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With the emergence and rapid spreading of NDM-1 and existence of clinically relevant VIM-1 and IMP-1, discovery of pan inhibitors targeting metallo-beta-lactamases (MBLs) became critical in our battle against bacterial infection. Concurrent with our fragment and high-throughput screenings, we performed a knowledge-based search of known metallo-beta-lactamase inhibitors (MBLIs) to identify starting points for early engagement of medicinal chemistry. A class of compounds exemplified by

Supplementary Table 1 from A Fully Human Insulin-Like Growth Factor-I Receptor Antibody SCH 717454 (Robatumumab) Has Antitumor Activity as a Single Agent and in Combination with Cytotoxics in Pediatric Tumor Xenografts

Supplementary Figure Legends 1-3, Table Legend 1 from A Fully Human Insulin-Like Growth Factor-I Receptor Antibody SCH 717454 (Robatumumab) Has Antitumor Activity as a Single Agent and in Combination with Cytotoxics in Pediatric Tumor Xenografts

The insulin-like growth factor-I receptor (IGF-IR) and its ligands (IGF-I and IGF-II) have been implicated in the growth, survival, and metastasis of a broad range of malignancies including pediatric tumors. Blocking the IGF-IR action is a potential cancer treatment. A fully human neutralizing monoclonal antibody, SCH 717454 (19D12, robatumumab), specific to IGF-IR, has shown potent antitumor effects in ovarian cancer in vitro and in vivo. In this study, SCH 717454 was evaluated in several pediatric solid tumors including neuroblastoma, osteosarcoma, and rhabdomyosarcoma. SCH 717454 is shown here to downregulate IGF-IR as well as inhibit IGF-IR and insulin receptor substrate-1 phosphorylation in pediatric tumor cells. IGF-IR and insulin receptor substrate-1 phosphorylation in the tumor cells. In vivo, SCH 717454 exhibits activity as a single agent and significantly inhibited growth of neuroblastoma, osteosarcoma, and rhabdomyosarcoma tumor xenografts. Combination of SCH 717454 with cisplatin or cyclophosphamide enhanced both the degree and the duration of the in vivo antitumor activity compared with single-agent treatments. Furthermore, SCH 717454 treatment markedly reduced Ki-67 expression and blood vessel formation in tumor xenografts, showing that the in vivo activity is derived from its inhibition of tumor cell proliferation and angiogenesis activity. Mol Cancer Ther; 9(2); 410–8

Supplementary Figure 1 from A Fully Human Insulin-Like Growth Factor-I Receptor Antibody SCH 717454 (Robatumumab) Has Antitumor Activity as a Single Agent and in Combination with Cytotoxics in Pediatric Tumor Xenografts

Supplementary Tables 1, 2, and 3 and Supplementary Figures 1 and 2

Supplementary Figure 3 from A Fully Human Insulin-Like Growth Factor-I Receptor Antibody SCH 717454 (Robatumumab) Has Antitumor Activity as a Single Agent and in Combination with Cytotoxics in Pediatric Tumor Xenografts

Supplementary Figure 2 from A Fully Human Insulin-Like Growth Factor-I Receptor Antibody SCH 717454 (Robatumumab) Has Antitumor Activity as a Single Agent and in Combination with Cytotoxics in Pediatric Tumor Xenografts

Kinesin spindle protein (KSP) is a mitotic kinesin required for the formation of the bipolar mitotic spindle, and inhibition of this motor protein results in mitotic arrest and cell death. KSP inhibitors show preclinical antitumor activity and are currently undergoing testing in clinical trials. These agents have been dosed intravenously using various dosing schedules. We sought to identify a KSP inhibitor that could be delivered orally and thus provide convenience of dosing as well as the ability to achieve more continuous exposure via the use of dose-dense administration. We discovered SCH 2047069, a potent KSP inhibitor with oral bioavailability across species and the ability to cross the blood-brain barrier. The compound induces mitotic arrest characterized by a monaster spindle and is associated with an increase in histone H3 and mitotic protein monoclonal 2 phosphorylation both in vitro and in vivo. SCH 2047069 showed antitumor activity in a variety of preclinical models as a single agent and in combination with paclitaxel, gemcitabine, or vincristine. Mol Cancer Ther; 9(11); 2993–3002. ©2010 AACR.

We describe our optimization efforts to improve the physicochemical properties, solubility, and off-target profile of 1, an inhibitor of TarO, an early stage enzyme in the biosynthetic pathway for wall teichoic acid (WTA) synthesis. Compound 1 displayed a TarO IC50 of 125 nM in an enzyme assay and possessed very high lipophilicity (clogP = 7.1) with no measurable solubility in PBS buffer. Structure–activity relationship (SAR) studies resulted in a series of compounds with improved lipophilic ligand efficiency (LLE) consistent with the reduction of clogP. From these efforts, analog 9 was selected for our initial in vivo study, which in combination with subefficacious dose of imipenem (IPM) robustly lowered the bacterial burden in a neutropenic Staphylococci murine infection model. Concurrent with our in vivo optimization effort using 9, we further improved LLE as exemplified by a much more druglike analog 26.

Tedizolid phosphate, the prodrug of the active antibiotic tedizolid, is an oxazolidinone for the treatment of acute bacterial skin and skin structure infections. Studies in a mouse thigh infection model demonstrated that tedizolid has improved potency and pharmacokinetics/pharmacodynamics (PK/PD) compared with those of linezolid. Subsequent studies showed that the efficacy of tedizolid was enhanced in immunocompetent (IC) mice compared with neutropenic (immunosuppressed [IS]) mice, with stasis at clinically relevant doses being achieved only in the presence of granulocytes. The tedizolid label therefore contains a warning about its use in neutropenic patients. This study reevaluated the PK/PD of tedizolid and linezolid in the mouse thigh infection model in IC and IS mice using a methicillin-resistant Staphylococcus aureus (MRSA) strain (ATCC 33591) and a methicillin-susceptible S. aureus (MSSA) strain (ATCC 29213). The antistaphylococcal effect of doses ranging from 1 to 150 mg/kg of body weight tedizolid (once daily) or linezolid (twice daily) was determined at 24, 48, and 72 h after initiating treatment. In IC mice, stasis was achieved in the absence of antibiotics, and both tedizolid and linezolid reduced the burden further beyond a static effect. In IS mice, tedizolid achieved stasis against MRSA ATCC 33591 and MSSA ATCC 29213 at 72 h at a human clinical dose of 200 mg, severalfold lower than that in earlier studies. Linezolid achieved a static effect against MRSA ATCC 33591 in IS mice at a dose lower than that used clinically. This study demonstrates that, with time, both tedizolid and linezolid at clinically relevant exposures achieve stasis in neutropenic mice with an MRSA or MSSA thigh infection.

Imidazo-[1, 2-a]pyrazine 1 is a potent inhibitor of Aurora A and B kinase in vitro and is effective in in vivo tumor models, but has poor oral bioavailbility and is unsuitable for oral dosing. We describe herein our effort to improve oral exposure in this class, resulting ultimately in the identification of a potent Aurora inhibitor 16, which exhibited good drug exposure levels across species upon oral dosing, and showed excellent in vivo efficacy in a mouse xenograft tumor model when dosed orally.

The widespread emergence of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically eroded the efficacy of current β-lactam antibiotics and created an urgent need for new treatment options. We report an S. aureus phenotypic screening strategy involving chemical suppression of the growth inhibitory consequences of depleting late-stage wall teichoic acid biosynthesis. This enabled us to identify early-stage pathway-specific inhibitors of wall teichoic acid biosynthesis predicted to be chemically synergistic with β-lactams. We demonstrated by genetic and biochemical means that each of the new chemical series discovered, herein named tarocin A and tarocin B, inhibited the first step in wall teichoic acid biosynthesis (TarO). Tarocins do not have intrinsic bioactivity but rather demonstrated potent bactericidal synergy in combination with broad-spectrum β-lactam antibiotics against diverse clinical isolates of methicillin-resistant staphylococci as well as robust efficacy in a murine infection model of MRSA. Tarocins and other inhibitors of wall teichoic acid biosynthesis may provide a rational strategy to develop Gram-positive bactericidal β-lactam combination agents active against methicillin-resistant staphylococci.

The imidazo-[1,2-a]-pyrazine (1) is a dual inhibitor of Aurora kinases A and B with modest cell potency (IC50 = 250 nM) and low solubility (5 μM). Lead optimization guided by the binding mode led to the acyclic amino alcohol 12k (SCH 1473759), which is a picomolar inhibitor of Aurora kinases (TdF K d Aur A = 0.02 nM and Aur B = 0.03 nM) with improved cell potency (phos-HH3 inhibition IC50 = 25 nM) and intrinsic aqueous solubility (11.4 mM). It also demonstrated efficacy and target engagement in human tumor xenograft mouse models.

Abrogation of p53 function occurs in almost all human cancers, with more than 50% of cancers harboring inactivating mutations in p53 itself. Mutation of p53 is indicative of highly aggressive cancers and poor prognosis. The vast majority of mutations in p53 occur in its core DNA binding domain (DBD) and result in inactivation of p53 by reducing its thermodynamic stability at physiological temperature. Here, we report a small molecule, SCH529074, that binds specifically to the p53 DBD in a saturable manner with an affinity of 1-2 microm. Binding restores wild type function to many oncogenic mutant forms of p53. This small molecule reactivates mutant p53 by acting as a chaperone, in a manner similar to that previously reported for the peptide CDB3. Binding of SCH529074 to the p53 DBD is specifically displaced by an oligonucleotide with a sequence derived from the p53-response element. In addition to reactivating mutant p53, SCH529074 binding inhibits ubiquitination of p53 by HDM2. We have also developed a novel variant of p53 by changing a single amino acid in the core domain of p53 (N268R), which abolishes binding of SCH529074. This amino acid change also inhibits HDM2-mediated ubiquitination of p53. Our novel findings indicate that through its interaction with p53 DBD, SCH529074 restores DNA binding activity to mutant p53 and inhibits HDM2-mediated ubiquitination.

Clostridium difficile is the causative agent of C. difficile -associated diarrhea (CDAD), with increased risk in elderly populations. Kibdelomycin, a novel natural-product inhibitor of type II topoisomerase enzymes, was evaluated for activity against C. difficile and gastrointestinal anaerobic organisms. Toxigenic C. difficile isolates ( n = 168) from U.S. hospitals and anaerobic Gram-positive and Gram-negative organisms ( n = 598) from Chicago-area hospitals were tested. Kibdelomycin showed potent activity against toxigenic C. difficile (MIC 90 = 0.25 μg/ml) and most Gram-positive aerobic organisms but had little activity against Bacteroides species (MIC 50 > 32 μg/ml; n = 270). Potent anti- C. difficile activity was also observed in the hamster model of C. difficile colitis. Dosing at 1.6 mg/kg (twice-daily oral dose) resulted in protection from a lethal infection and a 2-log reduction in C. difficile cecal counts. A 6.25-mg/kg twice-daily oral dose completely eliminated detectable C. difficile counts in cecal contents. A single 6.25-mg/kg oral dose showed that cecal contents were exposed to the drug at >2 μM (eightfold higher than the MIC), with no significant plasma exposure. These findings support further exploration of kibdelomycin for development of an anti- C. difficile agent.

CD45 is a receptor-type protein-tyrosine phosphatase (PTP) that is required for antigen-specific stimulation and proliferation in lymphocytes. This study was designed to determine the nature of specific kinases in lymphocytes that phosphorylate CD45 and to determine the effect of phosphorylation on CD45 PTP activity. A major cytoplasmic lymphocyte kinase that phosphorylated CD45 was identified as casein kinase 2 (CK2) by use of an in-gel kinase assay in combination with immunoprecipitation, immunodepletion, and specific inhibition. Mutational analysis of CK2 consensus sites showed that the target for CK2 was in an acidic insert of 19 amino acids in the D2 domain, and Ser to Ala mutations at amino acids 965, 968, 969, and 973 abrogated CK2 phosphorylation of CD45. CK2 phosphorylation increased CD45 activity 3-fold toward phosphorylated myelin basic protein, and this increase was reversible by PP2A treatment. Mutation of Ser to Glu at the CK2 sites had the same effect as phosphorylation and also tripled the Vmax of CD45. CD45 isolatedin vivo was highly phosphorylated and could not be phosphorylated by CK2 without prior dephosphorylation with phosphatase PP2A. We conclude that CK2 is a major lymphocyte kinase that is responsible for in vivo phosphorylation of CD45, and phosphorylation at specific CK2 sites regulates CD45 PTP activity.

Summary Innovative strategies are needed to combat drug resistance associated with methicillin-resistant Staphylococcus aureus (MRSA). Here, we investigate the potential of wall teichoic acid (WTA) biosynthesis inhibitors as combination agents to restore β-lactam efficacy against MRSA. Performing a whole-cell pathway-based screen, we identified a series of WTA inhibitors (WTAIs) targeting the WTA transporter protein, TarG. Whole-genome sequencing of WTAI-resistant isolates across two methicillin-resistant Staphylococci spp. revealed TarG as their common target, as well as a broad assortment of drug-resistant bypass mutants mapping to earlier steps of WTA biosynthesis. Extensive in vitro microbiological analysis and animal infection studies provide strong genetic and pharmacological evidence of the potential effectiveness of WTAIs as anti-MRSA β-lactam combination agents. This work also highlights the emerging role of whole-genome sequencing in antibiotic mode-of-action and resistance studies.

Kinesin spindle protein (KSP) is a mitotic kinesin required for the formation of the bipolar mitotic spindle, and inhibition of this motor protein results in mitotic arrest and cell death. KSP inhibitors show preclinical antitumor activity and are currently undergoing testing in clinical trials. These agents have been dosed intravenously using various dosing schedules. We sought to identify a KSP inhibitor that could be delivered orally and thus provide convenience of dosing as well as the ability to achieve more continuous exposure via the use of dose-dense administration. We discovered SCH 2047069, a potent KSP inhibitor with oral bioavailability across species and the ability to cross the blood-brain barrier. The compound induces mitotic arrest characterized by a monaster spindle and is associated with an increase in histone H3 and mitotic protein monoclonal 2 phosphorylation both in vitro and in vivo. SCH 2047069 showed antitumor activity in a variety of preclinical models as a single agent and in combination with paclitaxel, gemcitabine, or vincristine. Mol Cancer Ther; 9(11); 2993–3002. ©2010 AACR.

Aurora kinases are required for orderly progression of cells through mitosis, and inhibition of these kinases by siRNA or small molecule inhibitors results in cell death. We previously reported the synthesis of SCH 1473759, a novel sub-nanomolar Aurora A/B inhibitor. We utilized SCH 1473759 and a panel of tumor cell lines and xenograft models to gain knowledge about optimal dosing schedule and chemotherapeutic combinations for Aurora A/B inhibitors. SCH 1473759 was active against a large panel of tumor cell lines from different tissue origin and genetic backgrounds. Asynchronous cells required 24-h exposure to SCH 1473759 for maximal induction of >4 N DNA content and inhibition of cell growth. However, following taxane- or KSP inhibitor-induced mitotic arrest, less than 4-h exposure induced >4 N DNA content. This finding correlated with the ability of SCH 1473759 to accelerate exit from mitosis in response to taxane- and KSP inhibitor-induced arrest. We tested various dosing schedules in vivo and demonstrated SCH 1473759 dose- and schedule-dependent anti-tumor activity in four human tumor xenograft models. Further, the efficacy was enhanced in combination with taxanes and found to be most efficacious when SCH 1473759 was dosed 12-h post-taxane treatment. SCH 1473759 demonstrated potent mechanism-based activity, and activity was shown to be enhanced in combination with taxanes and KSP inhibitors. This information may be useful for optimizing the clinical efficacy of Aurora inhibitors.

We report a series of potent imidazo[1,2-a]pyrazine-based Aurora kinase inhibitors. Optimization of the solvent accessible 8-position led to improvements in both oral bioavailability and off-target kinase inhibition. Compound 25 demonstrates anti-tumor activity in an A2780 ovarian tumor xenograft model.

The insulin-like growth factor-I receptor (IGF-IR) and its ligands (IGF-I and IGF-II) have been implicated in the growth, survival, and metastasis of a broad range of malignancies including pediatric tumors. Blocking the IGF-IR action is a potential cancer treatment. A fully human neutralizing monoclonal antibody, SCH 717454 (19D12, robatumumab), specific to IGF-IR, has shown potent antitumor effects in ovarian cancer in vitro and in vivo. In this study, SCH 717454 was evaluated in several pediatric solid tumors including neuroblastoma, osteosarcoma, and rhabdomyosarcoma. SCH 717454 is shown here to downregulate IGF-IR as well as inhibit IGF-IR and insulin receptor substrate-1 phosphorylation in pediatric tumor cells. IGF-IR and insulin receptor substrate-1 phosphorylation in the tumor cells. In vivo, SCH 717454 exhibits activity as a single agent and significantly inhibited growth of neuroblastoma, osteosarcoma, and rhabdomyosarcoma tumor xenografts. Combination of SCH 717454 with cisplatin or cyclophosphamide enhanced both the degree and the duration of the in vivo antitumor activity compared with single-agent treatments. Furthermore, SCH 717454 treatment markedly reduced Ki-67 expression and blood vessel formation in tumor xenografts, showing that the in vivo activity is derived from its inhibition of tumor cell proliferation and angiogenesis activity. Mol Cancer Ther; 9(2); 410–8

CD45 contains tandem repeated protein tyrosine phosphatase (PTP) domains and is essential for the initiation of the earliest activation events resulting from Ag ligation of the TCR. The second PTP domain (D2) contains four CK2 phosphorylation sites in a unique 19-aa insert, which are targets of CK2 phosphorylation. This study was designed to evaluate the roles of these Ser residues in T cell activation. Transient transfection of the CD45− T cell line, J45.01, with CD45 cDNA incorporating four Ser to Ala (S/A) mutations in the 19-aa insert did not affect the magnitude of NF-AT activation resulting from TCR ligation. However, the basal level of NF-AT activity in unstimulated cells expressing the CD45 S/A mutation was elevated 9- to 10-fold. Increased basal NF-AT was dependent on extracellular Ca2+ stores as judged by EGTA treatment. In additional experiments, isolation of stable clones derived from transfection of the CD45 S/A mutant into CD45− H45.01 cells showed sustained calcium flux after TCR engagement. The sustained calcium flux returned to baseline levels after addition of EGTA, suggesting that the expression of the CD45 S/A mutant may have prevented deactivation of plasma membrane calcium channels. Consideration of both transient and stable transfection systems suggests that in addition to being essential for initial events in T cell triggering, the intact CD45 D2, 19-aa insert is necessary for regulation of TCR-mediated calcium signaling pathways.

Despite the need for new antibiotics to treat drug-resistant bacteria, current clinical combinations are largely restricted to β-lactam antibiotics paired with β-lactamase inhibitors. We have adapted a Staphylococcus aureus antisense knockdown strategy to genetically identify the cell division Z ring components-FtsA, FtsZ, and FtsW-as β-lactam susceptibility determinants of methicillin-resistant S. aureus (MRSA). We demonstrate that the FtsZ-specific inhibitor PC190723 acts synergistically with β-lactam antibiotics in vitro and in vivo and that this combination is efficacious in a murine model of MRSA infection. Fluorescence microscopy localization studies reveal that synergy between these agents is likely to be elicited by the concomitant delocalization of their cognate drug targets (FtsZ and PBP2) in MRSA treated with PC190723. A 2.0 A crystal structure of S. aureus FtsZ in complex with PC190723 identifies the compound binding site, which corresponds to the predominant location of mutations conferring resistance to PC190723 (PC190723(R)). Although structural studies suggested that these drug resistance mutations may be difficult to combat through chemical modification of PC190723, combining PC190723 with the β-lactam antibiotic imipenem markedly reduced the spontaneous frequency of PC190723(R) mutants. Multiple MRSA PC190723(R) FtsZ mutants also displayed attenuated virulence and restored susceptibility to β-lactam antibiotics in vitro and in a mouse model of imipenem efficacy. Collectively, these data support a target-based approach to rationally develop synergistic combination agents that mitigate drug resistance and effectively treat MRSA infections.