A Randomized Phase 2 Study Comparing the Combination of Ficlatuzumab and Gefitinib with Gefitinib Alone in Asian Patients with Advanced Stage Pulmonary Adenocarcinoma.

INTRODUCTION: A randomized phase 2 study was designed to compare the combination of ficlatuzumab (AV-299), a humanized hepatocyte growth factor-neutralizing monoclonal antibody, plus gefitinib versus gefitinib monotherapy in a pulmonary adenocarcinoma population clinically enriched for EFGR tyrosine kinase inhibitor-sensitizing mutations. METHODS: A total of 188 patients were randomized 1:1 to receive either gefitinib or ficlatuzumab plus gefitinib treatment. Patients who demonstrated disease control in the single-agent gefitinib arm were allowed to cross over to ficlatuzumab plus gefitinib treatment upon disease progression. Molecular analyses included tumor EGFR mutation status and retrospective proteomic testing using VeriStrat, a multivariate test based on mass spectrometry. RESULTS: The addition of ficlatuzumab to gefitinib did not provide significant improvement over gefitinib monotherapy for the primary end point of overall response rate or the secondary end points of progression-free survival and overall survival. In the subgroup classified as VeriStrat poor, the addition of ficlatuzumab to gefitinib showed significant improvement in both progression-free survival and overall survival in both the intent-to-treat population and the subgroup with EGFR tyrosine kinase inhibitor-sensitizing mutations. For all patients, the most frequent adverse events were diarrhea, dermatitis acneiform, and paronychia. CONCLUSIONS: Although the trial showed no significant benefit from the addition of ficlatuzumab to gefitinib in the overall population of Asian patients with advanced-stage pulmonary adenocarcinoma, the biomarker data suggest that patients classified as VeriStrat poor may benefit from ficlatuzumab combination therapy.

Chk1 inhibition and Wee1 inhibition combine synergistically to impede cellular proliferation.

Inhibition of the checkpoint kinase Chk1, both as a monotherapy and in combination with DNA damaging cytotoxics, is a promising therapeutic approach for the treatment of a wide array of human cancers. However, much remains to be elucidated in regard to the patient populations that will respond best to a Chk1 inhibitor and the optimal therapeutics to combine with a Chk1 inhibitor. In an effort to discover sensitizing mutations and novel combination strategies for Chk1 inhibition, an siRNA screen was performed in combination with the selective Chk1 inhibitor AR458323. This screen employed a custom made library of siRNAs targeting 195 genes, most of which are involved in cell-cycle control or DNA damage repair. One of the most prominent and consistent hits across runs of the screen performed in three different cancer cell lines was Wee1 kinase. MK-1775 is a small molecule inhibitor of Wee1 that is currently in early stage clinical trials. In confirmation of the results obtained from the siRNA screen, AR458323 and MK-1775 synergistically inhibited proliferation in multiple cancer cell types. This antiproliferative effect correlated with a synergistic induction of apoptosis. In cellular mechanistic studies, the combination of the two molecules resulted in dramatic decreases in inhibitory phosphorylation of cyclin-dependent kinases, an increase in DNA damage, alterations in cell-cycle profile, and collapse of DNA synthesis. In conclusion, the clinical combination of a Chk1 inhibitor and a Wee1 inhibitor holds promise as an effective treatment strategy for cancer.

Triterpene based compounds with potent anti-maturation activity against HIV-1.

Efforts towards developing orally bioavailable HIV-1 maturation inhibitors starting from betulinic acid 1 are described. SAR resulted in improved potency, physicochemical properties, and enhanced oral absorption in rats.

The human endosomal sorting complex required for transport (ESCRT-I) and its role in HIV-1 budding.

Efficient human immunodeficiency virus type 1 (HIV-1) budding requires an interaction between the PTAP late domain in the viral p6(Gag) protein and the cellular protein TSG101. In yeast, Vps23p/TSG101 binds both Vps28p and Vps37p to form the soluble ESCRT-I complex, which functions in sorting ubiquitylated protein cargoes into multivesicular bodies. Human cells also contain ESCRT-I, but the VPS37 component(s) have not been identified. Bioinformatics and yeast two-hybrid screening methods were therefore used to identify four novel human proteins (VPS37A-D) that share weak but significant sequence similarity with yeast Vps37p and to demonstrate that VPS37A and VPS37B bind TSG101. Detailed studies produced four lines of evidence that human VPS37B is a Vps37p ortholog. 1) TSG101 bound to several different sites on VPS37B, including a putative coiled-coil region and a PTAP motif. 2) TSG101 and VPS28 co-immunoprecipitated with VPS37B-FLAG, and the three proteins comigrated together in soluble complexes of the correct size for human ESCRT-I ( approximately 350 kDa). 3) Like TGS101, VPS37B became trapped on aberrant endosomal compartments in the presence of VPS4A proteins lacking ATPase activity. 4) Finally, VPS37B could recruit TSG101/ESCRT-I activity and thereby rescue the budding of both mutant Gag particles and HIV-1 viruses lacking native late domains. Further studies of ESCRT-I revealed that TSG101 mutations that inhibited PTAP or VPS28 binding blocked HIV-1 budding. Taken together, these experiments define new components of the human ESCRT-I complex and characterize several TSG101 protein/protein interactions required for HIV-1 budding and infectivity.

HIV Gag mimics the Tsg101-recruiting activity of the human Hrs protein.

The HIV-1 Gag protein recruits the cellular factor Tsg101 to facilitate the final stages of virus budding. A conserved P(S/T)AP tetrapeptide motif within Gag (the "late domain") binds directly to the NH2-terminal ubiquitin E2 variant (UEV) domain of Tsg101. In the cell, Tsg101 is required for biogenesis of vesicles that bud into the lumen of late endosomal compartments called multivesicular bodies (MVBs). However, the mechanism by which Tsg101 is recruited from the cytoplasm onto the endosomal membrane has not been known. Now, we report that Tsg101 binds the COOH-terminal region of the endosomal protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs; residues 222-777). This interaction is mediated, in part, by binding of the Tsg101 UEV domain to the Hrs 348PSAP351 motif. Importantly, Hrs222-777 can recruit Tsg101 and rescue the budding of virus-like Gag particles that are missing native late domains. These observations indicate that Hrs normally functions to recruit Tsg101 to the endosomal membrane. HIV-1 Gag apparently mimics this Hrs activity, and thereby usurps Tsg101 and other components of the MVB vesicle fission machinery to facilitate viral budding.

Functional surfaces of the human immunodeficiency virus type 1 capsid protein.

The human immunodeficiency virus type 1 initially assembles and buds as an immature particle that is organized by the viral Gag polyprotein. Gag is then proteolyzed to produce the smaller capsid protein CA, which forms the central conical capsid that surrounds the RNA genome in the mature, infectious virus. To define CA surfaces that function at different stages of the viral life cycle, a total of 48 different alanine-scanning surface mutations in CA were tested for their effects on Gag protein expression, processing, particle production and morphology, capsid assembly, and infectivity. The 27 detrimental mutations fall into three classes: 13 mutations significantly diminished or altered particle production, 9 mutations failed to assemble normal capsids, and 5 mutations supported normal viral assembly but were nevertheless reduced more than 20-fold in infectivity. The locations of the assembly-defective mutations implicate three different CA surfaces in immature particle assembly: one surface encompasses helices 4 to 6 in the CA N-terminal domain (NTD), a second surrounds the crystallographically defined CA dimer interface in the C-terminal domain (CTD), and a third surrounds the loop preceding helix 8 at the base of the CTD. Mature capsid formation required a distinct surface encompassing helices 1 to 3 in the NTD, in good agreement with a recent structural model for the viral capsid. Finally, the identification of replication-defective mutants with normal viral assembly phenotypes indicates that CA also performs important nonstructural functions at early stages of the viral life cycle.

Mechanisms of enveloped RNA virus budding.

To spread infection, enveloped viruses must bud from infected host cells. Recent research indicates that HIV and other enveloped RNA viruses bud by appropriating the cellular machinery that is normally used to create vesicles that bud into late endosomal compartments called multivesicular bodies. This new model of virus budding has many potential implications for cell biology and viral pathogenesis.