We recently reported the discovery of AM-8553 (1), a potent and selective piperidinone inhibitor of the MDM2-p53 interaction. Continued research investigation of the N-alkyl substituent of this series, focused in particular on a previously underutilized interaction in a shallow cleft on the MDM2 surface, led to the discovery of a one-carbon tethered sulfone which gave rise to substantial improvements in biochemical and cellular potency. Further investigation produced AMG 232 (2), which is currently being evaluated in human clinical trials for the treatment of cancer. Compound 2 is an extremely potent MDM2 inhibitor (SPR KD = 0.045 nM, SJSA-1 EdU IC50 = 9.1 nM), with remarkable pharmacokinetic properties and in vivo antitumor activity in the SJSA-1 osteosarcoma xenograft model (ED50 = 9.1 mg/kg).

Structure-based rational design led to the discovery of novel inhibitors of the MDM2-p53 protein-protein interaction. The affinity of these compounds for MDM2 was improved through conformational control of both the piperidinone ring and the appended N-alkyl substituent. Optimization afforded 29 (AM-8553), a potent and selective MDM2 inhibitor with excellent pharmacokinetic properties and in vivo efficacy.

The agouti-related protein gene (Agrp) plays an important role in body weight regulation. The mature human protein is a single polypeptide chain of 112 amino acid residues, consisting of an N-terminal acidic region and a unique C-terminal cysteine-rich domain. The disulfide structure of recombinant human AGRP was determined by chemical methods using partial reduction with tris(2-carboxyethyl)phosphine under acidic conditions, followed by direct alkylation with N-ethylmaleimide or fluorescein-5-maleimide. Partial reduction and alkylation provided several forms of AGRP that were modified in a stepwise fashion. The resulting proteins were characterized by peptide mapping, sequence analysis, and mass spectrometry, showing that AGRP contained a highly reducible disulfide bond, C85-C109, followed by less reactive ones, C90-C97, C74-C88, C67-C82, and C81-C99, respectively. The chemically defined disulfide connectivity of the recombinant human AGRP was homologous to that of omega-agatoxin IVB except for an additional disulfide bond, C85-C109.

The wbp gene cluster, encoding the B-band lipopolysaccharide O antigen of Pseudomonas aeruginosa serotype O5 strain PAO1, was previously shown to contain a wzy (rfc) gene encoding the O-antigen polymerase. This study describes the molecular characterization of the corresponding wzz (rol) gene, responsible for modulating O-antigen chain length. P. aeruginosa O5 Wzz has 19 to 20% amino acid identity with Wzz of Escherichia coli, Salmonella enterica, and Shigella flexneri. Knockout mutations of the wzz gene in serotypes O5 and O16 (which has an O antigen structurally related to that of O5) yielded mutants expressing O antigens with a distribution of chain lengths differing markedly from that of the parent strains. Unlike enteric wzz mutants, the P. aeruginosa wzz mutants continued to display some chain length modulation. The P. aeruginosa O5 wzz gene complemented both O5 and O16 wzz mutants as well as an E. coli wzz mutant. Coexpression of E. coli and P. aeruginosa wzz genes in a rough strain of E. coli carrying the P. aeruginosa wbp cluster resulted in the expression of two populations of O-antigen chain lengths. Sequence analysis of the region upstream of wzz led to identification of the genes rpsA and himD, encoding 30S ribosomal subunit protein S1 and integration host factor, respectively. This finding places rpsA and himD adjacent to wzz and the wbp cluster at 37 min on the PAO1 chromosomal map and completes the delineation of the O5 serogroup-specific region of the wbp cluster.Current models for polymerization and assembly of lipopolysaccharide (LPS) O antigens on lipid A core include three pathways, one of which requires the O-antigen polymerase, Wzy (Rfc [24,49]). Most Wzy-independent O antigens are homopolymers which are assembled processively on the cytoplasmic face of the cytoplasmic membrane. Following assembly, they are transferred via an ATP-binding cassette-type twocomponent transporter to the periplasmic face of the cytoplasmic membrane for attachment to lipid A core (49). Recently Keenleyside and Whitfield (24) described a novel Wzy-independent pathway for synthesis of O polysaccharide in Salmonella enterica sv. Borreze, which does not appear to required a two-component transporter for export of the homopolymer.In contrast, Wzy-dependent O antigens are generally heteropolymers, composed of repeats of O-antigen units which are synthesized individually on the cytoplasmic face of the cytoplasmic membrane and translocated by Wzx (RfbX [32]) to the periplasmic face of the cytoplasmic membrane, where they are polymerized by Wzy (49). Wzz (Rol [regulator of O-antigen length] or Cld [chain length determinant]) controls the Wzy-dependent polymerization of O antigen (2, 3) and some lipid A-core-linked capsular antigens (13, 15). When separated on polyacrylamide gels, LPS typically displays a bimodal or multimodal distribution of O-antigen chain lengths which is specific for a particular strain or serotype (19,21). This modal distribution reflects a preference for particular chain lengths, which leads to thei...

Structural analysis of both the MDM2-p53 protein-protein interaction and several small molecules bound to MDM2 led to the design and synthesis of tetrasubstituted morpholinone 10, an MDM2 inhibitor with a biochemical IC50 of 1.0 μM. The cocrystal structure of 10 with MDM2 inspired two independent optimization strategies and resulted in the discovery of morpholinones 16 and 27 possessing distinct binding modes. Both analogues were potent MDM2 inhibitors in biochemical and cellular assays, and morpholinone 27 (IC50 = 0.10 μM) also displayed suitable PK profile for in vivo animal experiments. A pharmacodynamic (PD) experiment in mice implanted with human SJSA-1 tumors showed p21(WAF1) mRNA induction (2.7-fold over vehicle) upon oral dosing of 27 at 300 mg/kg.

In matrix-assisted laser desorption/ionization of proteins, there exists a certain amount of fast metastable decay immediately after laser irradiation. The fragment ions thus formed can be resolved and their m/z values measured accurately by employing delayed extraction linear time-of-flight mass spectrometry. At higher than threshold laser fluences, proteins exhibit a series of fragment ions providing useful sequence information. We also observe that when moderate amounts of salts are present in the sample with sinapinic acid being the matrix, the intensities of cn ions (N-terminal fragments) are enhanced compared to other types of fragment ions. This enhancement in cn ion signals allows direct sequencing of proteins. The cn ions are completely absent when Xxx-Pro bonds are encountered and are of lower intensity when Xxx-Gly bonds are involved. Further, the cn ion series is interrupted at Xxx-Cys, when the cysteine is involved in a disulfide bond. Upon reduction of the disulfide bonds, the series continues and information is available for longer stretches. Using 10-20 pmol of recombinant proteins, sometimes contiguous sequence information up to 70 residues is obtained in a matter of minutes. Applications of the technique to some recombinant proteins with intra- or interchain disulfide linkages are presented.

It has been proposed that the insulin-like growth factors (IGFs) can act as autocrine and/or paracrine growth promoters in breast cancer. To investigate this hypothesis, we infected early passage MCF-7 cells with a retroviral vector containing the coding sequence for the IGF-II preprohormone along with a constitutive cytomegalovirus promoter sequence. These cells do not normally express IGF-I or IGF-II. After infection with the retroviral vector, several single cell clones were analyzed. Seven of nine isolated clones expressed very high levels of IGF-II mRNA. Biologically active IGF-II protein was easily detectable in the medium conditioned by the IGF-II-expressing clones, and IGF receptors were down-regulated in these. All IGF-II-expressing clones showed marked morphological changes in anchorage-dependent culture, growing in large clumps and as free-floating colonies. The cells also cloned in soft agar in the absence of estrogen, while the wild-type MCF-7 cells and control cells infected with an irrelevant DNA sequence showed none of these properties. alpha IR-3, an antibody that blocks the type I IGF receptor, inhibited the growth of IGF-II-expressing clones in serum-free medium. This model demonstrates that IGF-II can serve as an autocrine growth stimulant in breast cancer epithelial cells and that IGF-II overexpression may be capable of mediating malignant progression in human breast cancer.

Natural products serve as chemical blueprints for the majority of antibiotics in our clinical arsenal. The evolutionary process by which these molecules arise is inherently accompanied by the co-evolution of resistance mechanisms that shorten the clinical lifetime of any given class 1 . Virginiamycin acetyltransferases (Vats) are resistance proteins that provide protection against streptogramins 2 , potent Gram-positive antibiotics that inhibit the bacterial ribosome 3 . Due to the challenge of selectively modifying the chemically complex, 23-membered macrocyclic scaffold of group A streptogramins, analogs that overcome Vat resistance have not been previously accessed 2 . Here we report the design, synthesis, and antibacterial evaluation of group A streptogramin antibiotics with unprecedented structural variability. Using cryo-electron microscopy and forcefield-based refinement, we characterize the binding of eight analogs to the bacterial ribosome at high resolution, revealing new binding interactions that extend into the peptidyl tRNA binding site and towards synergistic binders that occupy the nascent peptide exit tunnel (NPET). One of these analogs has excellent activity against several streptogramin-resistant strains of S. aureus , exhibits decreased acetylation rates in vitro , and is effective at lowering bacterial load in a mouse model of infection. Our results demonstrate that the combination of rational design and modular chemical synthesis can revitalize classes of antibiotics that are limited by naturally arising resistance mechanisms.