adavosertib drug combination. Conclusion: Our data suggest that in addition to the well-studied PARP1 targeting mechanism, PARP16, a non-canonical member of the PARP family, is likely involved in talazoparib’s overall mechanism of action and may constitute a novel actionable target in SCLC. Citation Format: Vinayak Palve, Clare Knezevic, Yunting Luo, Xueli Li, Slivia Novakova, Eric Welsh, Bin Fang, Fumi Kinose, Eric B Haura, Alvaro N Monteiro, John M Koomen, Harshani R Lawrence, Uwe Rix. PARP16 is a novel target of talazoparib which contributes to synergy with adavosertib in SCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B021. doi:10.1158/1535-7163.TARG-19-B021

$('.header-date').hide();$('#titleAuthors').hide();$('#abstractHeader').hide(); Hua Yang 1 , Harshani R. Lawrence 3 , Aslamuzzaman Kazi 1 , Harsukh Gevariya 1 , Ronil Patel 1 , Yunting Luo 3 , Uwe Rix 1 , 2 , 4 , Ernst Schonbrunn 1 , 2 , 4 , Nicholas Lawrence 1 , 2 , 4 , Said M. Sebti 1 , 2 , 4 1 Drug Discovery Department, University of South Florida, Tampa, FL, USA. 2 Chemical Biology and Molecular Medicine Program, University of South Florida, Tampa, FL, USA. 3 Chemical Biology Core, Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, FL, USA. 4 Departments of Molecular Medicine and Oncologic Sciences, University of South Florida, Tampa, FL, USA. Correspondence: Said M. Sebti, e-mail: said.sebti@moffitt.org Conflict of Interests The Authors have no conflict of interests. Received : November 20, 2013 Accepted : March 19, 2014 Published : March 22, 2014 ABSTRACT Aurora A and JAK2 kinases are involved in cell division and tumor cell survival, respectively. Here we demonstrate that ectopic expression of Aurora A and JAK2 together is more effective than each alone at inducing non-transformed cells to grow in an anchorage-independent manner and to invade. Furthermore, siRNA silencing or pharmacological inhibition of Aurora A and JAK2 with Alisertib and Ruxolitinib, respectively, is more effective than blocking each kinase alone at suppressing anchorage-dependent and –independent growth and invasion as well as at inducing apoptosis. Importantly, we have developed dual Aurora and JAK inhibitors, AJI-214 and AJI-100, which potently inhibit Aurora A, Aurora B and JAK2 in vitro. In human cancer cells, these dual inhibitors block the auto-phosphorylation of Aurora A (Thr-288) and the phosphorylation of the Aurora B substrate histone H3 (Ser-10) and the JAK2 substrate STAT3 (Tyr-705). Furthermore, AJI-214 and AJI-100 inhibit anchorage dependent and independent cell growth and invasion and induce G2/M cell cycle accumulation and apoptosis. Finally, AJI-100 caused regression of human tumor xenografts in mice. Taken together, our genetic and pharmacological studies indicate that targeting Aurora A and JAK2 together is a more effective approach than each kinase alone at inhibiting malignant transformation and warrant further advanced pre clinical investigations of dual Aurora A/JAK2 inhibitors as potential anti tumor agents.

Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) are a promising class of targeted cancer drugs, but their individual target profiles beyond the PARP family, which could result in differential clinical use or toxicity, are unknown. Using an unbiased, mass spectrometry-based chemical proteomics approach, we generated a comparative proteome-wide target map of the four clinical PARPi, olaparib, veliparib, niraparib, and rucaparib. PARPi as a class displayed high target selectivity. However, in addition to the canonical targets PARP1, PARP2, and several of their binding partners, we also identified hexose-6-phosphate dehydrogenase (H6PD) and deoxycytidine kinase (DCK) as previously unrecognized targets of rucaparib and niraparib, respectively. Subsequent functional validation suggested that inhibition of DCK by niraparib could have detrimental effects when combined with nucleosideanalog pro-drugs. H6PD silencing can cause apoptosis and further sensitize cells to PARPi, suggesting that H6PD may be, in addition to its established role in metabolic disorders, a new anticancer target.

Screening efforts led to the identification of PI-8182 (1), an inhibitor of the chymotrypsin-like (CT-L) activity of the proteasome. Compound 1 contains a hydronaphthoquinone pharmacophore with a thioglycolic acid side chain at position 2 and thiophene sulfonamide at position 4. An efficient synthetic route to the hydronaphthoquinone sulfonamide scaffold was developed, and compound 1 was synthesized in-house to confirm the structure and activity (IC(50) = 3.0 ± 1.6 μM [n = 25]). Novel hydronaphthoquinone derivatives of 1 were designed, synthesized, and evaluated as proteasome inhibitors. The structure-activity relationship (SAR) guided synthesis of more than 170 derivatives revealed that the thioglycolic acid side chain is required and the carboxylic acid group of this side chain is critical to the CT-L inhibitory activity of compound 1. Furthermore, replacement of the carboxylic acid with carboxylic acid isosteres such as tetrazole or triazole greatly improves potency. Compounds with a thio-tetrazole or thio-triazole side chain in position 2, where the thiophene was replaced by hydrophobic aryl moieties, were the most active compounds with up to 20-fold greater CT-L inhibition than compound 1 (compounds 15e, 15f, 15h, 15j, IC(50) values around 200 nM, and compound 29, IC(50) = 150 nM). The synthetic iterations described here not only led to improving potency in vitro but also resulted in the identification of compounds that are more active such as 39 (IC(50) = 0.44 to 1.01 μM) than 1 (IC(50) = 3.54 to 7.22 μM) at inhibiting the proteasome CT-L activity in intact breast cancer cells. Treatment with 39 also resulted in the accumulation of ubiquitinated cellular proteins and inhibition of tumor cell proliferation of breast cancer cells. The hit 1 and its analogue 39 inhibited proteasome CT-L activity irreversibly.

The protein tyrosine phosphatase (PTP) Shp2 (PTPN11) is an attractive target for anticancer drug discovery because it mediates growth factor signaling and its gain-of-function mutants are causally linked to leukemias. We previously synthesized SPI-112 from a lead compound of Shp2 inhibitor, NSC-117199. In this study, we demonstrated that SPI-112 bound to Shp2 by surface plasmon resonance (SPR) and displayed competitive inhibitor kinetics to Shp2. Like some other compounds in the PTP inhibitor discovery efforts, SPI-112 was not cell permeable, precluding its use in biological studies. To overcome the cell permeation issue, we prepared a methyl ester SPI-112 analog (SPI-112Me) that is predicted to be hydrolyzed to SPI-112 upon entry into cells. Fluorescence uptake assay and confocal imaging suggested that SPI-112Me was taken up by cells. Incubation of cells with SPI-112Me inhibited epidermal growth factor (EGF)-stimulated Shp2 PTP activity and Shp2-mediated paxillin dephosphorylation, Erk1/2 activation, and cell migration. SPI-112Me treatment also inhibited Erk1/2 activation by a Gab1-Shp2 chimera. Treatment of Shp2(E76K) mutant-transformed TF-1 myeloid cells with SPI-112Me resulted in inhibition of Shp2(E76K)-dependent cell survival, which is associated with inhibition of Shp2(E76K) PTP activity, Shp2(E76K)-induced Erk1/2 activation, and Bcl-XL expression. Furthermore, SPI-112Me enhanced interferon-gamma (IFN-gamma)-stimulated STAT1 tyrosine phosphorylation, ISRE-luciferase reporter activity, p21 expression, and the anti-proliferative effect. Thus, the SPI-112 methyl ester analog was able to inhibit cellular Shp2 PTP activity.

To explore the clinical efficacy and safety of acupuncture and moxibustion for copracrasia.By prospective live randomized controlled trial, 40 cases with copracrasia were randomly divided into an acupuncture-moxibustion group and a medication group, 20 cases in each one. In the acupuncture-moxibustion group, acupuncture was applied at Ciliao (BL 32), Changqiang (GV 1) and Tianshu (ST 25) and mild moxibustion was used at Qihai (CV 6). Treatment was given for 12 weeks and 32 times, 3 times a week in the front 8 weeks, 2 times a week in the latter 4 weeks. In the medication group, conventional symptomatic treatment, support therapy, and complications preventing and treating were adopted for 12 weeks. Anal incontinence score (Vaizey incontinence score), effective rate and self-rating score for satisfaction were observed before and after treatment and in the follow-up period.After 12 weeks' treatment in the two groups, Vaizey incontinence' scores were both decreased (both P0. 05), and after treatment and in the follow-up period the scores in the acupuncture-moxibustion group were lower than those in the corresponding period in the medication group (both P0. 05). The effective rate of the acupuncture-moxibustion group was 80. 0% (16/20), which was statistically different from 50. 0% (10/20) in the medication group (P0. 05). The effective rate in the follow-up period of the acupuncture-moxibustion group was 90. 0% (18/20) and it was not statistically different from 80. 0% (16/20) in the medication group (P0. 05). The self-rating scores for satisfaction in the acupuncture-moxibustion group were superior to those in the medication group after treatment and in the follow-up period (both P0. 05).Acupuncture and moxibustion could improve copracrasia and the acupuncture-moxibustion rules and characteristics for the disorder should be paid attention to in the further research.

The tyrosine kinase ACK1, a critical signal transducer regulating survival of hormone-refractory cancers, is an important therapeutic target, for which there are no selective inhibitors in clinical trials to date. This work reports the discovery of novel and potent inhibitors for ACK1 tyrosine kinase (also known as TNK2) using an innovative fragment-based approach. Focused libraries were designed and synthesized by selecting fragments from reported ACK inhibitors to create hybrid structures in a mix and match process. The hybrid library was screened by enzyme-linked immunosorbent assay-based kinase inhibition and 33P HotSpot assays. Systematic structure–activity relationship studies led to the identification of compound (R)-9b, which shows potent in vitro (IC50 = 56 nM, n = 3, 33P HotSpot assay) and in vivo (IC50 < 2 μM, human cancer cell lines) ACK1 inhibition. Both (R)-9b and (S)-9b were stable in human plasma and displayed a long half-life (t1/2 > 6 h).

The ortho-carboxylic acid substituted bisanilinopyrimidine 1 was identified as a potent hit (Aurora A IC50 = 6.1 ± 1.0 nM) from in-house screening. Detailed structure activity relationship (SAR) studies indicated that polar substituents at the para position of the B-ring are critical for potent activity. X-ray crystallography studies revealed that compound 1 is a type-I inhibitor that binds the Aurora kinase active site in a DFG-in conformation. Structure activity guided replacement of the A-ring carboxylic acid with halogens and incorporation of fluorine at the pyrimidine 5-position led to highly potent inhibitors of Aurora A that bind in a DFG-out conformation. B-ring modifications were undertaken to improve the solubility and cell permeability. Compounds such as 9m with water-solubilizing moieties at the para-position of the B-ring inhibited the autophosphorylation of Aurora A in MDA-MB-468 breast cancer cells.

Most protein kinases share a DFG (Asp-Phe-Gly) motif in the ATP site which can assume two distinct conformations, the active DFG-in and the inactive DFG-out states. Small molecule inhibitors able to induce the DFG-out state have received considerable attention in kinase drug discovery. Using a typical DFG-in inhibitor scaffold of Aurora A, a kinase involved in the regulation of cell division, we found that halogen and nitrile substituents directed at the N-terminally flanking residue Ala273 induced global conformational changes in the enzyme, leading to DFG-out inhibitors that are among the most potent Aurora A inhibitors reported to date. The data suggest an unprecedented mechanism of action, in which induced-dipole forces along the Ala273 side chain alter the charge distribution of the DFG backbone, allowing the DFG to unwind. As the ADFG sequence and three-dimensional structure is highly conserved, DFG-out inhibitors of other kinases may be designed by specifically targeting the flanking alanine residue with electric dipoles.

BACKGROUND Androgen receptor (AR) plays a critical role in the progression of both androgen-dependent and androgen-independent prostate cancer (AIPC). Ligand-independent activation of AR in AIPC or castration resistant prostate cancer (CRPC) is often associated with poor prognosis. Recently, tyrosine kinase Ack1 has been shown to regulate AR activity by phosphorylating it at tyrosine 267 and this event was shown to be critical for AIPC growth. However, whether a small molecule inhibitor that can mitigate Ack1 activation is sufficient to abrogate AR activity on AR regulated promoters in androgen-depleted environment is not known. METHODS We have generated two key resources, antibodies that specifically recognize pTyr267-AR and synthesized a small molecule inhibitor of Ack1, 4-amino-5,6-biaryl-furo[2,3-d]pyrimidine (named here as AIM-100) to test whether AIM-100 modulates ligand-independent AR activity and inhibits prostate cell growth. RESULTS Prostate tissue microarray analysis indicates that Ack1 Tyr284 phosphorylation correlates positively with disease progression and negatively with the survival of prostate cancer patients. Interestingly, neither pTyr267-AR expression nor its transcriptional activation was affected by anti-androgens in activated Ack1 expressing or EGF stimulated prostate cells. However, the Ack1 inhibitor, AIM-100, not only inhibited Ack1 activation but also able to suppress pTyr267-AR phosphorylation, binding of AR to PSA, NKX3.1, and TMPRSS2 promoters, and inhibit AR transcription activity. CONCLUSION Ack1 Tyr284 phosphorylation is prognostic of progression of prostate cancer and inhibitors of Ack1 activity could be novel therapeutic agents to treat AIPC. Prostate 70:1274–1285, 2010. © 2010 Wiley-Liss, Inc.

Screening of the NCI Diversity Set-1 identified PI-083 (NSC-45382) a proteasome inhibitor selective for cancer over normal cells. Focused libraries of novel compounds based on PI-083 chloronaphthoquinone and sulfonamide moieties were synthesized to gain a better understanding of the structure-activity relationship responsible for chymotrypsin-like proteasome inhibitory activity. This led to the demonstration that the chloronaphthoquinone and the sulfonamide moieties are critical for inhibitory activity. The pyridyl group in PI-083 can be replaced with other heterocyclic groups without significant loss of activity. Molecular modeling studies were also performed to explore the detailed interactions of PI-083 and its derivatives with the beta5 and beta6 subunits of the 20S proteasome. The refined model showed an H-bond interaction between the Asp-114 and the sulfonamide moiety of the PI-083 in the beta6 subunit.

Screening of the NCI diversity set of compounds has led to the identification of 5 (NSC-117199), which inhibits the protein tyrosine phosphatase (PTP) Shp2 with an IC50 of 47 microM. A focused library incorporating an isatin scaffold was designed and evaluated for inhibition of Shp2 and Shp1 PTP activities. Several compounds were identified that selectively inhibit Shp2 over Shp1 and PTP1B with low to submicromolar activity. A model for the binding of the active compounds is proposed.

Due to the clinical promise of PARP inhibitors (PARPi) in both breast and ovarian cancers, an understanding of the full protein target profile of each clinical PARPi is important for understanding the differential activity within this drug class. Modified versions of clinical PARPi niraparib, olaparib, rucaparib, and veliparib containing propylamine linkers were synthesized to allow for coupling to solid supports. The modified PARPi were immobilized onto NHS-ester sepharose beads which were then incubated with CAL51 breast cancer cell lysate. Free unmodified drug was added to the bead/lysate mixtures as a competition control to aid in the identification of specific drug-protein interactions. After washing, elution, and trypsin digest, bound proteins were identified by LC-MS/MS and analyzed by Significance Analysis of Interactome (SAINT) Express. Immunoprecipitation was performed by pre-binding Protein A/G beads to a polyclonal PARP-1 antibody before incubation with lysate. H6PD activity was assessed by monitoring consumption of NADP in H6PD-overexpressing HEK293 cell lysate. Cell viability was measured by CellTiter Glo after incubation with drug for three days. Target profiles of PARPi were generated by unbiased chemical proteomics. Target candidates were identified using both SAINT and a score-based analysis method. Within the PARP protein family, most PARPi interacted with PARP 1 and 2 as well as tankyrase 1 and 2. SAINT Express analysis of the chemical proteomics data revealed deoxycytidine kinase (DCK) as a potential target of niraparib and hexose-6-phosphate dehydrogenase (H6PD) as a potential target of rucaparib. Analysis of H6PD activity in cellular lysate of H6PD-overexpressing cells revealed inhibition of H6PD by rucaparib. In whole cell culture, niraparib reduces the cytotoxicity of cytarabine, an anticancer agent that is activated by DCK-mediated phosphorylation. Inosine monophosphate dehydrogenase 2 (IMPDH2), which was initially identified as a candidate target of all PARPi, was co-immunoprecipitated from CAL51 lysate with PARP-1, confirming its PARP-1 binding abilities. Each clinical PARPi's molecular target profile was characterized with chemical proteomics. The clinical implications of inhibition of H6PD are currently unclear, but H6PD is of interest for its role in glucocorticoid activation and is a possible therapeutic target for diabetes. Rucaparib is the only small molecule known to inhibit H6PD and as such rucaparib may provide a starting point for the development of chemical tool compounds and therapeutics. Inhibition of DCK by niraparib suggests that combination strategies with niraparib and either cytarabine or gemcitabine should not be pursued. Understanding the full protein target profiles of these promising cancer drugs is important for their clinical usage and these results will enable a more complete understanding of their clinical activity. Citation Format: Claire Knezevic, Gabriela Wright, Brent Kuenzi, Lily Remsing Rix, Yunting Luo, Harshani Lawrence, Uwe Rix. Differential proteome-wide target profiles of clinical poly(ADP-ribose) polymerase inhibitors in breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A71.

The Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2), a widely expressed cytoplasmic tyrosine phosphatase with two src-homology 2 (SH2) domains, has received much attention in the signal transduction field. SHP2 is a transducer of growth factor, cytokine, integrin, and hormone signaling pathways that regulate processes such as cell proliferation, differentiation, adhesion, migration, and apoptosis and plays a pivotal role in growth factor and cytokine signaling. Gain-of-function SHP2 mutations are associated with several types of leukemias, and solid tumors; this makes SHP2 is an attractive target for cancer therapy Several small molecules have been developed for inhibition of this important phosphatase, which serve as chemical tools to probe the role of SHP2 in disease and lead compounds for optimization. Nevertheless it is challenging to improve these lead compounds with better potency, selectivity and cell permeability. To gain structural insights for development of potent and selective SHP2 inhibitors, we synthesized a non-hydrolyzable phosphotyrosine peptide mimetic based on reported SHP2 substrate peptide sequence (10-mer). The non-hydrolyzable phosphopeptide, containing the phosphotyrosyl surrogate difluoromethylphosphonate, was synthesized using solid phase peptide synthesis methods from the known building block N-FMOCF2Pmp and we improved the coupling reaction conditions to generate a peptide library of difluoromethylphosphonate to understand the SHP2 affinity. We will present the synthesis of the peptide library, analysis of SHP2 phosphatase inhibition, and the binding mode (X-ray crystal structure) of the phosphopeptide mimic to SHP2. Citation Format: Harshani R. Lawrence, Yunting Luo, Steven Gunawan, Andreas Becker, Yuan Ren, Ernst Schonbrunn, Jie Wu, Nicholas J. Lawrence. Development of a focused non-hydrolyzable phosphopeptide library based on a high affinity SHP2 substrate. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3697. doi:10.1158/1538-7445.AM2015-3697

The Shp2 protein tyrosine phosphatase mediates signal transduction of growth factor receptors and regulates cellular activities critical to tumor growth and metastasis. Shp2-activating mutations are associated with several types of leukemias and solid tumors, making Shp2 an attractive target for anticancer development. While there are reports of Shp2 inhibitors, there is a need for compounds with higher potency and improved selectivity. One approach to the discovery of new Shp2 inhibitors is via high throughput screening (HTS) of small molecule libraries. The small molecule probe 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) is currently a widely used substrate for phosphatases and has been used to assay Shp2 activity. However, DIFMUP binds only the catalytic site of the phosphatase and is unlikely to reveal compounds that are selective for Shp2 in an HTS. There is clearly a need to develop probes that are more sensitive and more specific for a particular phosphatase to identify Shp2 selective inhibitors and to measure Shp2 activity. We have developed a new phosphopeptide-like Shp2 substrate that becomes fluorescent upon enzyme-mediated hydrolysis, based on a phosphopeptide sequence that has high affinity for Shp2. We will report details of the novel Shp2 activity probe and its use as a reagent for HTS and determination of Shp2 inhibitor potency. We will also report on its suitability for HTS and preliminary results from screening and its potential for the discovery of novel Shp2 inhibitors. We will discuss the binding mode of the probe, based on novel co-crystal structures of related peptides with Shp2. Citation Format: Steven Gunawan, Yunting Luo, Yuan Ren, Harshani R. Lawrence, Jerry Wu, Nicholas J. Lawrence. Preparation of peptide-based Shp2 substrates with phosphatase activity-dependent fluorescence. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4547. doi:10.1158/1538-7445.AM2015-4547

Inhibition of mutant H-Ras farnesylation with farnesyltransferase inhibitors (FTIs) blocks its binding to membranes and its ability to activate oncogenic signaling. In contrast, inhibition of K-Ras farnesylation with FTIs leads to its prenylation by geranylgeranyltransferase I (GGT-1), and therefore, inhibition of K-Ras prenylation and oncogenic function requires blocking both FT and GGT-1. Furthermore, several proteins downstream of K-Ras that mediate its malignant transforming activity also require farnesylation (e.g.Rheb) or gernaylgeranylation (e.g. Ral). In addition, the ability of mutant K-Ras to induce lung cancer in mouse models is severely hampered when both FT and GGT-1 are conditionally deficient. These observations prompted us to design small molecule dual FT and GGT-1 inhibitors. In this presentation, the development of FGTI-2734 as a novel therapeutic for K-Ras dependent cancers will be described. The presentation will focus on the effect of this dual inhibitor as compared to the selective FTI-2148 and GGTI-2418 on K-ras prenylation, oncogenic signaling and malignant transformation in cancer cells that depend on K-Ras. Citation Format: Kazi Aslamuzzaman, Xiaolei Zhang, Yunting Luo, Ronil Patel, Steven Fletcher, Christopher Cummings, Harshani Lawrence, Andrew Hamilton, Said M. Sebti. Dual inhibitors of FT and GGT-1 as novel therapeutic agents for K-Ras-dependent tumors. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B35. doi: 10.1158/1557-3125.RASONC14-B35

Our ongoing efforts to validate kinases for cancer therapy led to the novel finding that ectopic expression of JAK2 and Aurora A together is more effective than each alone at inducing normal cells to grow in an anchorage-independent manner and to invade. In addition, siRNA silencing or pharmacological inhibition of JAK2 and Aurora A with Ruxolitinib and Alisertib, respectively, is more effective than blocking each kinase alone at suppressing anchorage-dependent and -independent growth and invasion as well as at inducing apoptosis. This led us to develop dual Aurora and JAK inhibitors, AJI-214 and AJI-100, which potently inhibit the activities of Aurora A, Aurora B and JAK2 in vitro. In human cancer cells, these dual inhibitors block the auto-phosphorylation of Aurora A (Thr-288) and the phosphorylation of the Aurora B substrate histone H3 (Ser-10) and the JAK2 substrate STAT3 (Tyr-705). Furthermore, AJI-214 and AJI-100 inhibit anchorage dependent and independent cell growth and invasion and induce G2/M cell cycle accumulation and apoptosis. Finally, the more soluble AJI-100 was effective at inducing tumor regression of human xenografts in mice. Taken together, our genetic and pharmacological studies indicate that targeting Aurora A and JAK2 together is a more effective approach than each alone at inhibiting malignant transformation and warrant further advanced pre clinical investigations of dual Aurora A/JAK2 inhibitors as potential anti tumor agents. Citation Format: Said M. Sebti, Hua Yang, Harshani Lawrence, Aslamuzzaman Kazi, Harsukh Gervaria, Ronil Patel, Yunting Luo, Uwe Rix, Ernst Schonbrunn, Nicholas Lawrence. Combined blockade of Aurora A and JAK2 kinase is highly effective at inhibiting malignant transformation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1750. doi:10.1158/1538-7445.AM2014-1750

The Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) is a transducer of growth factor, cytokine, integrin, and hormone signaling pathways that regulate processes such as cell proliferation, differentiation, adhesion, migration, and apoptosis and plays a pivotal role in growth factor and cytokine signaling. Gain-of-function SHP2 mutations are associated with several types of leukemias, and solid tumors. This makes SHP2 an attractive target for developing new anticancer therapy. Several small molecules have been developed for inhibition of this important phosphatase, which serve as chemical tools to probe the role of SHP2 in disease and lead compounds for optimization. Nevertheless improvements are required to improve these lead compounds with better potency, selectivity and cell permeability. To gain structural insights for development of potent and selective Shp2 inhibitors, we synthesized a non-hydrolysable phosphotyrosine peptide mimetic, based on reported Shp2 substrate peptide sequences. The non-hydrolyzable phosphopeptide, containing the phosphotyrosyl surrogate difluoromethylphosphonate, was prepared by solid phase peptide synthesis methods from the known building block N-FMOCF2Pmp. We will present the synthesis of the peptide, analysis of Shp2 phosphatase inhibition, and binding of the phosphopeptide mimic to SHP2 by micro isothermal calorimetry. We will present a model for the binding mode of the peptide and its implications for the design of selective Shp2 inhibitors. Citation Format: Harshani R. Lawrence, Yiyu Ge, Andreas Becker, Yuan Ren, Yunting Luo, Jie Wu and Nicholas J. Lawrence. Development of a non-hydrolysable phosphotyrosine mimetic peptide based on a high affinity SHP2 substrate. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2518. doi:10.1158/1538-7445.AM2014-2518

Lysophosphatidic acid acyltransferase (LPAAT-β), a phosphatidic acid (PA)-generating enzyme, plays an essential role in triglyceride synthesis. A less familiar role for LPAAT-β is that of a regulator of cell growth and differentiation by means of the function of its product, PA, which plays a vital role in the activation of key signal transduction proteins including Raf, PKC-ζ and mTOR. It is in this role that LPAAT-β presents as a potential therapeutic target in cancer. Accordingly, we developed a library of small molecule inhibitors of LPAAT-β with nanomolar activity against LPAAT-β enzyme activity in vitro (2012 Annual Meeting, Abstract #2470). We have now performed additional SAR and lead optimization to produce a potent small molecule that inhibits proliferation, mTOR signaling pathways, and total cellular PA levels at low micromolar concentrations. The lead compound also inhibits anchorage-independent growth of pancreatic cancer cell lines at nanomolar concentrations. This compound demonstrates effects on cellular signaling that are similar to the effects of LPAAT-β siRNA. Citation Format: Michelle A. Blaskovich, Yunting Luo, Yiyu Ge, Saïd M. Sebti, Harshani R. Lawrence, Nicholas J. Lawrence, Gregory M. Springett. A novel small molecule inhibitor of lysophosphatidic acid acyltransferase-beta inhibits pancreatic cancer cell proliferation and mTOR signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1640. doi:10.1158/1538-7445.AM2014-1640

ACK1, is a non-receptor tyrosine kinase that is aberrantly activated, over expressed or mutated in many cancer cell types. It interacts with several important ligand-activated receptor tyrosine kinases (RTKs), for example, EGFR, MerTK, HER2, PDGFR and insulin receptor to initiate intracellular signaling cascades. ACK1 activation has been reported in various cancers including prostate, breast and pancreatic tumors. We have shown that the androgen receptor (AR) and AKT are two major downstream effectors of ACK1. ACK1 directly phosphorylates AKT at Tyr176 resulting in AKT membrane localization and activation. We have also found that in prostate cancer cells ACK1 phosphorylates AR at Tyr-267 in an androgen-independent manner. Tyr284-phosphorylated-ACK1, Tyr176-phosphorylated-AKT and Tyr267-phosphorylated-AR levels are positively correlated with the severity of disease progression, and inversely correlated with the survival of patients with prostate cancer. Similarly, ACK1 mediated AKT tyrosine phosphorylation was found to correlate positively with breast cancer progression. Further, we have shown that a 5,6-biaryl-furo[2,3-d]pyrimidine inhibitor of ACK1 (AIM-100) inhibits ACK1 activation and suppresses pTyr267-AR phosphorylation and AKT Tyr176-phosphorylation, inhibiting AR and AKT activity. These findings indicate that Ack1 is prognostic of progression of prostate, breast and pancreatic cancer and inhibitors of ACK1 activity have potential as novel cancer therapeutic agents. We will discuss the development of new potent ACK1 inhibitors by fragment-recombination and structure-based design by incorporating fragments of key ACK1 inhibitors. Focused chemical libraries were developed and structure activity relationships will be described. The work described has produced compounds capable of inhibiting ACK1 in vitro at low nanomolar concentrations. We have used an ELISA based assay that measures the ability of ACK1 to phosphorylate a peptide derived from AKT, as a primary screening assay. ACK1 inhibition of hits was confirmed in the 33P ATP “Hotspot” assay platform. Selected compounds from the library have been shown to inhibit ACK1 autophosphorylation and the phosphorylation of AR at Tyr267, a surrogate for ACK1 inhibition in vivo. Furthermore we will show that ACK1 is a promising drug target for cancer therapy. Citation Format: Harshani R. Lawrence, Yunting Luo, Daniel Zhang, Nathan Tindall, Sevil Ozcan, Miles Huseyin, Sakib Kazi, Sayantani Bandyopadhyay, Kiran Mahajan, Nupam P. Mahajan, Nicholas J. Lawrence. New inhibitors the tyrosine kinase ACK1/TNK2 active in prostate, breast and pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2511. doi:10.1158/1538-7445.AM2014-2511

The Aurora kinases are a family of serine-threonine kinases that play an important role in the regulation of cell division. The three members of the Aurora family Aurora A, B and C share a high degree of structural homology in their kinase domain but each kinase plays a different role in the control of mitosis. Aurora A and B have received the most attention to date as anticancer targets. Detailed studies have shown Aurora A and B are over expressed in a variety of cancers and are implicated in many aspects of tumor development. Aurora A is frequently over-expressed in tumors and cancer cell lines and has characteristics of an oncogene. Aurora B is also over expressed in many cancer types but does not have oncogenic properties. Aurora B plays important roles in M phase to ensure correct chromosome-microtubule alignment and attachment and chromosomal cytokinesis. Therefore, inhibition of Aurora kinase A and B is emerging as target-based therapy in cancer treatment. From our previous work HLM-8598 was identified from an in-house chemical library as a potent and highly selective inhibitor for Aurora A (IC50 value of 0.073 ± 0.002 μM) over Aurora B (IC50 = 5.4 ± 1.8 μM). X-ray crystallography studies of HLM-8598 bound to Aurora A confirmed HLM-8598 is a type-1 kinase inhibitor that interacts with the ATP binding site of the enzyme. The HLM-8598 bound to Aurora A structure-assisted synthesis of new analogs with IC50 < 2.5 nM affinity. We will present the synthesis of new analogs that contain an ortho-halogen substituted phenyl moiety, structure activity relationship studies as a part of our efforts to identify unusually potent Aurora A kinase inhibitors. We will discuss the use of kinase structural elements to rationally guide compound synthesis and improve design strategy. The in vitro and in vivo activities of ortho-halogen phenyl substituted pyrimidines will be presented as unusually potent Aurora A inhibitors and potential anti-cancer agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3902. doi:1538-7445.AM2012-3902

Many low molecular weight GTPases such as Ras, Ral and Rho require posttranslational farnestylation or geranylgeranylation for mediating malignant transformation. This prompted the development of farnesyltransferase (FT) inhibitors (FTI) and geranylgeranyltransferase-I (GGT-1) inhibitors (GGTI) as potential anticancer agents. However, when cancer cells are treated with FTIs, K-Ras becomes geranylgeranylated suggesting that inhibition of both FT and GGT-1 may be required to inhibit tumors harboring K-Ras mutations. Recently our group has developed a series of dual FT and GGT-1 inhibitors based on an ethylenediamine scaffold. Among these dual inhibitors FGTI-2734 was most potent in vitro (FT IC50 = 250±190 nM and GGT-1 IC50 = 520±90 nM). In human cancer cells FGTI-2734 inhibits potently HDJ-2 farnesylation, Rap 1A geranylgeranylation and K-Ras prenylation. Furthermore, FGTI-2734 inhibits the phosphorylation of Erk1/2, Akt and S6K, induces p27 accumulation and inhibits potently tumor cell growth. Finally, FGTI-2734 significantly reduces survivin protein levels, and reduction of survivin levels is associated with induction of apoptosis. Thus, this single molecule with dual FT and GGT-1 inhibitory activities may have distinct advantage over selective FTIs and GGTIs and warrants further advanced pre-clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 620. doi:10.1158/1538-7445.AM2011-620

Protein tyrosine phosphorylation affects cellular activities that control tumor growth and progression. Protein tyrosine phosphorylation, a key regulatory process of signal transduction pathways, is controlled by the action protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Kinases are now a clinically proven anticancer drug target. The development of PTP inhibitors is, by comparison, a relatively new field. Since PTPs do not require a small molecule cofactor they generally show a low propensity to bind a small molecule inhibitor. Nevertheless there are now examples of highly potent PTP inhibitors that provide promising proof-of-principle that PTPs are druggable. Most PTP inhibitors have a charged or highly polar functional group that mimics the phosphotyrosine residue, that often leads to poor ADME and cell permeability properties. We will report our efforts to address the problems of cell permeability we have encountered in our Shp2 PTP inhibitor program. We have previously reported a series of isatin based Shp2 inhibitors based on a compound (NSC-117199) identified from screen of the NCI Diversity Set. We synthesized >100 analogs of NSC-117199 in our lead optimization effort, which yielded several compounds with approximately 50-fold increase in potency and > 10-fold increase in selectivity (Shp2 versus Shp1). However, these Shp2 PTP inhibitors have either a polar nitro or carboxyl group and have no detectable cellular activity. We will report three approaches to address this problem. The first approach was to prepare neutral ester prodrugs. Several compounds have clearly demonstrable activity against Shp2 in cells. A series of esters has also been designed to also improve the water solubility of the PTP inhibitor. A second approach taken was to further modify the isatin scaffold that does not necessitate the use of a prodrug strategy. We will report the discovery of new isatins related to NSC-117199 that have new modifications that result in cell active Shp2 inhibitors. Finally we will report the discovery and preliminary optimization of new classes of cell active Shp2 inhibitors based on alternative scaffolds. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 761.

Protein tyrosine phosphorylation, which is controlled by tyrosine kinase (PTK) and tyrosine phosphatase (PTP), regulates numerous cellular processes, including proliferation, survival, differentiation, migration and apoptosis. Accumulating evidence has shown that defective function of PTPs leads to aberrant protein phosphorylation, which is associated with many humane diseases, including cancer. Src homology 2 phosphatase-2 (Shp2) is a cytoplasmic, nonreceptor protein tyrosine phophatase. It is the first proto-oncogene identified in the PTP superfamily. The structure of Shp2 consists of two SH2 (N-SH2 and C-SH2) domains, a single PTP region and a C-terminal hydrophilic tail. In the basal state, Shp2 displays a low catalytic activity due to close interactions between the N-SH2 and PTP domains that keep the phosphatase in an autoinhibited closed conformation. Upon growth factor or cytokine stimulation, the SH2 domains of Shp2 binds to tyrosine phosphorylated docking proteins such as Gab1 and Gab2, which activates Src, Ras, and the Erk1/2 (Erk) mitogen-activated protein (MAP) kinase pathway. Shp2 gain-of-function mutations are found in leukemias and solid tumors and are linked to Noonan syndrome. Although Shp2 is involved in pathogenesis of human cancers, it is not clear how Shp2 mediates tumorigenesis. To aid chemical biology studies and ultimately experimental therapy, we have been actively engaged in the search potent and selective Shp2 inhibitors. We will describe the results of a fragment-based screen as a method for identifying Shp2 inhibitors. The fragment libraries were both purchased and constructed in-house with a bias towards compounds that would potentially function as phosphotyrosine mimics. This has revealed compounds with moderate to low activity (IC50 200-500 µM) that have served as the starting pints for fragment-to-lead development. The emerging structure-activity relationships will be discussed as well as synthesis, modeling and X-ray studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 734.