Coumarin-Based Dual Inhibitors of Human Carbonic Anhydrases and Monoamine Oxidases Featuring Amino Acyl and (Pseudo)-Dipeptidyl Appendages: In Vitro and Computational Studies.

The involvement of human carbonic anhydrase (hCA) IX/XII in the pathogenesis and progression of many types of cancer is well acknowledged, and more recently human monoamine oxidases (hMAOs) A and B have been found important contributors to tumor development and aggressiveness. With a view of an enzymatic dual-blockade approach, in this investigation, new coumarin-based amino acyl and (pseudo)-dipeptidyl derivatives were synthesized and firstly evaluated in vitro for inhibitory activity and selectivity against membrane-bound and cytosolic hCAs (hCA IX/XII over hCA I/II), as well as the hMAOs, to estimate their potential as anticancer agents. De novo design of peptide-coumarin conjugates was subsequently carried out and involved the combination of the widely explored coumarin nucleus with the unique biophysical and structural properties of native or modified peptides. All compounds displayed nanomolar inhibitory activities towards membrane-anchored hCAs, whilst they were unable to block the ubiquitous CA I and II isoforms. Structural features pertinent to potent and selective CA inhibitory activity are discussed, and modeling studies were found to support the biological data. Lower potency inhibition of the hMAOs was observed, with most compounds showing preferential inhibition of hMAO-A. The binding of the most potent ligands (6 and 16) to the hydrophobic active site of hMAO-A was investigated in an attempt to explain selectivity on the molecular level. Calculated Ligand Efficiency values indicate that compound 6 has the potential to serve as a lead compound for developing innovative anticancer agents based on the dual inhibition strategy. This information may help design new coumarin-based peptide molecules with diverse bioactivities.

Biofilm and Quorum Sensing inhibitors: the road so far.

INTRODUCTION: Biofilm is a complex aggregation of microorganisms characterized by the presence of a dynamic, adhesive and protective extracellular matrix composed of polysaccharides, proteins and nucleic acids. It is estimated that the vast majority of human infections are related to the biofilm in which the microorganisms reside and communicate with each other (Quorum Sensing), surviving in hostile environmental conditions. AREAS COVERED: This review provides a comprehensive focus on the development state of promising strategies against biofilm production and eradication describing chemical structures, results, administration routes, pharmaceutical compositions, and SARs as well as their shortcomings within the 2019-2020 range. EXPERT OPINION: New pharmacological targets have been explored in the past years, allowing a broader therapeutic arsenal against biofilm-related pathologies. The Quorum Sensing system was targeted as well in order to avoid the development of intrinsically antibiotic-resistant bacteria and to enhance a proper host defense.

Virtual screening identification and chemical optimization of substituted 2-arylbenzimidazoles as new non-zinc-binding MMP-2 inhibitors.

Matrix metalloproteinases (MMPs) are a large family of zinc-dependent endoproteases known to exert multiple regulatory roles in tumor progression and invasiveness. This encouraged over the years the approach of MMP, and particularly MMP-2, targeting for anticancer treatment. Early generations of MMP inhibitors, based on aspecific zinc binding groups (ZBGs) assembled on (pseudo)peptide scaffolds, have been discontinued due to the clinical emergence of toxicity and further drawbacks, giving the way to inhibitors with alternative zinc-chelator moieties or not binding the catalytic zinc ion. In the present paper, we continue the search for new non-zinc binding MMP-2 inhibitors: exploiting previously identified compounds, a virtual screening (VS) campaign was carried out and led to the identification of a new class of ligands. The structure-activity relationship (SAR) of the benzimidazole scaffold was explored by synthesis of several analogues whose inhibition activity was tested with enzyme inhibition assays. By performing the molecular simplification approach, we disclosed different sets of single-digit micromolar inhibitors of MMP-2, with up to a ten-fold increase in inhibitory activity and ameliorated selectivity towards off-target MMP-8, compared to selected lead compound. Molecular dynamics calculations conducted on complexes of MMP-2 with docked privileged structures confirmed that analyzed inhibitors avoid targeting the zinc ion and dip inside the S1' pocket. Present results provide a further enrichment of our insights for the design of novel MMP-2 selective inhibitors.

Novel Cyclic Biphalin Analogues by Ruthenium-Catalyzed Ring Closing Metathesis: in Vivo and in Vitro Biological Profile.

In this work we report the application of the ring-closing metathesis (RCM) to the preparation of two cyclic olefin-bridged analogues of biphalin (Tyr-d-Ala-Gly-Phe-NH-NH ← Phe ← Gly ← d-Ala ← Tyr), using the second generation Grubbs' catalyst. The resulting cis- and trans-cyclic isomers were identified, fully characterized, and tested in vitro at µ (ΜΟR), δ (DOR), and κ (KOR) opioid receptors and in vivo for antinociceptive activity. Both were shown to be full agonists at MOR and potential partial antagonists at DOR, with low potency KOR agonism. They also share a strong antinociceptive effect after intracerebroventricular (i.c.v.) and intravenous (i.v.) administration, higher than that of the cyclic biphalin analogues containing a disulfide bridge between the side chains of two d-Cys or d-Pen residues, previously described by our group.

Biochemical and pharmacological investigation of novel nociceptin/OFQ analogues and N/OFQ-RYYRIK hybrid peptides.

The endogenous ligand nociceptin (N/OFQ) and a positively charged synthetic peptide RYYRIK are both selective for the nociceptin opioid receptor (NOPr). Despite their structural dissimilarity, N/OFQ and RYYRIK compete for the same binding site of NOP receptor possessing full and partial agonistic character, respectively. In the view of the message-address concept, hybrid peptide constructs were probed for the NOP receptor combining different regions of N/OFQ and RYYRIK related peptide sequences. Nine novel nociceptin- or Ac-RYYRIK-NH2 peptide variants or hybrid peptides were synthesized and characterized. Peptides P2 and P8 contain fragments of native N/OFQ. The other seven analogues (P1, P3-7, P9) are composed of Ac-RYYRIK-NH2 fragments and parts of the original nociceptin sequence. The analogues were characterized in receptor binding assays and G-protein activation experiments on rat brain membranes, as well as by electrically stimulated mouse vas deferens bioassay. In receptor binding assays ligands P2, P4, P6 (Ki 0.37 nM) and P7 showed higher affinity (Ki 0.65 nM, 0.6 nM, 0.37 nM and 0.44 nM, respectively) for NOP receptor than their parent compounds N/OFQ (Ki 2.8 nM) or Ac-RYYRIK-NH2 (Ki 4.2 nM). In [35S]GTPγS binding experiments P2 and P3 behaved as full agonists. The other variants exhibited partial agonist properties characterized by submaximal stimulatory effects. In mouse vas deferens bioassay only P2 showed agonist activity. P4, P5, P6 inhibited the biological activity of N/OFQ more effectively than the NOP receptor selective antagonist JTC-801. In summary, hybrid peptides P4, P5 and P6 proved to be NOP receptor partial agonists even antagonists, while P2 peptide retained the full agonist property.

Discovery of arginine-containing tripeptides as a new class of pancreatic lipase inhibitors.

AIM: The inhibition of pancreatic lipase (PL) represents one of the most promising strategies in the search for novel antiobesity drugs. We propose here a pioneering course by exploring tripeptide scaffolds in the way to selective PL inhibitors. METHODOLOGY/RESULTS: The peptide series exhibited good PL inhibitory properties in vitro, with all the strongest inhibitors sharing a central arginine, shown in silico to be relevant for the active site-directed activity. The compounds were found devoid of inhibitory properties on acetylcholinesterase. CONCLUSION: Present results disclosed that basic tripeptides are able to interact efficiently with the PL-binding pocket, where they adopt a binding pose suitable for functional-to-inhibition interactions with key amino acids. Main inhibitor MALA4 may be selected as lead for further optimization.

Anti-Oxidant and Tyrosinase Inhibitory In Vitro Activity of Amino Acids and Small Peptides: New Hints for the Multifaceted Treatment of Neurologic and Metabolic Disfunctions.

Oxidative damage is among the factors associated with the onset of chronic pathologies, such as neurodegenerative and metabolic diseases. Several classes of anti-oxidant compounds have been suggested as having a protective role against cellular stressors, but, in this perspective, peptides' world represents a poorly explored source. In the present study, the free radical scavenging properties, the metal ion reducing power, and the metal chelating activity of a series of sulfurated amino acids and tripeptides were determined in vitro through canonical assays (DPPH, ABTS, CUPRAC, FRAP, PM, and EECC) and estimated in comparison with the corresponding activities of synthetic peptide semicarbazones, incorporating the peculiar non-proteinogenic amino acid, tert-leucine (tLeu). The compounds exhibited remarkable anti-oxidant properties. As expected, sulfurated compounds 1⁻5 were found to be the most efficient radical scavengers and strongest reductants. Nevertheless, tLeu-containing peptides 7 and 8 disclosed notable metal reducing and chelating activities. These unprecedented results indicate that tLeu-featuring di- and tripeptide backbones, bearing the semicarbazone chelating moiety, are compatible with the emergence of an anti-oxidant potential. Additionally, when tested against a panel of enzymes usually targeted for therapeutic purposes in neurodegenerative and metabolic disorders, all samples were found to be good inhibitors of tyrosinase.

Activation of ß- and γ-carbonic anhydrases from pathogenic bacteria with tripeptides.

Six tripeptides incorporating acidic amino acid residues were prepared for investigation as activators of ß- and γ-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic bacteria Vibrio cholerae, Mycobacterium tuberculosis, and Burkholderia pseudomallei. The primary amino acid residues that are involved in the catalytic mechanisms of these CA classes are poorly understood, although glutamic acid residues near the active site appear to be involved. The tripeptides that contain Glu or Asp residues can effectively activate VchCAß and VchCAγ (enzymes from V. cholerae), Rv3273 CA (mtCA3, a ß-CA from M. tuberculosis) and BpsCAγ (γ-CA from B. pseudomallei) at 0.21-18.1 µM levels. The position of the acidic residues in the peptide sequences can significantly affect bioactivity. For three of the enzymes, tripeptides were identified that are more effective activators than both l-Glu and l-Asp. The tripeptides are also relatively selective because they do not activate prototypical α-CAs (human carbonic anhydrases I and II). Because the role of CA activators in the pathogenicity and life cycles of these infectious bacteria are poorly understood, this study provides new molecular probes to explore such processes.

Dual targeting of cancer-related human matrix metalloproteinases and carbonic anhydrases by chiral N-(biarylsulfonyl)-phosphonic acids.

A series of nanomolar phosphonate matrix metalloproteinase (MPP) inhibitors was tested for inhibitory activity against a panel of selected human carbonic anhydrase (CA, EC 4.2.1.1) isozymes, covering the cancer-associated CA IX and XII. None of the reported sulfonyl and sulfonylamino-derivatives sensitively affected the catalytic activity of the cytosolic isoforms CA I and II, which are considered off-target isoforms in view of their physiological role. The most active inhibitors were in the series of chiral N-(sulfonyl)phosphovaline derivatives, which showed good to excellent inhibitory activity over target CAs, with compound 15 presenting the best isoform-selectivity toward CA IX. We suggest here that the phosphonates have the potential as dual inhibitors of MMPs and CAs, both involved in tumor formation, invasion and metastasis.

Fluorescent-labeled bioconjugates of the opioid peptides biphalin and DPDPE incorporating fluorescein-maleimide linkers.

AIM: The conjugation of fluorescent labels to opioid peptides is an extremely challenging task, which needs to be overcome to create new classes of probes for biological assays. MATERIALS & METHODS: Three opioid peptide analogs of biphalin and [D-Pen2,5]-Enkephalin (DPDPE) containing a fluorescein-maleimide motif were synthesized. RESULTS & DISCUSSION: The biphalin analog 17 binds to opioid receptors with Kiµ = 530 ± 90 nM and Kiδ = 69.8 ± 16.4 nM. We then tested the ability of the compounds to stimulate G-protein-coupling, 17 activated µ-receptor expressing cells (EC50 = 16.7 ± 6.7 nM, EMax = 76 ± 4%) as well as δ-receptor expressing cells (EC50 = 42 ± 10 nM, EMax = 34 ± 8%). However, 17 was not able to fluorescently label receptor in live or fixed cells. CONCLUSION: Our data suggest that the biphalin scaffold could be employed to develop fluorescent ligands with the appropriate fluorescent motif, and suggest a means for further probe development.

Nitrobenzoxadiazole-based GSTP1-1 inhibitors containing the full peptidyl moiety of (pseudo)glutathione.

CONTEXT: The inhibition of glutathione S-transferase P1-1 (GSTP1-1) is a sound strategy to overcome drug resistance in oncology practice. OBJECTIVE: The nitrobenzoxadiazolyl (NBD) S-conjugate of glutathione and the corresponding γ-oxa-glutamyl isostere (compounds 1 and 5, respectively) have been disclosed as GST inhibitors. The rationale of their design is discussed in juxtaposition to non-peptide NBD thioethers. MATERIALS AND METHODS: Synthesis of derivatives 1 and 5 and in vitro evaluation on human GSTP1-1 and M2-2 are reported. RESULTS: Conjugates 1 and 5 were found to be low micromolar inhibitors of both isoforms. Furthermore, they display a threefold reduction in selectivity for GSTM2-2 over the P1-1 isozyme in comparison with the potent non-peptide inhibitor nitrobenzoxadiazolyl-thiohexanol (NBDHEX). DISCUSSION AND CONCLUSIONS: Spectroscopic data are congruent with the formation of a stable sigma-complex between GSH and the inhibitors in the protein active site. Conjugate 5 is suitable for in vivo modulation of GST activity in cancer treatment.

Bioactive compounds of Crocus sativus L. and their semi-synthetic derivatives as promising anti-Helicobacter pylori, anti-malarial and anti-leishmanial agents.

Crocus sativus L. is known in herbal medicine for the various pharmacological effects of its components, but no data are found in literature about its biological properties toward Helicobacter pylori, Plasmodium spp. and Leishmania spp. In this work, the potential anti-bacterial and anti-parasitic effects of crocin and safranal, two important bioactive components in C. sativus, were explored, and also some semi-synthetic derivatives of safranal were tested in order to establish which modifications in the chemical structure could improve the biological activity. According to our promising results, we virtually screened our compounds by means of molecular modeling studies against the main H. pylori enzymes in order to unravel their putative mechanism of action.

Ac-tLeu-Asp-H is the minimal and highly effective human caspase-3 inhibitor: biological and in silico studies.

Caspase-3 displays a pivotal role as an executioner of apoptosis, hydrolyzing several proteins including the nuclear enzyme poly(ADP-ribose)polymerase (PARP). Ac-Asp-Glu-Val-Asp-H (K i° = 2.3 × 10(-10) M at pH 7.5 and 25.0 °C), designed on the basis of the cleavage site of PARP, has been reported as a highly specific human caspase-3 inhibitor. Here, di- and tri-peptidyl aldehydes 11-13 and 27-29 have been synthesized to overcome the susceptibility to proteolysis, the intrinsic instability, and the scarce membrane permeability of the current inhibitors. Compounds 11-13, 27-29 inhibit in vitro human caspase-3 competitively, values of K i° ranging between 6.5 (±0.82) × 10(-9) M and 1.1 (±0.04) × 10(-7) M (at pH 7.4 and 25.0 °C). Moreover, the most effective caspase-3 inhibitor 11 impairs apoptosis in human DLD-1 colon adenocarcinoma cells. Furthermore, the binding mode of 11-13 and 27-29 to human caspase-3 has been investigated in silico. The comparative analysis of human caspase-3 inhibitors indicates that (1) aldehyde 11 is the minimal highly effective inhibitor, (2) the tLeu-Asp sequence is pivotal for satisfactory enzyme inhibition, and (3) the occurrence of the tLeu residue at the inhibitor P2 position is fundamental for enzyme/inhibitor recognition. Moreover, calculations suggest that the tLeu residue reduces the conformational flexibility of the inhibitor that binds to the enzyme with a lower energetic penalty.

Synthesis and neuromodulatory effects of TRH-related peptides: inhibitory activity on catecholamine release in vitro.

BACKGROUND: A detailed comprehension of central mechanisms underlying feeding behavior holds considerable promise for the treatment of alimentary disorders. METHODS: In order to elucidate the tight interrelationships occurring at the hypothalamic neuronal endings between aminergic neurotransmitters and co-localized appetite modulators, we initially studied the effects of two anorexigenic peptides structurally related to thyrotropin-releasing hormone (TRH, 1), namely cyclo(His-Pro) (CHP, 2) and pGlu-His-Gly-OH (3), on [(3)H]-norepinephrine and [(3)H]-dopamine release from perfused rat hypothalamic synaptosomes. Furthermore, a number of TRH and CHP analogues were synthesized and tested for their ability to influence neurotransmitter release in the selected neuronal model. RESULTS: Peptide 3 showed only a slight inhibitory activity on norepinephrine release, whereas no effect was observed for compound 2. TRH analogue 8, metabolically stabilized by the replacement of pyroglutamate with the pyrohomocysteic acid (pHcs), was found to be inactive. Conversely, a significant inhibitory effect on dopamine and norepinephrine release was observed for the CHP-related diketopiperazines cyclo(Leu-Pro) (11) and cyclo(His-Gly) (14). CONCLUSIONS: These results suggest a potential role for cyclo-dipeptides 11 and 14 in the hypothalamic modulation of appetite suppressant circuitry.

Hybrid peptides endomorphin-2/DAMGO: design, synthesis and biological evaluation.

Endomorphin-2 [Tyr-Pro-Phe-Phe-NH2] and DAMGO [Tyr-D-Ala-Gly-(N-Me)Phe-Gly-ol] are natural (EM2) and synthetic (DAMGO) opioid peptides both selective for µ opioid receptor with high analgesic activity. In this work we report synthesis, in vitro and in vivo biological evaluation of five new hybrid EM2/DAMGO analogues, with the aim to obtain compounds with high affinity at µ-opioid receptor, high activity in animal nociception tests (hot plate and tail flick) and improved enzymatic stability. Double N-methylation on both Phe residues and C-terminal ethanolamide led to analogue 6e, which possesses a good in vitro µ affinity (Kiµ=34 nM), combined with a remarkable in vivo antinociceptive activity.

Human caspase-3 inhibition by Z-tLeu-Asp-H: tLeu(P2) counterbalances Asp(P4) and Glu(P3) specific inhibitor truncation.

Caspase-3 is responsible for the cleavage of several proteins including the nuclear enzyme poly(ADP-ribose) polymerase (PARP). Designed on the cleavage site of PARP, Ac-Asp-Glu-Val-Asp-H has been reported as a highly specific inhibitor. To overcome the susceptibility to proteolysis, the intrinsic instability, and the scarce membrane permeability of tetra-peptidyl aldehydes, di- and tri-peptidyl caspase-3 inhibitors have been synthesized. Here, the synthesis and the inhibition properties of peptidyl aldehydes Z-tLeu-Asp-H, Z-tLeu-Val-Asp-H, and Z-Val-tLeu-Asp-H are reported. Z-tLeu-Asp-H, Z-tLeu-Val-Asp-H, and Z-Val-tLeu-Asp-H inhibit competitively human caspase-3 activity in vitro with K(i)(0)=3.6nM, 18.2nM, and 109nM, respectively (pH 7.4 and 25 degrees C). Moreover, Z-tLeu-Asp-H impairs apoptosis in human DLD-1 colon adenocarcinoma cells without affecting caspase-8. Therefore, Ac-Asp-Glu-Val-Asp-H can be truncated to Z-tLeu-Asp-H retaining nanomolar inhibitory activity in vitro and displaying action in whole cells, these properties reflect the unprecedented introduction of the bulky and lipophilic tLeu residue at the P(2) position.

Transition state isosteres of the gamma-glutamyl peptide bond hydrolysis: synthesis and characterization of the psi (CH2NH) pseudopeptide analogue of glutathione.

The fully deprotected glutathione analogue containing the aminomethylene unit as transition state isostere of the gamma-Glu-Cys peptide bond was synthesized for the first time and characterized in both the reduced and oxidized forms.

Synthesis and preliminary pharmacological evaluation of trans-2-amino-5(6)-chloro-6(5)-hydroxy-1-phenyl-2,3-dihydro-1H-indenes as dopamine receptor ligands.

A series of trans-2-amino-5(6)-chloro-6(5)-hydroxy-1-phenyl-2,3-dihydro-1H-indenes were synthesized and evaluated for their binding affinity toward D1-like and D2-like dopamine (DA) receptors. The affinity and selectivity of these compounds were measured in a test involving displacement of [3H]SCH 23390 or [3H]YM-09-151-2, respectively, from homogenates of porcine striatal membranes. All tested compounds were poorly effective at DA receptors (Ki nM > 1000). The results suggest that introduction of chlorine substituent in five or six position of previously synthesized trans-2-amino-6(5)-hydroxy-1-phenyl-2,3-dihydro-1H-indenes decreases both D1-like and D2-like receptor affinity.