Analgesic Properties of Opioid/NK1 Multitarget Ligands with Distinct in Vitro Profiles in Naive and Chronic Constriction Injury Mice.

The lower efficacy of opioids in neuropathic pain may be due to the increased activity of pronociceptive systems such as substance P. We present evidence to support this hypothesis in this work from the spinal cord in a neuropathic pain model in mice. Biochemical analysis confirmed the elevated mRNA and protein level of pronociceptive substance P, the major endogenous ligand of the neurokinin-1 (NK1) receptor, in the lumbar spinal cord of chronic constriction injury (CCI)-mice. To improve opioid efficacy in neuropathic pain, novel compounds containing opioid agonist and neurokinin 1 (NK1) receptor antagonist pharmacophores were designed. Structure-activity studies were performed on opioid agonist/NK1 receptor antagonist hybrid peptides by modification of the C-terminal amide substituents. All compounds were evaluated for their affinity and in vitro activity at the mu opioid (MOP) and delta opioid (DOP) receptors, and for their affinity and antagonist activity at the NK1 receptor. On the basis of their in vitro profiles, the analgesic properties of two new bifunctional hybrids were evaluated in naive and CCI-mice, representing models for acute and neuropathic pain, respectively. The compounds were administered to the spinal cord by lumbar puncture. In naive mice, the single pharmacophore opioid parent compounds provided better analgesic results, as compared to the hybrids (max 70% MPE), raising the acute pain threshold close to 100% MPE. On the other hand, the opioid parents gave poor analgesic effects under neuropathic pain conditions, while the best hybrid delivered robust (close to 100% MPE) and long lasting alleviation of both tactile and thermal hypersensitivity. The results presented emphasize the potential of opioid/NK1 hybrids in view of analgesia under nerve injury conditions.

Exploring the biological consequences of conformational changes in aspartame models containing constrained analogues of phenylalanine.

The dipeptide aspartame (Asp-Phe-OMe) is a sweetener widely used in replacement of sucrose by food industry. 2',6'-Dimethyltyrosine (DMT) and 2',6'-dimethylphenylalanine (DMP) are two synthetic phenylalanine-constrained analogues, with a limited freedom in χ-space due to the presence of methyl groups in position 2',6' of the aromatic ring. These residues have shown to increase the activity of opioid peptides, such as endomorphins improving the binding to the opioid receptors. In this work, DMT and DMP have been synthesized following a diketopiperazine-mediated route and the corresponding aspartame derivatives (Asp-DMT-OMe and Asp-DMP-OMe) have been evaluated in vivo and in silico for their activity as synthetic sweeteners.

Exploring new Probenecid-based carbonic anhydrase inhibitors: Synthesis, biological evaluation and docking studies.

Novel Probenecid-based amide derivatives, incorporating different natural amino acids, were synthesized and assayed to test their effect on the human carbonic anhydrase (hCA, EC 4.2.1.1) transmembrane isoforms hCA IX and XII over the ubiquitous isoforms hCA I and II. Most of them presented a complete loss of hCA II inhibition (K(i)s > 10,000 nM) and strong inhibitory activity against hCA IX and XII in the nanomolar range with respect to the parent compound. A residual activity against hCA I was observed for some of them. These biological results have been explained by docking studies within the active sites of the four studied human carbonic anhydrases (with or without the zinc-bound water) and helped us to better comprehend the rationale behind the design of tertiary sulfonamide compounds as potent but atypical inhibitors of specific isoforms of human carbonic anhydrase.

New Insight on the Synthesis of Neurotoxins Domoic Acid and Kainic Acid.

Mono or di-substituted prolines, namely proline chimeras of natural or synthetic origin, carry the side chain of other specific amino acids on the pyrrolidine ring. Thus, proline chimeras are useful tools for a wide range of chemical and biological applications as chiral synthons or building blocks for peptidomimetic design. We focused our attention on domoic acid and kainic acid and we report here a concise and up to date review on their stereoselective and asymmetric syntheses.

Cyclotides: a natural combinatorial peptide library or a bioactive sequence player?

In this perspective review, we focalized our attention on the application of cyclotides in drug discovery. To date, two principal approaches have been explored since now: (i) the use of cyclotides as scaffold in which bioactive peptides can be grafted to improve stability, oral bioactivity and binding to GPCRs; (ii) their application as natural peptides library. For these reasons, cyclotides probably represent today a step further in the development of new tools in drug design.

Design, Synthesis and Biological Evaluation of Two Opioid Agonist and Cav 2.2 Blocker Multitarget Ligands.

N-type voltage-dependent Ca(2+) channels (CaV 2.2) are located at nerve endings in the central and peripheral nervous systems and are strongly associated with the pathological processes of cerebral ischaemia and neuropathic pain. CaV 2.2 blockers such as the ω-conotoxin MVIIA (Prialt) are analgesic and have opioid-sparing effects. With the aim to develop new multitarget analgesic compounds, we designed the first ω-conotoxin/opioid peptidomimetics based on the enkephalin-like sequence Tyr-D-Ala-Gly-Phe (for the opioid portion) and two fragments derived from the loop-2 pharmacophore of ω-conotoxin MVIIA. Antinociceptive activity evaluated in vitro and in vivo revealed differential affinity for CaV 2.2 and opioid receptors and no significant synergistic activity.

DPP-4 inhibitors: a patent review (2012 - 2014).

INTRODUCTION: Dipeptidyl peptidase 4 (DPP-4) is a serine protease, which catalyzes the hydrolytic process of the amide bond X-Ala or X-Pro at the N-terminus of peptides. This enzyme is involved in the degradation of two incretin hormones glucagon-like peptide-1 and glucose-dependent-insulinotropic polypeptide, which increase the production and release of insulin. Therefore, DPP-4 inhibitors are considered as one of the well-established therapies available for type 2 diabetes mellitus. AREAS COVERED: In this review, the authors report all the patents related to DPP-4 inhibitors from 2012 to 2014. The chemical-related patents have been divided in three general families: i) peptidomimetics; ii) peptides; and iii) non-peptidomimetics compounds. The third group is the most consistent; thus the patents have been further divided on the basis of the chemical structure. In the last section, we briefly discuss the patents containing the formulations or associations of DPP-4 inhibitors with other drugs. EXPERT OPINION: In the last few years, the research on DPP-4 inhibitors has significantly grown leading to the development of heterocyclic scaffolds and non-peptidomimetic structures. Unfortunately, these compounds are not immune from side effects associated with the inhibition of other substrates, indicating that DPP-4 inhibitors are likely multi-target drugs. Therefore, their potential multi-target effects require more attention in clinical practice, even if at this moment, all the patents are focused only on diabetes.

Novel cyclic biphalin analogue with improved antinociceptive properties.

Two novel opioid analogues have been designed by substituting the native d-Ala residues in position 2,2' of biphalin with two residues of d-penicillamine or l-penicillamine and by forming a disulfide bond between the thiol groups. The so-obtained compound 9 containing d-penicillamines showed excellent µ/δ mixed receptor affinities (K i (δ) = 5.2 nM; K i (µ) = 1.9 nM), together with an efficacious capacity to trigger the second messenger and a very good in vivo antinociceptive activity, whereas product 10 was scarcely active. An explanation of the two different pharmacological behaviors of products 9 and 10 was found by studying their conformational properties.

Synthesis and anti-cancer activity of naturally occurring 2,5-diketopiperazines.

Three naturally occurring oxyprenylated diketopiperazines were synthesized and preliminarily tested as growth inhibitory agents in vitro against various cancer cell lines. The compounds were tested on six human cancer cell lines with different sensitivity to proapoptotic stimuli using the MTT colorimetric assay. The data revealed that of the chemicals under study only deoxymicelianamide (11) displayed the highest activity, recording mean IC50 growth inhibitory values ranging from 2 to 23 µM. A comparative study with the non-geranylated saturated derivative of (11) revealed the importance of the presence of the geranyloxy side chain and the exocyclic 2,5-DPK double bond moiety for the observed activity.

Potent biphalin analogs with µ/δ mixed opioid activity: in vivo and in vitro biological evaluation.

Biphalin [(Tyr-D-Ala-Gly-Phe-NH-)2 ] is an octapeptide with mixed µ/δ opioid activity. Its structure is based on two identical enkephalin-like portions linked "tail-to-tail" by a hydrazine bridge. This study presents the synthesis and in vitro and in vivo bioassays of two biphalin analogs that do not present the toxicity connected with the presence of the hydrazine moiety and are able to elicit a higher antinociceptive effect than biphalin.

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.

Biological active analogues of the opioid peptide biphalin: mixed α/ß(3)-peptides.

Natural residues of the dimeric opioid peptide Biphalin were replaced by the corresponding homo-ß(3) amino acids. The derivative 1 containing hß(3) Phe in place of Phe showed good µ- and δ-receptor affinities (Ki(δ) = 0.72 nM; Ki(µ) = 1.1 nM) and antinociceptive activity in vivo together with an increased enzymatic stability in human plasma.

Strategies for developing tuberculosis vaccines: emerging approaches.

The current vaccine against TB, bacille Calmette-Guèrin (BCG) fails to protect against the most prevalent disease form, the pulmonary TB in adults. Thus, it is not a satisfactory vaccine. Given that T cells are central to protection against TB, future vaccine design should focus on T-lymphocyte populations. Most vaccines do not prevent infection but instead disease, that if they allow establishment of the pathogen in the host but prevent its harmful effects. The development of synthetic peptide-based immunogens is emerging as a possible approach in human vaccination in the future, as a replacement for conventional vaccines that use killed or attenuated whole microorganisms. The advantages of such synthetic vaccines (high potency, low adverse reactions, low cross-reactivity and high stability) are offset somewhat by the poorer inherent immunogenicity of these constructs. There is a greater need therefore to develop adjuvant/carrier systems to increase the immunogenicity of these newer vaccine candidates.

Antinociceptive profile of potent opioid peptide AM94, a fluorinated analogue of biphalin with non-hydrazine linker.

AM94 is a fluorinated analog of biphalin with non-hydrazine linker that has an in vitro affinity for µ-opioid and δ-opioid receptors tenfold higher than biphalin. Furthermore, in vivo evaluation in rats showed that AM94 has in hot plate test - after both intracerebroventricular and intravenous administrations - a greater and more durable efficacy than biphalin. Here, the antinociceptive profile of AM94 is further evaluated by following two different administration routes, intrathecal and subcutaneous, and two different animal species, rats and mice. The analgesic potency of AM94 is compared with that of both the parent peptide biphalin and morphine. Results show that in rats (tail flick test) and in mice (formalin test), AM94 has a higher and more durable analgesic effect than biphalin after intrathecal and subcutaneous administrations. Conformational properties of biphalin and AM94 were also investigated by variable-temperature (1)H NMR and energy minimization.

Structure-activity relationships of biphalin analogs and their biological evaluation on opioid receptors.

Biphalin (Tyr-D-Ala-Gly-Phe-NH-NH < -Phe < -Gly < -D-Ala < -Tyr) is an opioid octapeptide with a dimeric structure based on two identical pharmacophore portions, derived from enkephalins, joined "tail to tail" by a hydrazide bridge. This particular structure enhances the antinociceptive activity of the native enkephalins with an unknown mechanism, probably based on a cooperative binding and improved enzymatic stability. Biphalin has excellent binding affinity for µ and δ receptors and it is a highly potent analgesic, as potent as or more than ethorphine. A definitive explanation of the extraordinary in vivo potency shown by this compound, which has pronounced efficacy in pain modulation, is still not available; it has been suggested, however, that the high agonist activity may be related to its binding mode at both µ and δ opioid receptors. Biphalin has significantly higher potency than other analgesics with novel biological profiles; in particular, most recent data show that biphalin is unlikely to produce dependency in chronic use. In the past 20 years, there have been many attempts to modify its structure to obtain products unaffected by the action of enkephalinases, to enhance its antinociceptive activity and to modify the BBB penetration. In addition, structure-activity relationship studies (SAR) were performed in order to understand the elements responsible for biphalin's high activity. The aim of the studies reported in this review was to clarify: i) the role of the hydrazide bridge, ii) the role of residues in position 4, 4' and 3, 3', iii) the consequences of molecular simplifications (truncation, delection), iv) the consequences of cyclization through a disulfide bridge, v) conjugation with PEG and fluorescet residues, and vi) radiolabeling on Tyr.

Role of formyl peptide receptors (FPR) in abnormal inflammation responses involved in neurodegenerative diseases.

Neurodegenerative disorders, such as multiple sclerosis, prion diseases, Alzheimer's disease and Parkinson's disease are often associated with inflammatory process, which involves various components of the immune system in the central nervous system, in particular astrocytes and microglial cells. Inflammation mediators such as cytokines, leukotrienes, superoxide radicals, eicosanoids, the complement cascade, and FPR agonists (formyl peptides) may play a significant role in pro-inflammatory responses, in which infiltration of activated mononuclear phagocytes at the sites of lesion is a common feature. To prevent long-term inflammation damage, the central nervous system could be treated with antinflammatory agents such as non-steroidal anti-inflammatory drugs (NSAIDs), but only few drugs were found to be effective and their therapeutic benefits is limited by side effects. Accumulating evidences suggest that targeting glia-neuron system might be a therapeutic approach in the treatment of neurodegenerative disease progression, in particular of Alzheimer's disease. Aminopyridazine derivative discovered in unbiased cell-based screens for new synthetic compounds, have proved to be able to suppress selective glial activation responses via mechanisms distinct from NSAIDs. In this review, we report the potential involvement of FPR receptors in inflammatory responses and the potential use of their antagonists to modulate the inflammatory responses of the microglia. Recent results demonstrate that targeting of inflammatory glia cytokine pathways, can suppress Aß-induced neuroinflammation in vivo, resulting in the attenuation of neuronal damage.

Synthesis and biological evaluation of new active For-Met-Leu-Phe-OMe analogues containing para-substituted Phe residues.

In the present study, we report synthesis and biological evaluation of the N-Boc-protected tripeptides 4a-l and N-For protected tripeptides 5a-l as new For-Met-Leu-Phe-OMe (fMLF-OMe) analogues. All the new ligands are characterized by the C-terminal Phe residue variously substituted at position 4 of the aromatic ring. The agonism of 5a-l and the antagonism of 4a-l (chemotaxis, superoxide anion production, lysozyme release as well as receptor binding affinity) have been examined on human neutrophils. No synthesized compounds has higher activity than the standard fMLF-OMe tripeptide to stimulate chemotaxis, although compounds 5a and 5c with -CH(3) and -C(CH(3))(3), respectively, in position 4 on the aromatic ring, are better than the standard tripeptide to stimulate the production of superoxide anion, in higher concentration. Compounds 4f and 4i, containing -F and -I in position 4, respectively, on the aromatic ring of phenylalanine, exhibit significant chemotactic antagonism. The influence of the different substitution at the position 4 on the aromatic ring of phenylalanine is discussed.