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Supplementary Table 1: Table provides information on the siRNA sequences and/or chemical structures of the non-FDA approved inhibitors for bromodomain proteins, transcription factors and kinases described in the current study. Supplementary Table 2: Sequences of primers used for qRT-PCR analysis of various targets described in the study. Supplementary Table 3: Sequences of primers used for ChIP analysis of various targets described in the study. Supplementary Table 4: Analysis of HOXB13 target gene expression in the GSE92721 data set. Supplementary Table 5: Analysis of HOTBIN10 gene expression in the GSE92721data set. Supplementary Table 6: Analysis of HOTBIN10 gene expression in the Moffitt TCC data set. Supplementary Table 7: Clinical parameters of the primary cancers in the Moffitt TCC data set. Supplementary Table 8: Clinical parameters of the metastatic cancers in the Moffitt TCC data set. Supplementary Table 9: Analysis of HOTBIN10 genes in MSKCC gene expression data set (GSE21034). Supplementary Table 10: Analysis of HOTBIN10 gene expression in the GSE67980 CTC data set.

Table S1: Crystallographic data collection and refinement statistics Table S2: Inhibitory activity against other BET bromodomains Table S3: Bromodomain profiling data Fig. S1: Detailed binding interactions of compounds 1 - 5 in BRD4-1 Fig. S2: Comparison of compound 2 and JQ1 in Brd4-1 Fig. S3: Chemical structures of other compounds used in this work. Fig. S4: Statistical evaluation of kinome profiling data using the Gini coefficient Fig. S5: Original flow cytometry data of MM1.S cells Fig. S6: MM1.S cells require stimulation with IL-6 to detect phospho-STAT3 levels Fig. S7: Effect of dual BRD4-kinase inhibitors on MV-4-11 cells. Fig. S8: Time dependent alteration of c-Myc and p21Cip1 levels in MM1.S cells. Fig. S9: Effect of dual BET-kinase inhibitors on ruxolitinib sensitive MPN cell lines Fig. S10: Dose-effect analysis of UKE-1 cells treated with drug combinations Fig. S11: Cell line sensitivity by tissue type

Synergistic action of kinase and BET bromodomain inhibitors in cell killing has been reported for a variety of cancers. Using the chemical scaffold of the JAK2 inhibitor TG101348, we developed and characterized single agents which potently and simultaneously inhibit BRD4 and a specific set of oncogenic tyrosine kinases including JAK2, FLT3, RET, and ROS1. Lead compounds showed on-target inhibition in several blood cancer cell lines and were highly efficacious at inhibiting the growth of hematopoietic progenitor cells from patients with myeloproliferative neoplasm. Screening across 931 cancer cell lines revealed differential growth inhibitory potential with highest activity against bone and blood cancers and greatly enhanced activity over the single BET inhibitor JQ1. Gene drug sensitivity analyses and drug combination studies indicate synergism of BRD4 and kinase inhibition as a plausible reason for the superior potency in cell killing. Combined, our findings indicate promising potential of these agents as novel chemical probes and cancer therapeutics. Mol Cancer Ther; 16(6); 1054–67. ©2017 AACR.

Supplementary Fig. 1. Epigenetic landscape and the transcription cofactor recruitment at the HOXB13 enhancer region under various treatments; Supplementary Figure 2. BRD4 is a target of the dual BET-kinase inhibitors in CRPCs; Supplementary Figure 3. Sensitivity of normal and AR negative cell lines to the novel BET-kinase inhibitors; Supplementary Figure 4. HOXB13 but not FOXA1 is under the epigenetic control of BRD4 in prostate cancer cells; Supplementary Figure S5. Haploinsufficiency of HOXB13 sensitizes CRPCs to the novel BET-kinase inhibitors; Supplementary Figure S6. AURKB expression correlates with HOXB13 in human CTCs from mCRPC patients following Abiraterone therapy; Supplementary Fig. S7. AURKB is a therapeutic vulnerability of HOXB13 positive prostate cancers

Table S4: Kinome profiling data (Microsoft Excel sheet) Table S5: Cell line screening data (Microsoft Excel sheet)

The discovery that aberrant activity of Janus kinase 2 (JAK2) is a driver of myeloproliferative neoplasms (MPNs) has led to significant efforts to develop small molecule inhibitors for this patient population. Ruxolitinib and fedratinib have been approved for use in MPN patients, while baricitinib, an achiral analogue of ruxolitinib, has been approved for rheumatoid arthritis. However, structural information on the interaction of these therapeutics with JAK2 remains unknown. Here, we describe a new methodology for the large-scale production of JAK2 from mammalian cells, which enabled us to determine the first crystal structures of JAK2 bound to these drugs and derivatives thereof. Along with biochemical and cellular data, the results provide a comprehensive view of the shape complementarity required for chiral and achiral inhibitors to achieve highest activity, which may facilitate the development of more effective JAK2 inhibitors as therapeutics.

Resistance to androgen receptor (AR) antagonists is a significant problem in the treatment of castration-resistant prostate cancers (CRPC). Identification of the mechanisms by which CRPCs evade androgen deprivation therapies (ADT) is critical to develop novel therapeutics. We uncovered that CRPCs rely on BRD4-HOXB13 epigenetic reprogramming for androgen-independent cell proliferation. Mechanistically, BRD4, a member of the BET bromodomain family, epigenetically promotes HOXB13 expression. Consistently, genetic disruption of HOXB13 or pharmacological suppression of its mRNA and protein expression by the novel dual-activity BET bromodomain-kinase inhibitors directly correlates with rapid induction of apoptosis, potent inhibition of tumor cell proliferation and cell migration, and suppression of CRPC growth. Integrative analysis revealed that the BRD4-HOXB13 transcriptome comprises a proliferative gene network implicated in cell-cycle progression, nucleotide metabolism, and chromatin assembly. Notably, although the core HOXB13 target genes responsive to BET inhibitors (HOTBIN10) are overexpressed in metastatic cases, in ADT-treated CRPC cell lines and patient-derived circulating tumor cells (CTC) they are insensitive to AR depletion or blockade. Among the HOTBIN10 genes, AURKB and MELK expression correlates with HOXB13 expression in CTCs of mCRPC patients who did not respond to abiraterone (ABR), suggesting that AURKB inhibitors could be used additionally against high-risk HOXB13-positive metastatic prostate cancers. Combined, our study demonstrates that BRD4-HOXB13-HOTBIN10 regulatory circuit maintains the malignant state of CRPCs and identifies a core proproliferative network driving ADT resistance that is targetable with potent dual-activity bromodomain-kinase inhibitors.

Upon binding to thalidomide and other immunomodulatory drugs, the E3 ligase substrate receptor cereblon (CRBN) promotes proteosomal destruction by engaging the DDB1–CUL4A–Roc1–RBX1 E3 ubiquitin ligase in human cells but not in mouse cells, suggesting that sequence variations in CRBN may cause its inactivation. Therapeutically, CRBN engagers have the potential for broad applications in cancer and immune therapy by specifically reducing protein expression through targeted ubiquitin-mediated degradation. To examine the effects of defined sequence changes on CRBN's activity, we performed a comprehensive study using complementary theoretical, biophysical, and biological assays aimed at understanding CRBN's nonprimate sequence variations. With a series of recombinant thalidomide-binding domain (TBD) proteins, we show that CRBN sequence variants retain their drug-binding properties to both classical immunomodulatory drugs and dBET1, a chemical compound and targeting ligand designed to degrade bromodomain-containing 4 (BRD4) via a CRBN-dependent mechanism. We further show that dBET1 stimulates CRBN's E3 ubiquitin–conjugating function and degrades BRD4 in both mouse and human cells. This insight paves the way for studies of CRBN-dependent proteasome-targeting molecules in nonprimate models and provides a new understanding of CRBN's substrate-recruiting function.

Background and aims: Anticoagulation regimens are now being considered for COVID-19. Anticoagulation is known to increase the risk for adverse bleeding events, of which intracerebral hemorrhage (ICH). Methods: Case Report. Results: We report a 57-year-old man was admitted to hospital due decreased oxygen saturation, increased blood glucose levels and RT-PCR of oropharyngeal swab confirmed COVID-19. The patient had a previous history of type 2 DM and ischemic stroke. The first 5 days, the patient was receiving a prophylactic dose of low molecular weight heparin, enoxaparin, then continued with unfractionated heparin. The patient also received antiplatelet clopidogrel and convalescent plasma. The laboratory results are leucocyte 18.9 K/μL, ureum 79 mg/dL, creatinine 2.0 mg/dL, D-Dimer 0.22 μg/mL, INR 1.03, NLR 15.82, ALC 547, HbA1C 9.2, blood glucose level 336 mg/dL, CRP 41.2 mg/dL. On day ten of hospitalization, the patient had sudden right-sided hemiplegia and Broca's aphasia. A CT head demonstrated an ICH in the left basal ganglia with a volume approximately 77cc. Three days later the patient's condition had decreased consciousness and a craniotomy was performed. After being intubated for 4 days post craniotomy, because his condition improved, he was extubated. On day nine after surgery, the patient's condition decreased to coma, a repeat chest X-ray showed a worsening of the bilateral infiltrates and pleural effusion. His oxygen requirement was progressively increasing, so he was shifted to mechanical ventilation again. Conclusions: The risk of intracerebral hemorrhage in patients with COVID-19 is of paramount importance to inform the risk-benefit assessment of the use of anticoagulation in this patient.

This article describes the action of iodine(III) reagents [diacetoxyiodobenzene, PhI(OAc)2, and iodosobenzene, (PhIO)n] in conjunction with TMSBr which act as functional bromine equivalents in unique oxidations of saturated, carbamate protected N-heterocycles. Interestingly, during this work, treatment of the same carbamates with molecular bromine alone afforded similar products, which were sequestered by the solvent methanol.

Background:Staghorn stones are mostly treated by percutaneous nephrolithotomy (PCNL), either with an upper-pole (UP) or non-upper (lower- or middle-) pole (NP) approach. NP access has a lower risk of bleeding and thoracic complications but may not be sufficient for complete stone clearance. UP access is advocated as the preferred approach, because of direct access to the collecting system. However, it is associated with a higher complications rate, including pneumothorax and hydrothorax, and a higher risk of bleeding. This meta-analysis aimed to describe the outcomes and safety of PCNL for staghorn stones using UP and NP approaches.Methods:A systematic literature review was conducted using several databases such as: PubMed; EBSCO; Science Direct; Cochrane and Google Scholar. Data from all selected articles were extracted by two independent reviewers. Relevant parameters explored using Review Manager V5.3.Results:Five comparative studies of staghorn stones involving 384 renal units were analyzed; 176 cases used the UP approach and 208 the NP approach. There was no significant difference in stone-free rate between these approaches, with 74.4% undergoing the UP approach and 71.1% the NP approach considered stone-free (OR: 1.55; 95% CI: 0.92-2.63; P=0.10). The rate of thoracic complications (hydrothorax and pneumothorax) did not differ significantly (OR: 3.14; 95% CI: 0.63-15.62; P=0.16). However, we noted that 5 of 176 patients that underwent the UP approach experienced thoracic complications. The incidence of post-procedural fever and sepsis is similar (OR: 1.18; 95% CI: 0.52-2.64; P=0.69). Neither post-procedural urine leakage (OR: 2.03; 95% CI: 0.70-5.85; P=0.19) nor requirement of blood transfusions (OR: 0.49; 95% CI: 0.14-1.76; P=0.27) differed significantly.Conclusion:PCNL with UP access for staghorn stone has a similar stone-free rate to the NP approach. Thoracic complication rate which was believed to be higher in the UP group is also deemed similar with NP access.

Dihydropyrazino-quinazolinedione chemotypes are complex and structurally challenging structures of biological interest, being found in the marine alkaloids such as brevianamide M–N and fumiquinazolines A–C. Herein we report the synthesis of this tricyclic system in three synthetic operations by means of an Ugi multi-component reaction (MCR) followed by a tandem N-acyliminium ion cyclization-intramolecular nucleophilic addition reaction sequence. Additional structural diversification for further library enrichment was also accomplished via sequential N-alkylation and N-acylation/sulfonation.

Graft-versus-host disease (GVHD) is a leading cause of nonrelapse mortality after allogeneic hematopoietic cell transplantation. T cell costimulation by CD28 contributes to GVHD, but prevention is incomplete when targeting CD28, downstream mammalian target of rapamycin (mTOR), or Aurora A. Likewise, interleukin-6 (IL-6)–mediated Janus kinase 2 (JAK2) signaling promotes alloreactivity, yet JAK2 inhibition does not eliminate GVHD. We provide evidence that blocking Aurora A and JAK2 in human T cells is synergistic in vitro, prevents xenogeneic GVHD, and maintains antitumor responses by cytotoxic T lymphocytes (CTLs). Aurora A/JAK2 inhibition is immunosuppressive but permits the differentiation of inducible regulatory T cells (iTregs) that are hyperfunctional and CD39 bright and efficiently scavenge adenosine triphosphate (ATP). Increased iTreg potency is primarily a function of Aurora A blockade, whereas JAK2 inhibition suppresses T helper 17 (TH17) differentiation. Inhibiting either Aurora A or JAK2 significantly suppresses TH1 T cells. However, CTL generated in vivo retains tumor-specific killing despite Aurora A/JAK2 blockade. Thus, inhibiting CD28 and IL-6 signal transduction pathways in donor T cells can increase the Treg/Tconv ratio, prevent GVHD, and preserve antitumor CTL.

Synergistic action of kinase and BET bromodomain inhibitors in cell killing has been reported for a variety of cancers. Using the chemical scaffold of the JAK2 inhibitor TG101348, we developed and characterized single agents which potently and simultaneously inhibit BRD4 and a specific set of oncogenic tyrosine kinases including JAK2, FLT3, RET, and ROS1. Lead compounds showed on-target inhibition in several blood cancer cell lines and were highly efficacious at inhibiting the growth of hematopoietic progenitor cells from patients with myeloproliferative neoplasm. Screening across 931 cancer cell lines revealed differential growth inhibitory potential with highest activity against bone and blood cancers and greatly enhanced activity over the single BET inhibitor JQ1. Gene drug sensitivity analyses and drug combination studies indicate synergism of BRD4 and kinase inhibition as a plausible reason for the superior potency in cell killing. Combined, our findings indicate promising potential of these agents as novel chemical probes and cancer therapeutics. Mol Cancer Ther; 16(6); 1054–67. ©2017 AACR.

A two-step strategy for the synthesis of arrays of tricyclic tetrazolo-fused benzodiazepines and benzodiazepinones has been investigated. The protocol uses ortho-N-Boc phenylisocyanides and phenylglyoxaldehydes or ethyl glyoxylate in the four-component Ugi-azide reaction to afford MCR (MultiComponent Reactions) derived adducts equipped with the desired diversity inputs. A subsequent acidic treatment (TFA/DCE) allows a simultaneous deprotectionecyclization leading to the final products. 2012 Elsevier Ltd. All rights reserved.

A concise two-step procedure for the synthesis of novel δ-lactam tetrazoles has been established via the Ugi-azide reaction using 5-oxohexanoic acid along with primary amines, isocyanides, and azidotrimethylsilane followed by 1,1'-carbonyldiimidazole-mediated intramolecular amide formation. Expansion to e-lactam tetrazole scaffolds was accomplished using methyl 6-oxoheptanoate via the same Ugi-azide reaction followed by basic hydrolysis and SOCl(2) activation to enable lactam formation.

A one-pot, two-step synthesis of bis-pyrrolidinone tetrazoles has been established via the Ugi-Azide reaction using methyl levulinate, primary amines, isocyanides and azidotrimethylsilane with subsequent acid treatment to catalyze the lactam formation. The efficiency of the protocol was established followed by a successful transition to library production in four 24-well plates.

The following article describes a concise synthesis of a collection of 4,5-dihydro-1H-benzo[e][1,4]diazepines fused to a hydantoin ring. Molecular complexity and biological relevance are high and structures are generated in a mere three steps, employing the Ugi reaction to assemble diversity reagents. The protocol represents a novel UDC (Ugi-deprotect-cyclize) strategy employed in the Ugi-5-component CO2-mediated condensation, followed by further cyclization under basic conditions, to afford the fused hydantoin. Mechanistic caveats, dependent on the aldehydes of choice will be revealed and a facile oxidation of the final products to imidazolidenetriones is briefly discussed.

SHP2 (PTPN11) mediates oncogenic signaling and gain-of-function (GOF) SHP2 mutations are associated with leukemias and other types of cancer. Recently, SHP099 was identified as a novel allosteric SHP2 inhibitor that displayed high potency and selectivity to the wildtype SHP2 in vitro and inhibited protein tyrosine kinase oncogene-driven cancer cells. However, it was unclear whether SHP099 inhibits SHP2 mutants. Using SHP2 mutant-dependent TF-1 leukemia cells, we determined sensitivities of four common oncogenic SHP2 mutants to SHP099. SHP2D61Y-, SHP2A72V-, and SHP2E76K-expressing TF-1 cells were resistant, whereas SHP2E69K-expressing TF-1 cells were sensitive to SHP099 with a potency similar to that of cytokine-stimulated TF-1 cells expressing the wildtype SHP2. Consistently, SHP099 reduced active ERK1/2 and Bcl-XL and induced apoptosis of SHP2E69K-dependent TF-1 cells, but had no effects on the other SHP2 mutant cell lines. SHP2 knockout cells were unresponsive to SHP099, demonstrating specificity. Like the wildtype SHP2, SHP2E69K PTP was inhibited by SHP099 in vitro with a sub-micromolar IC50, whereas micromolar IC50s of SHP099 were observed against SHP2D61Y, SHP2A72V, and SHP2E76K. These results indicate that SHP2D61Y, SHP2A72V, and SHP2E76K mutants are resistant to SHP099 and that SHP2E69K is a SHP099-sensitive oncogenic mutant. Citation Format: Xiaojun Sun, Yuan Ren, Steven Gunawan, Peng Teng, Zhengming Chen, Harshani R. Lawrence, Jianfeng Cai, Nicholas J. Lawrence, Jie Wu. Mutation selectivity of the allosteric SHP2 inhibitor SHP099 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4879.

Protein degraders have been developed that can aide in understanding gene function. These chemicals contain complementary bifunctional components that 1) interact with specific proteins, and 2) engage an E3 ligase which leads to polyubiquitination and intracellular degradation by the proteasome. Thalidomide, infamously known for its teratogenic effects, is central to this new technology since it binds cereblon (CRBN) which promiscuously recruits the DDB1/Cul4A/Rbx1 complex to trigger target destruction. Thalidomide, and especially its immunomodulatory derivatives lenalidomide and pomalidomide, induce NK and T-cell activation by degrading IKZF1 which is a transcription factor that suppresses IL-2. Currently, it is debated whether mouse cereblon has conserved function due to a single non-conserved amino acid in mouse CRBN, Ile390 (equivalent to Val388 in human CRBN). First, our work confirms that lenalidomide induces IL-2 and the ubiquitin-dependent degradation of IKZF1 only in human and not mouse T-cells treated with lenalidomide. To further test for species-related effects, a series of theoretical and physical binding assays using mutant CRBN proteins show that the binding of IMiD compounds is not impaired by amino acid differences in mouse CRBN. Using dBET1, a novel thalidomide-JQ1 protein degrader, we show for the first time that mouse CRBN can trigger CRBN-dependent degradation of BRD4 and confirm that the CRBN/DDB1/Cul4A/Rbx1 complex is functional in mouse T-cells. Collectively, our results suggest that IKZF1 has divergent regulation in mouse cells rather than the IMiD complex and provide information relevant to the development of chemical conjugates that induce targeted intracellular protein degradation.

Ugi-azide reaction of tethered aldo/keto—acids/esters followed by a ring closing step opens access to a variety of complex lactam—tetrazoles.

A two-step strategy for the synthesis of arrays of tricyclic tetrazolo-fused benzodiazepines and benzodiazepinones has been investigated. The protocol uses ortho-N-Boc phenylisocyanides and phenylglyoxaldehydes or ethyl glyoxylate in the four-component Ugi-azide reaction to afford MCR (MultiComponent Reactions) derived adducts equipped with the desired diversity inputs. A subsequent acidic treatment (TFA/DCE) allows a simultaneous deprotectionecyclization leading to the final products. 2012 Elsevier Ltd. All rights reserved.

This Letter presents a novel three step solution phase protocol to synthesize 3-(tetrazol-5-yl)quinoxalin-2(1H)-ones. The strategy utilizes ethyl glyoxalate and mono-N-Boc-protected-o-phenylenediamine derivatives in the Ugi-Azide multi-component reaction (MCR) to generate a unique 1,5-disubstituted tetrazole. Subsequent acid treatment stimulates a simultaneous Boc deprotection and intramolecular cyclization leading to bis-3,4-dihydroquinoxalinone tetrazoles. Direct oxidation using a stable solid-phase radical catalyst (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) with ceric ammonium nitrate (CAN) in catalytic fashion initiating aerobic oxidation, completes the entire procedure to generate a series of original unique bis-quinoxalinone tetrazoles. The method was also expanded to produce a bis-benzodiazepine tetrazole.

A concise two-step procedure for the synthesis of novel δ-lactam tetrazoles has been established via the Ugi-azide reaction using 5-oxohexanoic acid along with primary amines, isocyanides, and azidotrimethylsilane followed by 1,1'-carbonyldiimidazole-mediated intramolecular amide formation. Expansion to ε-lactam tetrazole scaffolds was accomplished using methyl 6-oxoheptanoate via the same Ugi-azide reaction followed by basic hydrolysis and SOCl(2) activation to enable lactam formation.

The title compound, C8H13Br2NO3, crystallizes as a non-merohedral twin with twin law −0.6 0 0.4/0 − 1 0 /1.6 0 0.6, and the structure has a refined twin domain ratio of 0.546 (5). The structure shows a compact conformation, with the ester unit roughly coplanar with a mean plane fitted through the non-H atoms of the pyrrolidine ring [dihedral angle = 8.23 (9)°]. In the crystal, inversion dimers linked by pairs of O—H...O hydrogen bonds generate an R22(12) motif.

The optimism of anti-JAK2 therapy for the treatment of MPNs is largely based on the ability of JAK inhibitors to improve MPN patient constitutional symptoms. Unfortunately, such inhibitors are generally unable to induce remission or even allele burden, suggesting anti-JAK2 based therapies need refinement and optimization. Combination therapies continue to be widely investigated, however, such approaches could have significant complications when translating to patients. These include the difficulty determining proper dosing of each drug in combination in patients, the cost of treatment, and issues with combining drugs developed by different pharmaceutical companies. Recently, we and others have identified anti-BET bromodomain inhibitory properties of various kinase inhibitors, including the JAK2 inhibitors TG101209 and TG101308. Bromodomains are protein-protein interaction motifs that bind to acetylated lysine and, for example, can play roles in regulating gene expression by binding acetylated histones. These dual kinase-BET inhibitors bind to the acetylated lysine-binding pocket of BET bromodomains and thus inhibit the function of such domains. Importantly, cancer cells appear to be particularly sensitive to BET inhibitors compared to normal cells. Combining a BET inhibitor and a JAK2 inhibitor has been shown to be more effective against MPN cells than JAK2 inhibition alone. Our recent identification of dual kinase-BET inhibitors allows for the rational design of drugs with polypharmacology to inhibit more than one class of targets. To this end, we have optimized small molecules for dual anti-JAK2 and anti-BET activity. MA2-014 is a chemical derivative of TG101209 that exhibits similar anti-JAK2 activity, but about ten-fold improved anti-BET activity than TG101209. In fact, the activity of MA2-014 to target BET domains is similar to the prototypical BET inhibitor JQ1. For example, the IC50s of MA2-014 and JQ1 against the second bromodomain of the BET family member BRD4 are each about 20 nM. MA2-014 retains comparable biochemical activity against JAK2 as TG101209 and ruxolitinib (IC50s of low single digit nM, ~0.5 to 3 nM). In MPN cells, however, the ability of MA2-014 to inhibit JAK2-V617F signaling in MPN cells, as measured by P-STAT5, is about ten-fold improved over TG101209, and is comparable to ruxolitinib. Likewise, the ability of MA2-014 to inhibit the expression of c-Myc, which is widely used as a biomarker for BET inhibition, is about ten-fold better than TG101209 in MPN cells. The IC50 for MA2-014 for growth of Uke1 MPN cells is about 200 nM, compared to 500 nM for TG101209. Taken together, these data suggest that MA2-014 is a dual JAK2-BET inhibitor that exhibits superior BET inhibitory activity with similar if not better cellular JAK2 inhibitory activity than TG101209. MA2-014 efficiently inhibited the erythropoietin independent erythroid colony formation of myeloid progenitors from MPN patients, which is a hallmark of these cells and is widely used to test MPN therapeutics. The IC50 of MA2-014 in this assay is 50 nM, essentially identical to ruxolitinib, the only FDA approved JAK2 inhibitor for MPNs. The IC50 of TG101209 to inhibit this colony formation of primary MPN cells is 200 nM, four times greater than MA2-014. Interestingly, JAK2-V617F-driven MPN model cells that are resistant to ruxolitinib retained sensitivity to MA2-014. The IC50s of ruxolitinib and MA2-014 against the growth of BaF3-JAK2-V617F cells are about 100 nM. However, the same cells that are resistant to ruxolitinib (IC50 >2000 nM) remain sensitive to MA2-014 (IC50 of 240 nM). Finally, in long-term culture assays, we have determined that JAK2-V617F driven MPN Uke1 cells are not able to become resistant to MA2-014 as readily as they do to TG101209 or ruxolitinib. These data suggest MA2-014 may be more resilient to drug resistance, the major hurdle in the clinical effectiveness of JAK2 inhibitors. Collectively, our work demonstrates that rationally-designed polypharmacology may be a novel approach to develop effective therapeutics for cancer, especially diseases that are driven by aberrant kinase signaling and are also sensitive to BET inhibition, as exemplified by MPNs. The use of such an optimized polypharmacologic therapeutic may provide the benefits of combination therapy with fewer complications associated with clinical development. Disclosures No relevant conflicts of interest to declare.

Bromodomain (BRD)-containing proteins are essential for the recognition of acetylated lysine residues of histones during transcriptional activation. The BRD-containing proteins have emerged as promising drug targets for a number of diseases, including many cancers, that are characterized by changes in the epigenetic cell signature. Recent reports have shown that targeting BRD4 with small molecules may represent a new way to treat prostate and breast cancer, acute myeloid leukemia and melanoma. We have identified many type 1 and type 2 kinase inhibitors which also inhibit BRD4 by robotic co-crystallization screening of kinase inhibitor libraries against BRD4. In each case the co-crystal structure unambiguously revealed the inhibitor bound to the acetyl lysine site of BRD4-1. The identified BRD4 ligands were subjected to differential scanning fluorimetry (DSF) and AlphaScreen assay to assess their binding and inhibitory potentials against BRD4. As shown previously for other BRD-inhibitor-protein complexes, the melting temperatures of BRD4-kinase inhibitor complexes were logarithmically proportional to their IC50 values. We now report the design, synthesis, structural analysis and biological evaluation of next-generation nanomolar BET-selective and nanomolar dual-activity BET-JAK2 inhibitors, based on the initial co-crystallization screening hits. Structure activity relationships were developed using both DSF and co-crystallization of the ligands with BRD4, to assess binding potential and binding modes, respectively. We report initial evaluation of the anticancer potential of compounds possessing dual potent BRD4 and JAK2 inhibitory properties. In addition to myeloma cell lines, this includes the evaluation of dual BRD4-JAK2 inhibitor compounds against JAK2-driven myeloproliferative neoplasm cell lines and primary cells from patients. Citation Format: Steven Gunawan, Ayaz Muhammad, Stuart W. J. Ember, Jin-Yi Zhu, Rebecca A. Jacobsen, Norbert Berndt, Que T. Lambert, Gary W. Reuther, Harshani R. Lawrence, Ernst Schonbrunn, Nicholas J. Lawrence. Targeting the acetyl-lysine binding site of BRD4 with dual nanomolar BET-JAK2 inhibitors: A new anticancer therapeutic strategy. [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 3643. doi:10.1158/1538-7445.AM2015-3643

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

1,5-disubstituted tetrazoles are an important drug-like scaffold known for their ability to mimic the cis-amide bond conformation. The scaffold is readily accessible via substitution of the carboxylic acid component of the Ugi multi-component reaction (MCR) with TMSN3 in what is herein denoted the Ugi-azide reaction. This full paper presents a concise, novel, general strategy to access a plethora of new heterocylic scaffolds utilizing tethered aldo/keto-acids/esters in the Ugi-azide reaction followed by a ring closing event that generates novel highly complex bis-heterocyclic lactam-tetrazoles.