Exploring new scaffolds for the dual inhibition of HIV-1 RT polymerase and ribonuclease associated functions

Current therapeutic protocols for the treatment of HIV infection consist of the combination of diverse anti-retroviral drugs in order to reduce the selection of resistant mutants and to allow for the use of lower doses of each single agent to reduce toxicity. However, avoiding drugs interactions and patient compliance are issues not fully accomplished so far. Pursuing on our investigation on potential anti HIV multi-target agents we have designed and synthesized a small library of biphenylhydrazo 4-arylthiazoles derivatives and evaluated to investigate the ability of the new derivatives to simultaneously inhibit both associated functions of HIV reverse transcriptase. All compounds were active towards the two functions, although at different concentrations. The substitution pattern on the biphenyl moiety appears relevant to determine the activity. In particular, compound 2-{3- [(2-{4-[4-(hydroxynitroso)phenyl]-1,3-thiazol-2-yl} hydrazin-1-ylidene) methyl]-4-methoxyphenyl} benzamide bromide (EMAC2063) was the most potent towards RNaseH (IC50 = 4.5 mM)- and RDDP (IC50 = 8.0 mM) HIV RT-associated function

Atomic-resolution monitoring of protein maturation in live human cells by NMR

We use NMR directly in live human cells to describe the complete post-translational maturation process of human superoxide dismutase 1 (SOD1). We follow, at atomic resolution, zinc binding, homodimer formation and copper uptake, and discover that copper chaperone for SOD1 oxidizes the SOD1 intrasubunit disulfide bond through both copper-dependent and copper-independent mechanisms. Our approach represents a new strategy for structural investigation of endogenously expressed proteins in a physiological (cellular) environment

Sequential protein expression and selective labeling for in-cell NMR in human cells

In-cell NMR is a powerful technique to investigate proteins in living human cells at atomic resolution. Ideally, when studying functional processes involving protein-protein interactions by NMR, only one partner should be isotopically labeled. Here we show that constitutive and transient protein expression can be combined with protein silencing to obtain selective protein labeling in human cells.We established a human cell line stably overexpressing the copper binding protein HAH1. A second protein (human superoxide dismutase 1, SOD1) was overexpressed by transient transfection and isotopically labeled. A silencing vector containing shRNA sequences against the HAH1 gene was used to decrease the rate of HAH1 synthesis during the expression of SOD1. The levels of HAH1 mRNA and protein were measured as a function of time following transfection by RT-PCR and Western Blot, and the final cell samples were analyzed by in-cell NMR.SOD1 was ectopically expressed and labeled in a time window during which HAH1 biosynthesis was strongly decreased by shRNA, thus preventing its labeling. In-cell NMR spectra confirmed that, while both proteins were present, only SOD1 was selectively labeled and could be detected by (1)H-(15)N heteronuclear NMR.We showed that controlling protein expression by specifically silencing a stably expressed protein is a useful strategy to obtain selective isotope labeling of only one protein. This approach relies on established techniques thus permitting the investigation of protein-protein interactions by NMR in human cells

The Casein Kinase 2-Dependent Phosphorylation of NS5A Domain 3 from Hepatitis C Virus Followed by Time-Resolved NMR Spectroscopy

Hepatitis C virus (HCV) chronically affects millions of individuals worldwide. The HCV nonstructural protein 5A (NS5A) plays a critical role in the viral assembly pathway. Domain 3 (D3) of NS5A is an unstructured polypeptide responsible for the interaction with the core particle assembly structure. Casein kinase 2 (CK2) phosphorylates NS5A-D3 at multiple sites that have mostly been predicted and only observed indirectly. In order to identify the CK2-dependent phosphorylation sites, we monitored the reaction between NS5A-D3 and CK2 in vitro by time-resolved NMR. We unambiguously identified four serine residues as substrates of CK2. The apparent rate constant for each site was determined from the reaction curves. Ser408 was quickly phosphorylated, whereas the three other serine residues reacted more slowly. These results provide a starting point from which to elucidate the role of phosphorylation in the mechanisms of viral assembly-and in the modulation of the viral activity-at the molecular level

Visualization of redox-controlled protein fold in living cells

none4noMost mitochondrial proteins are encoded by nuclear DNA, synthesized in the cytoplasm, and imported into mitochondria. Several proteins of the intermembrane space (IMS) are imported and localized through an oxidative process, being folded through the formation of structural disulfide bonds catalyzed by Mia40, and trapped in the IMS. To be imported, these proteins need to be reduced and unfolded; however, no structural information in situ exists on these proteins in the cytoplasm. In humans, Mia40 undergoes the same mechanism, although its folding state in the cytoplasm is unknown. We provide atomic-level details on the Mia40 folding state in the human cell cytoplasm through in-cell nuclear magnetic resonance. Overexpressed cytoplasmic Mia40 is folded, and its folding state depends on the glutaredoxin 1 (Grx1) and thioredoxin 1 (Trx1) systems. Specifically, increased Grx1 levels keep most Mia40 unfolded, while Trx1 is less effective.noneBANCI, LUCIA; BARBIERI, LETIZIA; LUCHINAT, ENRICO; SECCI, ERICABANCI, LUCIA; BARBIERI, LETIZIA; LUCHINAT, ENRICO; SECCI, ERIC

2H-chromene and 7H-furo-chromene derivatives selectively inhibit tumour associated human carbonic anhydrase IX and XII isoforms

AbstractTumour associated carbonic anhydrases (CAs) IX and XII have been recognised as potential targets for the treatment of hypoxic tumours. Therefore, considering the high pharmacological potential of the chromene scaffold as selective ligand of the IX and XII isoforms, two libraries of compounds, namely 2H-chromene and 7H-furo-chromene derivatives, with diverse substitution patterns were designed and synthesised. The structure of the newly synthesised compounds was characterised and their inhibitory potency and selectivity towards human CA off target isoforms I, II and cancer-associated CA isoforms IX and XII were evaluated. Most of the compounds inhibit CA isoforms IX and XII with no activity against the I and II isozymes. Thus, while the potency was influenced by the substitution pattern along the chromene scaffold, the selectivity was conserved along the series, confirming the high potential of both 2H-chromene and 7H-furo-chromene scaffolds for the design of isozyme selective inhibitors