Abstract: Reverse cholesterol transport (RCT) is believed to be the primary mechanism by which HDL and its major protein apoA-I protect against atherosclerosis. Starting from the inactive 22-amino acid peptide representing the consensus sequence of the class A amphipathic helical repeats of apoA-I, we designed novel peptides able to mobilize cholesterol from macrophages in vitro, and to stimulate the formation of 'nascent HDL' particles, with potency comparable to the entire apoA-I protein.
Abstract: A novel hexahydrobenzonaphthyridinone PARP-1 pharmacophore is reported, subsequent SAR exploration around this scaffold led to selective PARP-1 inhibitors with low nanomolar enzyme potency, displaying good cellular activity and promising rat PK properties.
Abstract: Reverse cholesterol transport promoted by HDL-apoA-I is an important mechanism of protection against atherosclerosis. We have previously identified apoA-I mimetic peptides by synthesizing analogs of the 22 amino acid apoA-I consensus sequence (apoA-I(cons)) containing non-natural aliphatic amino acids. Here we examined the effect of different aliphatic non-natural amino acids on the structure-activity relationship (SAR) of apoA-I mimetic peptides. These novel apoA-I mimetics, with long hydrocarbon chain (C(5-8)) amino acids incorporated in the amphipathic α helix of the apoA-I(cons), have the following properties: (i) they stimulate in vitro cholesterol efflux from macrophages via ABCA1; (ii) they associate with HDL and cause formation of pre-β HDL particles when incubated with human and mouse plasma; (iii) they associate with HDL and induce pre-β HDL formation in vivo, with a corresponding increase in ABCA1-dependent cholesterol efflux capacity ex vivo; (iv) at high dose they associate with VLDL and induce hypertriglyceridemia in mice. These results suggest our peptide design confers activities that are potentially anti-atherogenic. However a dosing regimen which maximizes their therapeutic properties while minimizing adverse effects needs to be established.
Abstract: Histone deacetylase (HDAC) inhibitors offer a promising strategy for cancer therapy and the first generation HDAC inhibitors are currently in clinical trials. A structurally novel series of HDAC inhibitors based on the natural cyclic tetrapeptide Apicidin is described. Selected screening of the sample collection looking for L-2-amino-8-oxodecanoic acid (L-Aoda) derivatives identified a small acyclic lead molecule 1 with the unusual ketone zinc binding group. SAR studies around this lead resulted in optimization to potent, low molecular weight, selective, non-hydroxamic acid HDAC inhibitors, equipotent to current clinical candidates.
Abstract: [reaction: see text] In the present paper, systematic studies revealed that Cu(I) salts in general and Cu(II) salts under certain circumstances promote effective reaction between peptide thiol esters and the N-terminal amino function of a second peptide segment to give the native amide bond for both solution- and solid-phase syntheses. Chiral integrity was retained. Reaction conditions were optimized and applied to the synthesis of a small protein, the identity of which was confirmed by NMR analysis.
Abstract: [reaction: see text] Fmoc-protected amino acid fluorides were found to be excellent reagents for the acylation of sulfonamide safety-catch linkers (SCL) suitable for the subsequent preparation of peptide C-terminal thioesters. High loadings were obtained on different types of resins with low levels of epimerization.
Abstract: A serine protease domain contained within the viral NS3 protein is a key player in the maturational processing of the hepatitis C virus polyprotein and a prime target for the development of antiviral drugs. In the present work, we describe a dansylated hexapeptide inhibitor of this enzyme. Active site occupancy by this compound could be monitored following fluorescence resonance energy transfer between the dansyl fluorophore and protein tryptophan residues and could be used to 1) unambiguously assess active site binding of NS3 protease inhibitors, 2) directly determine equilibrium and pre-steady-state parameters of enzyme-inhibitor complex formation, and 3) dissect, using site-directed mutagenesis, the contribution of single residues of NS3 to inhibitor binding in direct binding assays. The assay was also used to characterize the inhibition of the NS3 protease by its cleavage products. We show that enzyme-product inhibitor complex formation depends on the presence of an NS4A cofactor peptide. Equilibrium and pre-steady-state data support an ordered mechanism of ternary (enzyme-inhibitor-cofactor) complex formation, requiring cofactor complexation prior to inhibitor binding.
Abstract: Infection by Hepatitis C Virus (HCV) leads to a slowly progressing disease that over two decades can lead to liver cirrhosis or liver cancer. Currently, one of the most promising approaches to anti-HCV therapy is the development of inhibitors of the NS3/4A protease, which is essential for maturation of the viral polyprotein. Several substrate-derived inhibitors of NS3/4A have been described, all taking advantage of binding to the S subsite of the enzyme. Inspection of the S' subsite of NS3/4A shows binding pockets which might be exploited for inhibitor binding, but due to the fact that ground-state binding to the S' subsite is not used by the substrate, this does not represent a suitable starting point. We have now optimized S'-binding in the context of noncleavable decapeptides spanning P6-P4'. Binding was sequentially increased by introduction of the previously optimized P-region [Ingallinella et al. (1998) Biochemistry 37, 8906-8914], change of the P4' residue, and combinatorial optimization of positions P2'-P3'. The overall process led to an increase in binding of more than 3 orders of magnitude, with the best decapeptide showing IC(50) < 200 pM. The binding mode of the decapeptides described in the present work shares features with the binding mode of the natural substrates, together with novel interactions within the S' subsite. Therefore, these peptides may represent an entry point for a novel class of NS3 inhibitors.
Abstract: One of the most promising approaches to anti-hepatitis C virus drug discovery is the development of inhibitors of the virally encoded protease NS3. This chymotrypsin-like serine protease is essential for the maturation of the viral polyprotein, and processing requires complex formation between NS3 and its cofactor NS4A. Recently, we reported on the discovery of potent cleavage product-derived inhibitors [Ingallinella et al. (1998) Biochemistry 37, 8906-8914]. Here we study the interaction of these inhibitors with NS3 and the NS3/cofactor complex. Inhibitors bind NS3 according to an induced-fit mechanism. In the absence of cofactor different binding modes are apparent, while in the presence of cofactor all inhibitors show the same binding mode with a small rearrangement in the NS3 structure, as suggested by circular dichroism spectroscopy. These data are consistent with the hypothesis that NS4A complexation induces an NS3 structure that is already (but not entirely) preorganized for substrate binding not only for what concerns the S' site, as already suggested, but also for the S site. Inhibitor binding to the NS3/cofactor complex induces the stabilization of the enzyme structure as highlighted by limited proteolysis experiments. We envisage that this may occur through stabilization of the individual N-terminal and C-terminal domains where the cofactor and inhibitor, respectively, bind and subsequent tightening of the interdomain interaction in the ternary complex.
Abstract: Alpha-synuclein (alpha-syn) protein and a fragment of it, called NAC, have been found in association with the pathological lesions of a number of neurodegenerative diseases. Recently, mutations in the alpha-syn gene have been reported in families susceptible to an inherited form of Parkinson's disease. We have shown that human wild-type alpha-syn, mutant alpha-syn(Ala30Pro) and mutant alpha-syn(Ala53Thr) proteins can self-aggregate and form amyloid-like filaments. Here we report that aggregates of NAC and alpha-syn proteins induced apoptotic cell death in human neuroblastoma SH-SY5Y cells. These findings indicate that accumulation of alpha-syn and its degradation products may play a major role in the development of the pathogenesis of these neurodegenerative diseases.
