Cesare Giordano

Indolylarylsulfones Bearing Natural and Unnatural Amino Acids. Discovery of Potent Inhibitors of HIV-1 Non-Nucleoside Wild Type and Resistant Mutant Strains Reverse Transcriptase and Coxsackie B4 Virus

New potent indolylarylsulfone (IAS) HIV-1 NNRTIs were obtained by coupling natural and unnatural amino acids to the 2-carboxamide and introducing different electron-withdrawing substituents at position 4 and 5 of the indole nucleus. The new IASs inhibited the HIV-1 replication in human T-lymphocyte (CEM) cells at low/subnanomolar concentration and were weakly cytostatic. Against the mutant L100I, K103N, and Y181C RT HIV-1 strains in CEM cells, sulfones 3, 4, 19, 27, and 31 were comparable to EFV. The new IASs were inhibitors to Coxsackie B4 virus at low micromolar (2-9 microM) concentrations. Superimposition of PLANTS docked conformations of IASs 19 and 9 revealed different hydrophobic interactions of the 3,5-dimethylphenyl group, for which a staking interaction with Tyr181 aromatic side chain was observed. The binding mode of 19 was not affected by the L100I mutation and was consistent with the interactions reported for the WT strain.

PARP-1 Modulates Amyloid Beta Peptide-Induced Neuronal Damage

Amyloid beta peptide (Aβ) causes neurodegeneration by several mechanisms including oxidative stress, which is known to induce DNA damage with the consequent activation of poly (ADP-ribose) polymerase (PARP-1). To elucidate the role of PARP-1 in the neurodegenerative process, SH-SY5Y neuroblastoma cells were treated with Aβ25–35 fragment in the presence or absence of MC2050, a new PARP-1 inhibitor. Aβ25–35 induces an enhancement of PARP activity which is prevented by cell pre-treatment with MC2050. These data were confirmed by measuring PARP-1 activity in CHO cells transfected with amylod precursor protein and in vivo in brains specimens of TgCRND8 transgenic mice overproducing the amyloid peptide. Following Aβ25–35 exposure a significant increase in intracellular ROS was observed. These data were supported by the finding that Aβ25–35 induces DNA damage which in turn activates PARP-1. Challenge with Aβ25–35 is also able to activate NF-kB via PARP-1, as demonstrated by NF-kB impairment upon MC2050 treatment. Moreover, Aβ25–35 via PARP-1 induces a significant increase in the p53 protein level and a parallel decrease in the anti-apoptotic Bcl-2 protein. These overall data support the hypothesis of PARP-1 involvment in cellular responses induced by Aβ and hence a possible rationale for the implication of PARP-1 in neurodegeneration is discussed.

β-Sheet interfering molecules acting against β-amyloid aggregation and fibrillogenesis

β-Sheet aggregates and amyloid fibrils rising from conformational changes of proteins are observed in several pathological human conditions. These structures are organized in β-strands that can reciprocally interact by hydrophobic and π-π interactions. The amyloid aggregates can give rise to pathological conditions through complex biochemical mechanisms whose physico-chemical nature has been understood in recent times. This review focuses on the various classes of natural and synthetic small molecules able to act against β-amyloid fibrillogenesis and toxicity that may represent new pharmacological tools in Alzheimers diseases. Some peptides, named β-sheet breaker peptides, are able to hamper amyloid aggregation and fibrillogenesis by interfering with and destabilizing the non native β-sheet structures. Other natural compounds, like polyphenols or indolic molecules such as melatonin, can interfere with β-amyloid peptide pathogenicity by inhibiting aggregation and counteracting oxidative stress that is a key hallmark in Alzheimers disease.

2 Å X-ray structure of adamalysin II complexed with a peptide phosphonate inhibitor adopting a retro-binding mode

The search of reprolysin inhibitors offers the possibility of intervention against both matrixins and ADAMs. Here we report the crystal structure of the complex between adamalysin II, a member of the reprolysin family, and a phosphonate inhibitor modeled on an endogenous venom tripeptide. The inhibitor occupies the primed region of the cleavage site adopting a retro‐binding mode. The phosphonate group ligates the zinc ion in an asymmetric bidentate mode and the adjacent Trp indole system partly fills the primary specificity subsite S1′. An adamalysin‐based model of tumor necrosis factor‐α‐converting enzyme (TACE) reveals a smaller S1′ pocket for this enzyme.

Computational and Experimental Studies on β-Sheet Breakers Targeting Aβ1–40 Fibrils

Background: β-Amyloid aggregates are at the basis of Alzheimer disease development. Short synthetic peptides are seen to inhibit polymerization. Results: Various synthetic peptides have been studied by MD simulations and tested experimentally. Conclusion: Combined results indicate Ac-LPFFN-NH2 as an effective lead compound able to slow down Aβ1–40 aggregation. Significance: Designing potential Aβ aggregation inhibitors will help fight Alzheimer disease. In this work we present and compare the results of extensive molecular dynamics simulations of model systems comprising an Aβ1–40 peptide in water in interaction with short peptides (β-sheet breakers) mimicking the 17–21 region of the Aβ1–40 sequence. Various systems differing in the customized β-sheet breaker structure have been studied. Specifically we have considered three kinds of β-sheet breakers, namely Ac-LPFFD-NH2 and two variants thereof, one obtained by substituting the acetyl group with the sulfonic amino acid taurine (Tau-LPFFD-NH2) and a second novel one in which the aspartic acid is substituted by an asparagine (Ac-LPFFN-NH2). Thioflavin T fluorescence, circular dichroism, and mass spectrometry experiments have been performed indicating that β-sheet breakers are able to inhibit in vitro fibril formation and prevent the β sheet folding of portions of the Aβ1–40 peptide. We show that molecular dynamics simulations and far UV circular dichroism provide consistent evidence that the new Ac-LPFFN-NH2 β-sheet breaker is more effective than the other two in stabilizing the native α-helix structure of Aβ1–40. In agreement with these results thioflavin T fluorescence experiments confirm the higher efficiency in inhibiting Aβ1–40 aggregation. Furthermore, mass spectrometry data and molecular dynamics simulations consistently identified the 17–21 Aβ1–40 portion as the location of the interaction region between peptide and the Ac-LPFFN-NH2 β-sheet breaker.

Synthesis and activity of endomorphin-2 and morphiceptin analogues with proline surrogates in position 2.

The opioid agonists endomorphins (Tyr-Pro-Trp-Phe-NH(2); EM1 and Tyr-Pro-Phe-Phe-NH(2); EM2) and morphiceptin (Tyr-Pro-Phe-Pro-NH(2)) exhibit an extremely high selectivity for mu-opioid receptor. Here a series of novel EM2 and morphiceptin analogues containing in place of the proline at position 2 the S and R residues of beta-homologues of proline (HPro), of 2-pyrrolidinemethanesulphonic acid (HPrs) and of 3-pyrrolidinesulphonic acid (betaPrs) have been synthesized and their binding affinity and functional activity have been investigated. The highest micro-receptor affinity is shown by [(S)betaPrs(2)]EM2 analogue (6e) which represents the first example of a beta-sulphonamido analogue in the field of opioid peptides.

Synthesis and activity of fibrillogenesis peptide inhibitors related to the 17–21 β-amyloid sequence

Peptide derivatives 1-5, incorporating synthetic non-proteinogenic amino acids, related to the beta-amyloid 17-21 fragment of the amyloidogenic Abeta(1-40), and the N-protected decapeptide 6, corresponding to a dimeric sequence of the same fragment, have been synthesized. These compounds were designed by using Sotos pentapeptide Ac-LPFFD-NH(2) (iAbeta5p) as lead compound. Their activity as inhibitors of fibrillogenesis and stability against enzymatic degradation have been determined. Compounds 1, 5 and 6 are potent inhibitors in comparison to the lead compound. Exposure to chymotrypsin of peptide derivatives 1-5, all containing unnatural amino acids, shows increased stability as compared with iAbeta5p and 6. Conformational properties of the new compounds have been determined by CD and FT-IR spectroscopies.

Peptidyl and azapeptidyl methylketones as substrate analog inhibitors of papain and cathepsin B

Summary Peptidyl methylketones containing Phe, Tyr, Tyr(I), Tyr(I 2 ), Leu and Ile in P 2 were synthesized and tested as substrate analog reversible inhibitors of papain and bovine spleen cathepsin B. The most effective cathepsin B inhibitor contained Tyr(I 2 ) and displayed an inhibition constant of 4.7 μM at pH 6.8 and 25°C, while Leu or Ile gave practically inert analogs. Replacement of the amino acids in P 2 with the analogous α-azaamino acids, as well as the glycine in P 1 with α-azaglycine, led to complete loss of inhibiting activity. Introducing alkoxy substituents at the methyl adjacent to the ketone group generally resulted in more effective inhibitors, with inhibition constants in the micromolar range for both papain and cathepsin B.

INHIBITION OF ADAMALYSIN II AND MMPS BY PHOSPHONATE ANALOGUES OF SNAKE VENOM PEPTIDES

Phosphonate analogues of the peptidomimetic N-(Furan-2-yl)carbonyl-Leu-Trp-OH were prepared with the goal of evaluating the effect of phosphonate for carboxylate replacement on binding with snake venom metalloproteinases and MMPs. N-(Furan-2-yl)carbonyl-Leu-L-Trp(P)-(OH)2 showed a 75-fold increase of the inhibiting activity against adamalysin II, a snake venom metalloproteinase structurally related to MMPs and TACE. Both the phosphonate and carboxylate peptidomimetics fit into the active site adopting a retrobinding mode and provide the structural base for a new class of metalloproteinases inhibitors.

Synthesis and activity of HCO-Met-Leu-Phe-OMe analogues containing β-alanine or taurine at the central position

New synthetic analogues of the chemotactic N-formyltripeptide HCO-Met-Leu-Phe-OMe have been synthesized. The reported new models, namely Boc-Met-beta-Ala-Phe-OMe (1), HCO-Met-beta-Ala-Phe-OMe (2), Boc-Met-Tau-Phe-OMe (3), HCO-Met-Tau-Phe-OMe (4) and HCl.Met-Tau-Phe-OMe (5), are characterized by the presence at the central position of a residue of beta-alanine or 2-aminoethanesulfonic acid (taurine) replacing the native L-leucine. Whereas tripeptides 1 and 2 have been found quite inactive as chemoattractants, all the three models containing the Tau residue exhibit a remarkable activity. Superoxide anion production and lysozyme release have been also evaluated and the biological results are discussed together with the conformational preferences of the examined models.