Mirko Brunetti

A designed P1 cysteine mimetic for covalent and non-covalent inhibitors of HCV NS3 protease

The difluoromethyl group was designed by computational chemistry methods as a mimetic of the canonical P1 cysteine thiol for inhibitors of the hepatitis C virus NS3 protease. This modification led to the development of competitive, non-covalent inhibitor 4 (K(i) 30 nM) and reversible covalent inhibitors (6, K(i) 0.5 nM; and 8 K*(i) 10 pM).

Characterization of the Hepatitis C Virus NS2/3 Processing Reaction by Using a Purified Precursor Protein

ABSTRACT The NS2-NS3 region of the hepatitis C virus polyprotein encodes a proteolytic activity that is required for processing of the NS2/3 junction. Membrane association of NS2 and the autocatalytic nature of the NS2/3 processing event have so far constituted hurdles to the detailed investigation of this reaction. We now report the first biochemical characterization of the self-processing activity of a purified NS2/3 precursor. Using multiple sequence alignments, we were able to define a minimal domain, devoid of membrane-anchoring sequences, which was still capable of performing the processing reaction. This truncated protein was efficiently expressed and processed in Escherichia coli. The processing reaction could be significantly suppressed by growth in minimal medium in the absence of added zinc ions, leading to the accumulation of an unprocessed precursor protein in inclusion bodies. This protein was purified to homogeneity, refolded, and shown to undergo processing at the authentic NS2/NS3 cleavage site with rates comparable to those observed using an in vitro-translated full-length NS2/3 precursor. Size-exclusion chromatography and a dependence of the processing rate on the concentration of truncated NS2/3 suggested a functional multimerization of the precursor protein. However, we were unable to observe trans cleavage activity between cleavage-site mutants and active-site mutants. Furthermore, the cleavage reaction of the wild-type protein was not inhibited by addition of a mutant that was unable to undergo self-processing. Site-directed mutagenesis data and the independence of the processing rate from the nature of the added metal ion argue in favor of NS2/3 being a cysteine protease having Cys993 and His952 as a catalytic dyad. We conclude that a purified protein can efficiently reproduce processing at the NS2/3 site in the absence of additional cofactors.

Loss of Histone Deacetylase 4 Causes Segregation Defects during Mitosis of p53-Deficient Human Tumor Cells

We investigated the role of histone deacetylase 4 (HDAC4) using RNA interference (RNAi) and knockout cells to specifically address its role in cell cycle progression in tumor and normal cells. Ablation of HDAC4 led to growth inhibition in human tumor cells but not to detectable effects in normal human dermal fibroblasts (NHDF) or myelopoietic progenitors. HDAC4-/+ or HDAC4-/- murine embryonic fibroblasts showed no detectable growth defects. On the other hand, HDAC4 RNAi in HeLa cells produced mitotic arrest followed by caspase-dependent apoptosis. Mitotically arrested cells showed chromosome segregation defects. Even though the growth of both p53-wild-type and p53-null tumor cells were affected by HDAC4 ablation, segregation defects were observed only in p53-null cells. HDAC4 associates with the PP2A-B56 regulatory subunit, which is known to be involved in chromosome segregation, and RNAi of either the structural subunit A or the regulatory subunit B56 of PP2A also caused chromosome segregation defects. We conclude that HDAC4 is required for cell cycle progression of tumor cells by multiple mechanisms, one of which seems to be specific to p53-deficient cells through chromosome segregation defects. On the contrary, HDAC4 is not required for the progression of NHDF. We therefore suggest that systemic selective interference with the expression or function of HDAC4 is expected to have a significant therapeutic window, in particular, for p53-deficient tumors.

In Vivo Selection of Protease Cleavage Sites by Using Chimeric Sindbis Virus Libraries

ABSTRACT Identifying protease cleavage sites contributes to our understanding of their specificity and biochemical properties and can help in designing specific inhibitors. One route to this end is the generation and screening of random libraries of cleavage sites. Both synthetic and phage-displayed libraries have been extensively used in vitro. We describe a novel system based on recombinant Sindbis virus which can be used to identify cleavage sites in vivo, thus eliminating the need for a purified enzyme and overcoming the problem of choosing the correct in vitro conditions. As a model we used the serine protease of the hepatitis C virus (HCV). We engineered the gene coding for this enzyme and two specific cleavage sites in the Sindbis virus structural gene and constructed libraries of viral genomes with a random sequence at either of the cleavage sites. The system was designed so that only viral genomes coding for sequences cleaved by the protease would produce viable viruses. With this system we selected viruses containing sequences mirroring those of the natural HCV protease substrates which were cleaved with comparable efficiencies.

MK-4101 - a potent inhibitor of the hedgehog pathway - is highly active against medulloblastoma and basal cell carcinoma

Aberrant activation of the Hedgehog (Hh) signaling pathway is implicated in the pathogenesis of many cancers, including medulloblastoma and basal cell carcinoma (BCC). In this study, using neonatally irradiated Ptch1+/− mice as a model of Hh-dependent tumors, we investigated the in vivo effects of MK-4101, a novel SMO antagonist, for the treatment of medulloblastoma and BCC. Results clearly demonstrated a robust antitumor activity of MK-4101, achieved through the inhibition of proliferation and induction of extensive apoptosis in tumor cells. Of note, beside antitumor activity on transplanted tumors, MK-4101 was highly efficacious against primary medulloblastoma and BCC developing in the cerebellum and skin of Ptch1+/− mice. By identifying the changes induced by MK-4101 in gene expression profiles in tumors, we also elucidated the mechanism of action of this novel, orally administrable compound. MK-4101 targets the Hh pathway in tumor cells, showing the maximum inhibitory effect on Gli1. MK-4101 also induced deregulation of cell cycle and block of DNA replication in tumors. Members of the IGF and Wnt signaling pathways were among the most highly deregulated genes by MK-4101, suggesting that the interplay among Hh, IGF, and Wnt is crucial in Hh-dependent tumorigenesis. Altogether, the results of this preclinical study support a therapeutic opportunity for MK-4101 in the treatment of Hh-driven cancers, also providing useful information for combination therapy with drugs targeting pathways cooperating with Hh oncogenic activity. Mol Cancer Ther; 15(6); 1177–89. ©2016 AACR.