Sulfoximines as potent RORγ inverse agonists

Progress in the identification of suitable RORγ inverse agonists as clinical candidates has been hampered by the high lipophilicity that seems required for high potency on this nuclear receptor. In this context, we decided to focus on the replacement of the hydroxymethyl group found on known modulators to determine if more polarity could be tolerated in this position. SAR of the replacement of this moiety is presented in this article leading to the identification of sulfoximine derivatives as potent modulators with pharmacological activity in the in vivo mouse Imiquimod psoriasis model.     

Phosphorylated p40PHOX as a negative regulator of NADPH oxidase

The leukocyte NADPH oxidase catalyzes the production of O(2)(-) from oxygen at the expense of NADPH. Activation of the enzyme requires interaction of the cytosolic factors p47(PHOX), p67(PHOX), and Rac2 with the membrane-associated cytochrome b(558). Activation of the oxidase in a semirecombinant cell-free system in the absence of an amphiphilic activator can be achieved by phosphorylation of the cytosolic factor p47(PHOX) by protein kinase C. Another cytosolic factor, p40(PHOX), was recently shown to be phosphorylated on serine and threonine residues upon activation of NADPH oxidase, but both stimulatory and inhibitory roles were reported. In the present study, we demonstrate that the addition of phosphorylated p40(PHOX) to the cell-free system inhibits NADPH oxidase activated by protein kinase C-phosphorylated p47(PHOX), an effect not observed with the unphosphorylated p40(PHOX). Moreover phosphorylated p40(PHOX) inhibits the oxidase if added before or after full activation of the enzyme. Direct mutagenesis of protein kinase C consensus sites enables us to conclude that phosphorylation of threonine 154 is required for the inhibitory effect of p40(PHOX) to occur. Although the phosphorylated mutants and nonphosphorylated mutants are still able to interact with both p47(PHOX) and p67(PHOX) in pull-down assays, their proteolysis pattern upon thrombin treatment suggests a difference in conformation between the phosphorylated and nonphosphorylated mutants. We postulate that phosphorylation of p40(PHOX) on threonine 154 leads to an inhibitory conformation that shifts the balance toward an inhibitory role and blocks oxidase activation.     

Use of hydrophilic interaction chromatography for the study of tyrosine protein kinase specificity

A new HPLC method has been developed to assay tyrosine protein kinase activity. Using hydrophilic interaction chromatography, it is possible to resolve the four components of the incubation medium: substrate peptide, [³²P]phosphorylated peptide, unreacted [γ-³²P]ATP, and ³²P-labelled inorganic phosphate. ATP interacts so strongly with the stationary phase material that it can be removed selectively from the incubation medium with solid-phase extraction cartridges packed with the same type of material. The three remaining components of interest can then be resolved by reversed-phase or hydrophilic interaction HPLC. This procedure permits the evaluation of almost every type of peptide as a substrate of tyrosine protein kinase.     

Adipose Tissue Is a Neglected Viral Reservoir and an Inflammatory Site during Chronic HIV and SIV Infection

Two of the crucial aspects of human immunodeficiency virus (HIV) infection are (i) viral persistence in reservoirs (precluding viral eradication) and (ii) chronic inflammation (directly associated with all-cause morbidities in antiretroviral therapy (ART)-controlled HIV-infected patients). The objective of the present study was to assess the potential involvement of adipose tissue in these two aspects. Adipose tissue is composed of adipocytes and the stromal vascular fraction (SVF); the latter comprises immune cells such as CD4⁺ T cells and macrophages (both of which are important target cells for HIV). The inflammatory potential of adipose tissue has been extensively described in the context of obesity. During HIV infection, the inflammatory profile of adipose tissue has been revealed by the occurrence of lipodystrophies (primarily related to ART). Data on the impact of HIV on the SVF (especially in individuals not receiving ART) are scarce. We first analyzed the impact of simian immunodeficiency virus (SIV) infection on abdominal subcutaneous and visceral adipose tissues in SIVmac251 infected macaques and found that both adipocytes and adipose tissue immune cells were affected. The adipocyte density was elevated, and adipose tissue immune cells presented enhanced immune activation and/or inflammatory profiles. We detected cell-associated SIV DNA and RNA in the SVF and in sorted CD4⁺ T cells and macrophages from adipose tissue. We demonstrated that SVF cells (including CD4⁺ T cells) are infected in ART-controlled HIV-infected patients. Importantly, the production of HIV RNA was detected by in situ hybridization, and after the in vitro reactivation of sorted CD4⁺ T cells from adipose tissue. We thus identified adipose tissue as a crucial cofactor in both viral persistence and chronic immune activation/inflammation during HIV infection. These observations open up new therapeutic strategies for limiting the size of the viral reservoir and decreasing low-grade chronic inflammation via the modulation of adipose tissue-related pathways.     

Electrical protein array chips for the detection of staphylococcal virulence factors

A new approach for the detection of virulence factors of Staphylococcus aureus and Staphylococcus epidermidis using an electrical protein array chip technology is presented. The procedure is based on an enzyme-linked sandwich immunoassay, which includes recognition and binding of virulence factors by specific capture and detection antibodies. Detection of antibody-bound virulence factors is achieved by measuring the electrical current generated by redox recycling of an enzymatically released substance. The current (measured in nanoampere) corresponds to the amount of the target molecule in the analyzed sample. The electrical protein chip allows for a fast detection of Staphylococcus enterotoxin B (SEB) of S. aureus and immunodominant antigen A homologue (IsaA homologue) of S. epidermidis in different liquid matrices. The S. aureus SEB virulence factor could be detected in minimal medium, milk, and urine in a concentration of 1 ng/ml within less than 23 min. Furthermore, a simultaneous detection of SEB of S. aureus and IsaA homologue of S. epidermidis in a single assay could be demonstrated.     

CCM1–ICAP-1 complex controls β1 integrin–dependent endothelial contractility and fibronectin remodeling

The endothelial CCM complex regulates blood vessel stability and permeability. Loss-of-function mutations in CCM genes are responsible for human cerebral cavernous malformations (CCMs), which are characterized by clusters of hemorrhagic dilated capillaries composed of endothelium lacking mural cells and altered sub-endothelial extracellular matrix (ECM). Association of the CCM1/2 complex with ICAP-1, an inhibitor of β1 integrin, prompted us to investigate whether the CCM complex interferes with integrin signaling. We demonstrate that CCM1/2 loss resulted in ICAP-1 destabilization, which increased β1 integrin activation and led to increased RhoA-dependent contractility. The resulting abnormal distribution of forces led to aberrant ECM remodeling around lesions of CCM1- and CCM2-deficient mice. ICAP-1–deficient vessels displayed similar defects. We demonstrate that a positive feedback loop between the aberrant ECM and internal cellular tension led to decreased endothelial barrier function. Our data support that up-regulation of β1 integrin activation participates in the progression of CCM lesions by destabilizing intercellular junctions through increased cell contractility and aberrant ECM remodeling.     

Podosome-type adhesions and focal adhesions, so alike yet so different

Cell-matrix adhesions are essential for cell migration, tissue organization and differentiation, therefore playing central roles in embryonic development, remodeling and homeostasis of tissues and organs. Matrix adhesion-dependent signals cooperate with other pathways to regulate biological functions such as cell survival, cell proliferation, wound healing, and tumorigenesis. Cell migration and invasion are integrated processes requiring the continuous, coordinated assembly and disassembly of integrin-mediated adhesions. An understanding of how integrins regulate cell migration and invasiveness through the dynamic regulation of adhesions is fundamental to both physiological and pathological situations. A variety of cell-matrix adhesions has been identified, namely, focal complexes, focal adhesions, fibrillar adhesions, podosomes, and invadopodia (podosome-type adhesions). These adhesion sites contain integrin clusters able to develop specialized structures, which are different in their architecture and dynamics although they share almost the same proteins. Here we compare recent advances and developments in the elucidation of the organization and dynamics of focal adhesions and podosome-type adhesions, in order to understand how such subcellular sites - though closely related in their composition - can be structurally and functionally different. The underlying question is how their respective physiological or pathological roles are related to their distinct organization.     

Prospective Evaluation of First-Line Erlotinib in Advanced Non-Small Cell Lung Cancer (NSCLC) Carrying an Activating EGFR Mutation: A Multicenter Academic Phase II Study in Caucasian Patients (FIELT)

Introduction

Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibition is the preferred first-line treatment of advanced adenocarcinoma of the lung that harbors EGFR activating tyrosine kinase domain mutations. Most data available pertain to Asian populations in which such mutations are more prevalent. We report on the long-term results of first-line treatment with erlotinib in Caucasian patients with advanced adenocarcinoma of the lung that have a somatic EGFR mutation in their tumor.

Methods

Multicenter academic prospective phase II study with erlotinib in patients with an activating EGFR tyrosine kinase (TK) domain somatic mutation (any exon encoding the kinase domain) in the tumor and no prior treatment for their advanced disease.

Results

Phenotypic preselecting of 229 patients led to a high EGFR mutation detection rate of 24% of which 46 patients were included in the phase II study. With a progression free survival (PFS) of 81% at three months the study met its primary endpoint for presumed superiority over chemotherapy. With an overall median PFS of 11 months and a median overall survival (OS) of 23 months, the results compare favorably with results obtained in randomized studies using TKI in first line in EGFR mutation positive adenocarcinoma of the lung.

Conclusion

The present study reinforces the use of EGFR tyrosine kinase inhibition (TKI) as a first line treatment of choice for advanced adenocarcinoma of the lung carrying an activating EGFR mutation. The mutation rate in preselected Caucasian patients is higher than previously reported. Issues relevant for clinical practice are discussed.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00339586","term_id":"NCT00339586"}}NCT00339586