T he phosphoinositide-3-kinase (PI3K) family of lipid kinases is involved in a diverse set of cellular functions, including cell growth, proliferation, motility, differentiation, glucose transport, survival, intracellular trafficking, and membrane ruffling. 1 PI3K's can be categorized into class I, II, or III, depending on their subunit structure, regulation, and substrate selectivity. 2 Class IA PI3K's are activated by receptor tyrosine kinases and consist of a regulatory subunit (p85) and a catalytic subunit (p110). There are three catalytic isoforms: p110R, β, and δ. A single class IB PI3K, activated by GPCRs, consists of only one member: a p110γ catalytic subunit and a p101 regulatory subunit. The primary in vivo substrate of the class I PI3K's is phosphatidylinositol (4,5) diphosphate (PtdIns(4,5)P2), which upon phosphorylation at the 3-position of the inositol ring to form phosphatidylinositol triphosphate (3,4,5)P3 (PIP3) serves as a second messenger by activating a series of downstream effectors that mediate the cellular functions mentioned above. The PI3K isoforms have different distributions and share similar cellular functions, which are context dependent. In particular, p110R pathway deregulation has been demonstrated in ovarian, breast, colon, and brain cancers. 3,4 Inhibitors of PI3KR represent an intriguing therapeutic modality for these indications, and as such, there is much interest in generating suitable molecules to test this hypothesis in the clinic. 5À10 We have previously reported on a series of 6-hydroxyphenyl-2-morpholino pyrimidines, 11 as potent pan class I PI3K inhibitors that exhibit high selectivity toward protein kinases (serine/threonine and tyrosine kinases). We have further reported on non-phenol containing heterocyclic, morpholino pyrimidines 12 such as compound 1 which demonstrate in vivo PI3K pathway modulation and modest tumor growth inhibition. Described herein are our efforts to identify potent morpholino pyrimidinyl inhibitors of class I PI3Ks that exhibit potency and pharmacokinetic properties which allow for maximal pathway modulation in vivo and have druglike properties suitable for clinical development. These efforts culminated in the identification of 15, NVP-BKM120.Aminopyrimidine 1 and analogues such as 3 (Figure 1) exhibit low or sub-nanomolar biochemical potency and sub-micromolar cellular potency against PI3KR. Even with high rodent CL values, such analogues can demonstrate PI3K pathway modulation in mouse xenograft models. 12 During our exploration of the C 6 position, it was noted that C 6 aminopyridine analogue 4, while being less potent than 3 against PI3KR (>10Â potency loss), exhibited a markedly reduced (>9Â) rat CL value, increased %F, and increased oral AUC. Thus, superior pharmacokinetic properties were achievable within this scaffold and the challenge remaining was to retain this kind of pharmacokinetic profile while optimizing all the other attributes (potency, solubility, permeability, safety) necessary for advancement. To address this challenge, ...

BackgroundThere is a growing interest in using functional food components as therapy for cardiovascular diseases such as hypertension. We have previously characterized a tri-peptide IRW (Ile-Arg-Trp) from egg white protein ovotransferrin; this peptide showed anti-inflammatory, anti-oxidant and angiotensin converting enzyme (ACE) inhibitor properties in vitro. Given the pathogenic roles played by angiotensin, oxidative stress and inflammation in the spontaneously hypertensive rat (SHR), we tested the therapeutic potential of IRW in this well-established model of hypertension.Methods and Results16–17 week old male SHRs were orally administered IRW at either a low dose (3 mg/Kg BW) or a high dose (15 mg/Kg BW) daily for 18 days. Blood pressure (BP) and heart rate were measured by telemetry. Animals were sacrificed at the end of the treatment for vascular function studies and measuring markers of inflammation. IRW treatment attenuated mean BP by ~10 mmHg and ~40 mmHg at the low- and high-dose groups respectively compared to untreated SHRs. Heart rate was not affected. Reduction in BP was accompanied by the restoration of diurnal variations in BP, preservation of nitric oxide dependent vasorelaxation, as well as reduction of plasma angiotensin II, other inflammatory markers and tissue fibrosis.ConclusionOur results demonstrate anti-hypertensive effects of IRW in vivo likely mediated through ACE inhibition, endothelial nitric oxide synthase and anti-inflammatory properties.

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Neutrophil migration inhibition factor from T lymphocytes (NIF-T) is a lymphokine that acts to localize granulocytes. Medium conditioned by the Mo human T-lymphoblast cell line was used to purify NIF-T, a glycoprotein with a molecular weight of 22,000. The NIF-T was found to potently stimulate the growth of granulocyte and macrophage colonies from human bone marrow and colony formation by the KG-1 myeloid leukemia cell line. Thus a human lymphokine (NIF-T) that modulates the activities of mature neutrophilic granulocytes is also a colony-stimulating factor acting on precursors to induce growth and differentiation of new effector cells.

The ability to selectively degrade proteins with bifunctional small molecules has the potential to fundamentally alter therapy in a variety of diseases. However, the relatively large size of these chimeric molecules often results in challenging physico‐chemical properties (e. g., low aqueous solubility) and poor pharmacokinetics which may complicate their in vivo applications. We recently discovered an exquisitely potent chimeric BET degrader (GNE‐987) which exhibited picomolar cell potencies but also demonstrated low in vivo exposures. In an effort to improve the pharmacokinetic properties of this molecule, we discovered the first degrader‐antibody conjugate by attaching GNE‐987 to an anti‐CLL1 antibody via a novel linker. A single IV dose of the conjugate afforded sustained in vivo exposures that resulted in antigen‐specific tumor regressions. Enhancement of a chimeric protein degrader with poor in vivo properties through antibody conjugation thereby expands the utility of directed protein degradation as both a biological tool and a therapeutic possibility.

The migration and invasion of cells are necessary for many normal and pathological processes, including tissue remodeling, embryo implantation, angiogenesis, and tumor cell invasion and metastasis (1-4). Recent reports suggest that these processes require an active cell-surface proteolytic cascade (5, 6). Important components of this cascade are the plasminogen activator/plasmin system, as well as the matrix metalloproteinases (6). The requirement for both protease expression and a cell-surface protease binding protein has been demonstrated most clearly in the case of urokinase plasminogen activator (uPA) and the uPA receptor (uPAR) but has also been recently described for type IV collagenase (7,8). It has been shown in human colon and breast carcinomas that urokinase is expressed in stromal, fibroblast-like cells and uPAR is expressed on tumor epithelial cells or macrophages, respectively (9, 10). This suggests that a paracrine relationship between uPA and its receptor occurs in these pathological conditions. The in vitro observation that human tumor cell invasion is proportional to receptor-bound urokinase, not total urokinase synthesis, further supports the hypothesis that cell-surface protease localization is a key for invasion (11,12). Other results show that plasminogen activation is more efficient when both uPA and plasminogen are bound on a cell surface, and that cell-surface plasmin is resistant to inhibition by a2-antiplasmin (7 (23). We report here the identification and characterization of peptide antagonists with nanomolar affinity for the human uPAR by using a 15-mer peptide library. This extension of bacteriophage peptide display to cell-surfaceexpressed proteins expands the utility of the method to a wide variety of biologically interesting targets. MATERIALS AND METHODSReagets and Stains. Bacteriophage library construction and bacteriophage growth and isolation were performed as described by Devlin et al. (15). The Escherichia coli strains H249, a recA, sup0, F' derivative of MM294, and JM103 [F' traD36 proAB+ lacJq lacZAM15 A(pro-lac) supE hsdR endAI sbcBl5 thi-1 strAAi] were used for these experiments. Recombinant DNA manipulations were according to Sambrook et aL (24); electrocompetent E. coil HB101 (Stratagene) were used for subcloning unless otherwise noted. Restriction enzymes were from New England Biolabs; high molecularweight human uPA, plasminogen, and the anti-uPAR monoclonal antibody 3936 were from American Diagnostica (Greenwich, CT). Streptavidin was from Molecular Probes or Sigma, and bovine serum albumin (BSA) was from Sigma. Immulon-2 96-well plates were fom Dynatech. The plasmin substrate S-2251 was from Kabi Pharmacia Diagnostics (Piscataway, NJ). Linear synthetic peptides were prepared on an Applied Biosystems model 430A peptide synthesizer using 9-fluorenylmethoxycarbonyl-based chemistry and were purified by reversed-phase HPLC after trifluoroacetic acid cleavage. Alternatively, peptides were obtained from Chiron Mimotopes (Melbourne, Australia). The cyclic uPA peptide encom...

Erythropoietin is the primary physiological regulator of erythropoiesis; however, in vitro studies have identified another class of mediators which appear to be important in stimulating erythroid progenitors. These factors have generally been referred to as burst-promoting activities (BPA), because they stimulate the growth of early erythroid progenitors referred to as burst-forming units-erythroid (BFU-E) which give rise to colonies of up to thousands of haemoglobinized cells. We recently reported purification of a burst-promoting activity from medium conditioned by the Mo T-lymphoblast cell line infected with human T-cell lymphotropic virus type II (HTLV-II). This purified glycoprotein of relative molecular mass (Mr) 28,000 also stimulates colony formation by more mature erythroid precursors (CFU-E) and is therefore referred to as erythroid-potentiating activity (EPA). Purified EPA specifically stimulates human and murine cells of the erythroid lineage, unlike murine interleukin-3 (IL-3) which stimulates precursor cells from all haematopoietic lineages. We report here the isolation of a complementary DNA molecular clone encoding EPA and its use in producing EPA in COS (monkey) cells and CHO (Chinese hamster ovary) cells. We also define the organization of the EPA gene in human DNA.

The biological and medicinal impacts of proteolysis-targeting chimeras (PROTACs) and related chimeric molecules that effect intracellular degradation of target proteins via ubiquitin ligase-mediated ubiquitination continue to grow. However, these chimeric entities are relatively large compounds that often possess molecular characteristics, which may compromise oral bioavailability, solubility, and/or in vivo pharmacokinetic properties. We therefore explored the conjugation of such molecules to monoclonal antibodies using technologies originally developed for cytotoxic payloads so as to provide alternate delivery options for these novel agents. In this report, we describe the first phase of our systematic development of antibody–drug conjugates (ADCs) derived from bromodomain-containing protein 4 (BRD4)-targeting chimeric degrader entities. We demonstrate the antigen-dependent delivery of the degrader payloads to PC3-S1 prostate cancer cells along with related impacts on MYC transcription and intracellular BRD4 levels. These experiments culminate with the identification of one degrader conjugate, which exhibits antigen-dependent antiproliferation effects in LNCaP prostate cancer cells.