TP-0184 inhibits FLT3/ACVR1 to overcome FLT3 inhibitor resistance and hinder AML growth synergistically with venetoclax.

We identified activin A receptor type I (ACVR1), a member of the TGF-ß superfamily, as a factor favoring acute myeloid leukemia (AML) growth and a new potential therapeutic target. ACVR1 is overexpressed in FLT3-mutated AML and inhibition of ACVR1 expression sensitized AML cells to FLT3 inhibitors. We developed a novel ACVR1 inhibitor, TP-0184, which selectively caused growth arrest in FLT3-mutated AML cell lines. Molecular docking and in vitro kinase assays revealed that TP-0184 binds to both ACVR1 and FLT3 with high affinity and inhibits FLT3/ACVR1 downstream signaling. Treatment with TP-0184 or in combination with BCL2 inhibitor, venetoclax dramatically inhibited leukemia growth in FLT3-mutated AML cell lines and patient-derived xenograft models in a dose-dependent manner. These findings suggest that ACVR1 is a novel biomarker and plays a role in AML resistance to FLT3 inhibitors and that FLT3/ACVR1 dual inhibitor TP-0184 is a novel potential therapeutic tool for AML with FLT3 mutations.

A First-in-Class Inhibitor of ER Coregulator PELP1 Targets ER+ Breast Cancer.

Most patients with estrogen receptor alpha-positive (ER+) breast cancers initially respond to treatment but eventually develop therapy resistance with disease progression. Overexpression of oncogenic ER coregulators, including proline, glutamic acid, and leucine-rich protein 1 (PELP1), are implicated in breast cancer progression. The lack of small molecules that inhibits PELP1 represents a major knowledge gap. Here, using a yeast-two-hybrid screen, we identified novel peptide inhibitors of PELP1 (PIP). Biochemical assays demonstrated that one of these peptides, PIP1, directly interacted with PELP1 to block PELP1 oncogenic functions. Computational modeling of PIP1 revealed key residues contributing to its activity and facilitated the development of a small-molecule inhibitor of PELP1, SMIP34, and further analyses confirmed that SMIP34 directly bound to PELP1. In breast cancer cells, SMIP34 reduced cell growth in a dose-dependent manner. SMIP34 inhibited proliferation of not only wild-type (WT) but also mutant (MT) ER+ and therapy-resistant breast cancer cells, in part by inducing PELP1 degradation via the proteasome pathway. RNA sequencing analyses showed that SMIP34 treatment altered the expression of genes associated with estrogen response, cell cycle, and apoptosis pathways. In cell line-derived and patient-derived xenografts of both WT and MT ER+ breast cancer models, SMIP34 reduced proliferation and significantly suppressed tumor progression. Collectively, these results demonstrate SMIP34 as a first-in-class inhibitor of oncogenic PELP1 signaling in advanced breast cancer. SIGNIFICANCE: Development of a novel inhibitor of oncogenic PELP1 provides potential therapeutic avenues for treating therapy-resistant, advanced ER+ breast cancer.

SIK2 inhibition enhances PARP inhibitor activity synergistically in ovarian and triple-negative breast cancers.

Poly(ADP-ribose) polymerase inhibitors (PARP inhibitors) have had an increasing role in the treatment of ovarian and breast cancers. PARP inhibitors are selectively active in cells with homologous recombination DNA repair deficiency caused by mutations in BRCA1/2 and other DNA repair pathway genes. Cancers with homologous recombination DNA repair proficiency respond poorly to PARP inhibitors. Cancers that initially respond to PARP inhibitors eventually develop drug resistance. We have identified salt-inducible kinase 2 (SIK2) inhibitors, ARN3236 and ARN3261, which decreased DNA double-strand break (DSB) repair functions and produced synthetic lethality with multiple PARP inhibitors in both homologous recombination DNA repair deficiency and proficiency cancer cells. SIK2 is required for centrosome splitting and PI3K activation and regulates cancer cell proliferation, metastasis, and sensitivity to chemotherapy. Here, we showed that SIK2 inhibitors sensitized ovarian and triple-negative breast cancer (TNBC) cells and xenografts to PARP inhibitors. SIK2 inhibitors decreased PARP enzyme activity and phosphorylation of class-IIa histone deacetylases (HDAC4/5/7). Furthermore, SIK2 inhibitors abolished class-IIa HDAC4/5/7-associated transcriptional activity of myocyte enhancer factor-2D (MEF2D), decreasing MEF2D binding to regulatory regions with high chromatin accessibility in FANCD2, EXO1, and XRCC4 genes, resulting in repression of their functions in the DNA DSB repair pathway. The combination of PARP inhibitors and SIK2 inhibitors provides a therapeutic strategy to enhance PARP inhibitor sensitivity for ovarian cancer and TNBC.

Ligand-based Discovery of Novel Small Molecule Inhibitors of RON Receptor Tyrosine Kinase.

BACKGROUND: RON (Recepteur d'Origine Nantais) receptor tyrosine kinase is a promising target for anti-cancer therapeutics. The aim of this study was to identify new RON inhibitors using virtual screening methods. METHODS: To this end, a ligand-based virtual screening approach was employed for screening of ZINC database on the homology model of RON receptor. All the selected hits were inspected in terms of drug-likeness, ADME properties, and toxicity profiles. Ligand-based similarity searches along with further filtering criteria led to the identification of two compounds, TKI1 and TKI2 that were evaluated using in vitro cell-based RON inhibition assays. RESULTS: The results showed that TKI1 and TKI2 could reduce phosphorylation of RON. Both compounds showed inhibitory activity of the downstream mTOR pathway with no apparent effects on other signaling mediators in a dose-dependent manner. CONCLUSION: These compounds can provide a basis for developing novel anti-RON inhibitors applicable to cancer therapy using medicinal chemistry-oriented optimization strategies.

A computational study of structural differences of binding of NADP+ and G6P substrates to G6PD Mediterraneanc.563T, G6PD A-c.202A/c.376G, G6PD Cairoc.404C and G6PD Gazac.536A mutations.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common X-linked inherited enzymopathic disorder that may lead to transfusion-requiring acute hemolytic anemia (AHA) triggered by fava beans ingestion, infection or some drugs. The gene encoding for G6PD carries a large number of genetic variants that have varying pathogenicity. We reported on three G6PD variants in the Gaza Strip Palestinian population with differing clinical impacts and frequencies: G6PD Mediterraneanc.563T, African G6PD A-c.202A/c.376G, and G6PD Cairoc.404C. We also identified a novel G6PD missense (Ser179Asn) mutation c.536G > A "G6PD Gaza". In this work we explore the effect of these four genetic variants on the structural and substrate (NADP+ and G6P) binding characteristics of the G6PD enzyme using the Monte Carlo (MC) flexible docking and molecular dynamics (MD) simulation approaches. We report that G6PD A-c.202A/c.376G, G6PD Mediterraneanc.563T, G6PD Cairoc.404C and G6PD Gazac.536A mutations cause significant structural changes in G6PD enzyme to induce conformational instability leading to the loss of binding of one or both substrates and are causative of G6PD deficiency.

The Small Molecule BC-2059 Inhibits Wingless/Integrated (Wnt)-Dependent Gene Transcription in Cancer through Disruption of the Transducin ß-Like 1-ß-Catenin Protein Complex.

The central role of ß-catenin in the Wnt pathway makes it an attractive therapeutic target for cancers driven by aberrant Wnt signaling. We recently developed a small-molecule inhibitor, BC-2059, that promotes apoptosis by disrupting the ß-catenin/transducin ß-like 1 (TBL1) complex through an unknown mechanism of action. In this study, we show that BC-2059 directly interacts with high affinity for TBL1 when in complex with ß-catenin. We identified two amino acids in a hydrophobic pocket of TBL1 that are required for binding with ß-catenin, and computational modeling predicted that BC-2059 interacts at the same hydrophobic pocket. Although this pocket in TBL1 is involved in binding with NCoR/SMRT complex members G Protein Pathway Suppressor 2 (GSP2) and SMRT and p65 NFκB subunit, BC-2059 failed to disrupt the interaction of TBL1 with either NCoR/SMRT or NFκB. Together, our results show that BC-2059 selectively targets TBL1/ß-catenin protein complex, suggesting BC-2059 as a therapeutic for tumors with deregulated Wnt signaling pathway. SIGNIFICANCE STATEMENT: This study reports the mechanism of action of a novel Wnt pathway inhibitor, characterizing the selective disruption of the transducin ß-like 1/ß-catenin protein complex. As Wnt signaling is dysregulated across cancer types, this study suggests BC-2059 has the potential to benefit patients with tumors reliant on this pathway.

The novel reversible LSD1 inhibitor SP-2577 promotes anti-tumor immunity in SWItch/Sucrose-NonFermentable (SWI/SNF) complex mutated ovarian cancer.

Mutations of the SWI/SNF chromatin remodeling complex occur in 20% of all human cancers, including ovarian cancer. Approximately half of ovarian clear cell carcinomas (OCCC) carry mutations in the SWI/SNF subunit ARID1A, while small cell carcinoma of the ovary hypercalcemic type (SCCOHT) presents with inactivating mutations of the SWI/SNF ATPase SMARCA4 alongside epigenetic silencing of the ATPase SMARCA2. Loss of these ATPases disrupts SWI/SNF chromatin remodeling activity and may also interfere with the function of other histone-modifying enzymes that associate with or are dependent on SWI/SNF activity. One such enzyme is lysine-specific histone demethylase 1 (LSD1/KDM1A), which regulates the chromatin landscape and gene expression by demethylating proteins such as histone H3. Cross-cancer analysis of the TCGA database shows that LSD1 is highly expressed in SWI/SNF-mutated tumors. SCCOHT and OCCC cell lines have shown sensitivity to the reversible LSD1 inhibitor SP-2577 (Seclidemstat), suggesting that SWI/SNF-deficient ovarian cancers are dependent on LSD1 activity. Moreover, it has been shown that inhibition of LSD1 stimulates interferon (IFN)-dependent anti-tumor immunity through induction of endogenous retroviral elements and may thereby overcome resistance to checkpoint blockade. In this study, we investigated the ability of SP-2577 to promote anti-tumor immunity and T-cell infiltration in SCCOHT and OCCC cell lines. We found that SP-2577 stimulated IFN-dependent anti-tumor immunity in SCCOHT and promoted the expression of PD-L1 in both SCCOHT and OCCC. Together, these findings suggest that the combination therapy of SP-2577 with checkpoint inhibitors may induce or augment immunogenic responses of SWI/SNF-mutated ovarian cancers and warrants further investigation.

Discovery of Novel Inhibitors Targeting Multi-UDP-hexose Pyrophosphorylases as Anticancer Agents.

To minimize treatment toxicities, recent anti-cancer research efforts have switched from broad-based chemotherapy to targeted therapy, and emerging data show that altered cellular metabolism in cancerous cells can be exploited as new venues for targeted intervention. In this study, we focused on, among the altered metabolic processes in cancerous cells, altered glycosylation due to its documented roles in cancer tumorigenesis, metastasis and drug resistance. We hypothesize that the enzymes required for the biosynthesis of UDP-hexoses, glycosyl donors for glycan synthesis, could serve as therapeutic targets for cancers. Through structure-based virtual screening and kinetic assay, we identified a drug-like chemical fragment, GAL-012, that inhibit a small family of UDP-hexose pyrophosphorylases-galactose pyro-phosphorylase (GALT), UDP-glucose pyrophosphorylase (UGP2) and UDP-N-acetylglucosamine pyrophosphorylase (AGX1/UAP1) with an IC50 of 30 µM. The computational docking studies supported the interaction of GAL-012 to the binding sites of GALT at Trp190 and Ser192, UGP2 at Gly116 and Lys127, and AGX1/UAP1 at Asn327 and Lys407, respectively. One of GAL-012 derivatives GAL-012-2 also demonstrated the inhibitory activity against GALT and UGP2. Moreover, we showed that GAL-012 suppressed the growth of PC3 cells in a dose-dependent manner with an EC50 of 75 µM with no effects on normal skin fibroblasts at 200 µM. Western blot analysis revealed reduced expression of pAKT (Ser473), pAKT (Thr308) by 77% and 72%, respectively in the treated cells. siRNA experiments against the respective genes encoding the pyrophosphorylases were also performed and the results further validated the proposed roles in cancer growth inhibition. Finally, synergistic relationships between GAL-012 and tunicamycin, as well as bortezomib (BTZ) in killing cultured cancer cells were observed, respectively. With its unique scaffold and relatively small size, GAL-012 serves as a promising early chemotype for optimization to become a safe, effective, multi-target anti-cancer drug candidate which could be used alone or in combination with known therapeutics.

Development of High-Throughput Screening Assays for Inhibitors of ETS Transcription Factors.

ETS transcription factors from the ERG and ETV1/4/5 subfamilies are overexpressed in the majority of prostate cancer patients and contribute to disease progression. Here, we have developed two in vitro assays for the interaction of ETS transcription factors with DNA that are amenable to high-throughput screening. Using ETS1 as a model, we applied these assays to screen 110 compounds derived from a high-throughput virtual screen. We found that the use of lower-affinity DNA binding sequences, similar to those that ERG and ETV1 bind to in prostate cells, allowed for higher inhibition from many of these test compounds. Further pilot experiments demonstrated that the in vitro assays are robust for ERG, ETV1, and ETV5, three of the ETS transcription factors that are overexpressed in prostate cancer.

Fragment-based design, synthesis, biological evaluation, and SAR of 1H-benzo[d]imidazol-2-yl)-1H-indazol derivatives as potent PDK1 inhibitors.

In this work, we describe the use of the rule of 3 fragment-based strategies from biochemical screening data of 1100 in-house, small, low molecular weight fragments. The sequential combination of in silico fragment hopping and fragment linking based on S160/Y161/A162 hinge residues hydrogen bonding interactions leads to the identification of novel 1H-benzo[d]imidazol-2-yl)-1H-indazol class of Phosphoinositide-Dependent Kinase-1 (PDK1) inhibitors. Consequent SAR and follow-up screening data led to the discovery of two potent PDK1 inhibitors: compound 32 and 35, with an IC50 of 80 nM and 94 nM, respectively. Further biological evaluation showed that, at the low nanomolar concentration, the drug had potent ability to inhibit phosphorylation of AKT and p70S6, and selectively kill the cancer cells with mutations in both PTEN and PI3K. The microarray data showed that DUSP6, DUSP4, and FOSL1 were down-regulated in the sensitive cell lines with the compound treatment. The in vivo test showed that 35 can significantly inhibit tumor growth without influencing body weight growth. Our results suggest that these compounds, especially 35, merit further pre-clinical evaluation.

Ruthenium(II)- and copper(I)-catalyzed synthesis of click-xylosides and assessment of their glycosaminoglycan priming activity.

Xylosides are small molecules that serve as primers of glycosaminoglycan biosynthesis. Xyloside mediated modulation of biological functions depends on the extent of priming activity and fine structures of primed GAG chains. In earlier studies, copper (Cu) catalyzed synthesis of click-xylosides and their priming activity were extensively documented. In the current study, ruthenium (Ru) mediated catalysis was employed to synthesize xylosides with a 1,5-linkage between the xylose and the triazole ring instead of a 1,4-linkage as found in Cu-catalyzed click-xyloside synthesis. Mono- and bis-click-xylosides were synthesized using each catalytic method and their glycosaminoglycan priming activity was assessed in vitro using a cellular system. Ru-catalyzed click-xylosides showed a higher priming activity as measured by incorporation of radioactive sulfate into primed glycosaminoglycan chains. This study demonstrates that altering the linkage of the aglycone to the triazole ring changes the priming activity. Computational modeling provides a molecular rationale for higher priming ability of Ru-mediated click-xylosides. Higher GAG priming activity is attributed to the formation of more stable interactions between the 1,5-linked xylosides and ß-1,4-galactosyltransferase 7 (ß4GalT7).

Repurposing of Proton Pump Inhibitors as first identified small molecule inhibitors of endo-ß-N-acetylglucosaminidase (ENGase) for the treatment of NGLY1 deficiency, a rare genetic disease.

N-Glycanase deficiency, or NGLY1 deficiency, is an extremely rare human genetic disease. N-Glycanase, encoded by the gene NGLY1, is an important enzyme involved in protein deglycosylation of misfolded proteins. Deglycosylation of misfolded proteins precedes the endoplasmic reticulum (ER)-associated degradation (ERAD) process. NGLY1 patients produce little or no N-glycanase (Ngly1), and the symptoms include global developmental delay, frequent seizures, complex hyperkinetic movement disorder, difficulty in swallowing/aspiration, liver dysfunction, and a lack of tears. Unfortunately, there has not been any therapeutic option available for this rare disease so far. Recently, a proposed molecular mechanism for NGLY1 deficiency suggested that endo-ß-N-acetylglucosaminidase (ENGase) inhibitors may be promising therapeutics for NGLY1 patients. Herein, we performed structure-based virtual screening utilizing FDA-approved drug database on this ENGase target to enable repurposing of existing drugs. Several Proton Pump Inhibitors (PPIs), a series of substituted 1H-benzo [d] imidazole, and 1H-imidazo [4,5-b] pyridines, among other scaffolds, have been identified as potent ENGase inhibitors. An electrophoretic mobility shift assay was employed to assess the inhibition of ENGase activity by these PPIs. Our efforts led to the discovery of Rabeprazole Sodium as the most promising hit with an IC50 of 4.47±0.44µM. This is the first report that describes the discovery of small molecule ENGase inhibitors, which can potentially be used for the treatment of human NGLY1 deficiency.

A Novel Compound ARN-3236 Inhibits Salt-Inducible Kinase 2 and Sensitizes Ovarian Cancer Cell Lines and Xenografts to Paclitaxel.

Purpose: Salt-inducible kinase 2 (SIK2) is a centrosome kinase required for mitotic spindle formation and a potential target for ovarian cancer therapy. Here, we examine the effects of a novel small-molecule SIK2 inhibitor, ARN-3236, on sensitivity to paclitaxel in ovarian cancer.Experimental Design: SIK2 expression was determined in ovarian cancer tissue samples and cell lines. ARN-3236 was tested for its efficiency to inhibit growth and enhance paclitaxel sensitivity in cultures and xenografts of ovarian cancer cell lines. SIK2 siRNA and ARN-3236 were compared for their ability to produce nuclear-centrosome dissociation, inhibit centrosome splitting, block mitotic progression, induce tetraploidy, trigger apoptotic cell death, and reduce AKT/survivin signaling.Results: SIK2 is overexpressed in approximately 30% of high-grade serous ovarian cancers. ARN-3236 inhibited the growth of 10 ovarian cancer cell lines at an IC50 of 0.8 to 2.6 µmol/L, where the IC50 of ARN-3236 was inversely correlated with endogenous SIK2 expression (Pearson r = -0.642, P = 0.03). ARN-3236 enhanced sensitivity to paclitaxel in 8 of 10 cell lines, as well as in SKOv3ip (P = 0.028) and OVCAR8 xenografts. In at least three cell lines, a synergistic interaction was observed. ARN-3236 uncoupled the centrosome from the nucleus in interphase, blocked centrosome separation in mitosis, caused prometaphase arrest, and induced apoptotic cell death and tetraploidy. ARN-3236 also inhibited AKT phosphorylation and attenuated survivin expression.Conclusions: ARN-3236 is the first orally available inhibitor of SIK2 to be evaluated against ovarian cancer in preclinical models and shows promise in inhibiting ovarian cancer growth and enhancing paclitaxel chemosensitivity. Clin Cancer Res; 23(8); 1945-54. ©2016 AACR.

SIK2 Restricts Autophagic Flux To Support Triple-Negative Breast Cancer Survival.

Triple-negative breast cancer (TNBC) is a highly heterogeneous disease with multiple, distinct molecular subtypes that exhibit unique transcriptional programs and clinical progression trajectories. Despite knowledge of the molecular heterogeneity of the disease, most patients are limited to generic, indiscriminate treatment options: cytotoxic chemotherapy, surgery, and radiation. To identify new intervention targets in TNBC, we used large-scale, loss-of-function screening to identify molecular vulnerabilities among different oncogenomic backgrounds. This strategy returned salt inducible kinase 2 (SIK2) as essential for TNBC survival. Genetic or pharmacological inhibition of SIK2 leads to increased autophagic flux in both normal-immortalized and tumor-derived cell lines. However, this activity causes cell death selectively in breast cancer cells and is biased toward the claudin-low subtype. Depletion of ATG5, which is essential for autophagic vesicle formation, rescued the loss of viability following SIK2 inhibition. Importantly, we find that SIK2 is essential for TNBC tumor growth in vivo Taken together, these findings indicate that claudin-low tumor cells rely on SIK2 to restrain maladaptive autophagic activation. Inhibition of SIK2 therefore presents itself as an intervention opportunity to reactivate this tumor suppressor mechanism.

Design, Synthesis, and Biological Evaluation of a Series of Anthracene-9,10-dione Dioxime ß-Catenin Pathway Inhibitors.

The Wnt/ß-catenin signaling pathway plays a vital role in cell growth, the regulation, cell development, and the differentiation of normal stem cells. Constitutive activation of the Wnt/ß-catenin signaling pathway is found in many human cancers, and thus, it is an attractive target for anticancer therapy. Specific inhibitors of this pathway have been keenly researched and developed. Cell based screening of compounds library, hit-to-lead optimization, computational and structure-based design strategies resulted in the design and synthesis of a series of anthracene-9,10-dione dioxime series of compounds demonstrated potent inhibition of ß-catenin in vitro (IC50 < 10 nM, 14) and the growth of several cancer cell lines. This article discusses the potential of inhibiting the Wnt/ß-catenin signaling pathway as a therapeutic approach for cancer along with an overview of the development of specific inhibitors.

Inhibition of Nek2 by small molecules affects proteasome activity.

BACKGROUND: Nek2 is a serine/threonine kinase localized to the centrosome. It promotes cell cycle progression from G2 to M by inducing centrosome separation. Recent studies have shown that high Nek2 expression is correlated with drug resistance in multiple myeloma patients. MATERIALS AND METHODS: To investigate the role of Nek2 in bortezomib resistance, we ectopically overexpressed Nek2 in several cancer cell lines, including multiple myeloma lines. Small-molecule inhibitors of Nek2 were discovered using an in-house library of compounds. We tested the inhibitors on proteasome and cell cycle activity in several cell lines. RESULTS: Proteasome activity was elevated in Nek2-overexpressing cell lines. The Nek2 inhibitors inhibited proteasome activity in these cancer cell lines. Treatment with these inhibitors resulted in inhibition of proteasome-mediated degradation of several cell cycle regulators in HeLa cells, leaving them arrested in G2/M. Combining these Nek2 inhibitors with bortezomib increased the efficacy of bortezomib in decreasing proteasome activity in vitro. Treatment with these novel Nek2 inhibitors successfully mitigated drug resistance in bortezomib-resistant multiple myeloma. CONCLUSION: Nek2 plays a central role in proteasome-mediated cell cycle regulation and in conferring resistance to bortezomib in cancer cells. Taken together, our results introduce Nek2 as a therapeutic target in bortezomib-resistant multiple myeloma.

High-throughput virtual screening identifies novel N'-(1-phenylethylidene)-benzohydrazides as potent, specific, and reversible LSD1 inhibitors.

Lysine specific demethylase 1 (LSD1) plays an important role in regulating histone lysine methylation at residues K4 and K9 on histone H3 and is an attractive therapeutic target in multiple malignancies. Here we report a structure-based virtual screen of a compound library containing ∼2 million small molecular entities. Computational docking and scoring followed by biochemical screening led to the identification of a novel N'-(1-phenylethylidene)-benzohydrazide series of LSD1 inhibitors with hits showing biochemical IC50s in the 200-400 nM range. Hit-to-lead optimization and structure-activity relationship studies aided in the discovery of compound 12, with a Ki of 31 nM. Compound 12 is reversible and specific for LSD1 as compared to the monoamine oxidases shows minimal inhibition of CYPs and hERG and inhibits proliferation and survival in several cancer cell lines, including breast and colorectal cancer. Compound 12 may be used to probe LSD1's biological role in these cancers.

Use of a bacteriophage lysin to identify a novel target for antimicrobial development.

We identified an essential cell wall biosynthetic enzyme in Bacillus anthracis and an inhibitor thereof to which the organism did not spontaneously evolve measurable resistance. This work is based on the exquisite binding specificity of bacteriophage-encoded cell wall-hydrolytic lysins, which have evolved to recognize critical receptors within the bacterial cell wall. Focusing on the B. anthracis-specific PlyG lysin, we first identified its unique cell wall receptor and cognate biosynthetic pathway. Within this pathway, one biosynthetic enzyme, 2-epimerase, was required for both PlyG receptor expression and bacterial growth. The 2-epimerase was used to design a small-molecule inhibitor, epimerox. Epimerox prevented growth of several Gram-positive pathogens and rescued mice challenged with lethal doses of B. anthracis. Importantly, resistance to epimerox was not detected (<10(-11) frequency) in B. anthracis and S. aureus. These results describe the use of phage lysins to identify promising lead molecules with reduced resistance potential for antimicrobial development.

Discovery of Novel Putative Inhibitors of UDP-GlcNAc 2-Epimerase as Potent Antibacterial Agents.

We present the discovery and optimization of a novel series of inhibitors of bacterial UDP-N-acetylglucosamine 2-epimerase (called 2-epimerase in this paper). Starting from virtual screening hits, the activity of various inhibitory molecules was optimized using a combination of structure-based and rational design approaches. We successfully designed and identified a 2-epimerase inhibitor (compound 12-ES-Na, that we named Epimerox) which blocked the growth of methicillin-resistant Staphylococcus aureus (MRSA) at 3.9 µM MIC (minimum inhibitory concentration) and showed potent broad-range activity against all Gram-positive bacteria that were tested. Additionally a microplate coupled assay was performed to further confirm that the 2-epimerase inhibition of Epimerox was through a target-specific mechanism. Furthermore, Epimerox demonstrated in vivo efficacy and had a pharmacokinetic profile that is consonant with it being developed into a promising new antibiotic agent for treatment of infections caused by Gram-positive bacteria.

A novel EPAS1/HIF2A germline mutation in a congenital polycythemia with paraganglioma.

Congenital polycythemias have diverse etiologies, including mutations in the hypoxia sensing pathway. These include HIF2A at exon 12, VHL gene (Chuvash polycythemia), and PHD2 mutations, which in one family was also associated with recurrent pheochromocytoma/paraganglioma (PHEO/PGL). Over the past two decades, we have studied seven unrelated patients with sporadic congenital polycythemia who subsequently developed PHEO/PGL with, until now, no discernible molecular basis. We now report a polycythemic patient with a novel germline HIF2A (F374Y) (exon 9) mutation, inherited from his mother, who developed PHEO/PGL. We show that this is a gain-of-function mutation and demonstrate no loss-of-heterozygosity or additional somatic mutation of HIF2A in the tumor, indicating HIF2A (F374Y) may be predisposing rather than causative of PHEO/PGL. This report, in view of two other concomitantly reported PHEO/PGL patients with somatic mutations of HIF2A and polycythemia, underscores the PHEO/PGL-promoting potential of mutations of HIF2A that alone are not sufficient for PHEO/PGL development.