Martina Gobec

Synthesis of conformationally constrained γ-D-glutamyl-meso-diaminopimelic acid derivatives as ligands of nucleotide-binding oligomerization domain protein 1 (Nod1).

Nod1, an important member of the pattern recognition receptor family, remains a virtually unexploited target. Harnessing its innate immune stimulatory properties still remains an unfulfilled goal of medicinal chemistry. Nucleotide-binding oligomerization domain protein 1 (Nod1) agonists have been shown to boost the inflammatory responses against pathogenic microbes and could thus constitute a new class of broad spectrum antimicrobial agents. To gain additional insight into the structure/activity relationships of Nod1 agonistic compounds, a series of novel, conformationally constrained γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) analogs have been designed and synthesized. Ramos-Blue cells expressing Nod1 were used to screen and validate our compounds for their Nod1-agonist activity. Their immunomodulatory properties were subsequently determined in vitro, by evaluating their capacity to induce pro-inflammatory cytokine and chemokine production from human peripheral blood mononuclear cells (PBMC), by themselves and in synergy with lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) ligand. The synthesized iE-DAP analogs were shown to possess immuno-enhancing properties as a result of their potent and specific Nod1-agonistic effect. The activity of the compound exhibiting the greatest capacity to induce pro-inflammatory cytokine release from PBMC surpassed that of lauroyl-γ-D-glutamyl-meso-diaminopimelic acid (C12-iE-DAP).

Nonpeptidic Selective Inhibitors of the Chymotrypsin‐Like (β5 i) Subunit of the Immunoproteasome

Elevated expression of the immunoproteasome has been associated with autoimmune diseases, inflammatory diseases, and various types of cancer. Selective inhibitors of the immunoproteasome are not only scarce, but also almost entirely restricted to peptide-based compounds. Herein, we describe nonpeptidic reversible inhibitors that selectively block the chymotrypsin-like (β5i) subunit of the human immunoproteasome in the low micromolar range. The most potent of the reversibly acting compounds were then converted into covalent, irreversible, nonpeptidic inhibitors that retained selectivity for the β5i subunit. In addition, these inhibitors discriminate between the immunoproteasome and the constitutive proteasome in cell-based assays. Along with their lack of cytotoxicity, these data point to these nonpeptidic compounds being suitable for further investigation as β5i-selective probes for possible application in noncancer diseases related to the immunoproteasome.

Development of an in-vivo active reversible butyrylcholinesterase inhibitor.

Alzheimer’s disease (AD) is characterized by severe basal forebrain cholinergic deficit, which results in progressive and chronic deterioration of memory and cognitive functions. Similar to acetylcholinesterase, butyrylcholinesterase (BChE) contributes to the termination of cholinergic neurotransmission. Its enzymatic activity increases with the disease progression, thus classifying BChE as a viable therapeutic target in advanced AD. Potent, selective and reversible human BChE inhibitors were developed. The solved crystal structure of human BChE in complex with the most potent inhibitor reveals its binding mode and provides the molecular basis of its low nanomolar potency. Additionally, this compound is noncytotoxic and has neuroprotective properties. Furthermore, this inhibitor moderately crosses the blood-brain barrier and improves memory, cognitive functions and learning abilities of mice in a model of the cholinergic deficit that characterizes AD, without producing acute cholinergic adverse effects. Our study provides an advanced lead compound for developing drugs for alleviating symptoms caused by cholinergic hypofunction in advanced AD.

Antioxidant and anti-inflammatory properties of 1,2,4-oxadiazole analogs of resveratrol

The chemopreventive properties of resveratrol are ascribed mostly to its antioxidant activity, in particular its scavenging ability for reactive oxygen species (ROS), and to the inhibition of NF-κB pathway which has also been suggested as an important underlying mechanism of its reported properties. In present study, a small library of nine 1,2,4-oxadiazole-based structural analogs of resveratrol were assayed for their antioxidant and anti-inflammatory activities. Several compounds showed significant inhibitory activities against NF-κB and/or ROS production. Compound 2, incorporating two para-hydroxyphenyl moieties connected by the 1,2,4-oxadiazole ring, was the most active, its potency in inhibiting activation of NF-κB and ROS scavenging abilities surpassing that of resveratrol. Additionally, we elucidated the mechanisms underlying the NF-κB inhibitory activity of compound 2. Finally, in contrast to resveratrol, compound 2 significantly reduced the LPS-induced release of pro-inflammatory cytokines, indicating its prominent anti-inflammatory potential.

Design, synthesis and biological evaluation of novel desmuramyldipeptide analogs

A series of novel desmuramyldipeptides have been designed and synthesized as part of our search for therapeutically useful muramyldipeptide (MDP) analogs. Their immunomodulatory properties were initially assessed in vitro, evaluating their effect on lipopolysaccharide (LPS)-induced cytokine release in THP-1 cells. Following the initial screening, selected compounds were further investigated for immunomodulatory properties using LPS and phorbol 12-myristate 13-acetate (PMA)/ionomycin-stimulated human peripheral blood mononuclear cells. The results confirmed the immunomodulatory properties of some of the synthesized desmuramyldipeptide analogs. Taken together, presented data confirmed the immunostimulatory effect of compound 44, MDP derivative incorporating a pyrido-fused [1,2]-benzisothiazole moiety, while for compounds 32 and 39, indole scaffold-based derivatives of MDP, an immunosuppressive effect was observed. Further studies will be necessary to address their potential therapeutic use as immunomodulatory drugs, both as immunostimulants or anti-inflammatory agents.

Structure-Activity Relationships of Novel Tryptamine-Based Inhibitors of Bacterial Transglycosylase.

Penicillin-binding proteins represent well-established, validated, and still very promising targets for the design and development of new antibacterial agents. The transglycosylase domain of penicillin-binding proteins is especially important, as it catalyzes polymerization of glycan chains, using the peptidoglycan precursor lipid II as a substrate. On the basis of the previous discovery of a noncovalent small-molecule inhibitor of transglycosylase activity, we systematically explored the structure-activity relationships of these tryptamine-based inhibitors. The main aim was to reduce the nonspecific cytotoxic properties of the initial hit compound and concurrently to retain the mode of its inhibition. A focused library of tryptamine-based compounds was synthesized, characterized, and evaluated biochemically. The results presented here show the successful reduction of the nonspecific cytotoxicity, and the retention of the inhibition of transglycosylase enzymatic activity, as well as the ability of these compounds to bind to lipid II and to have antibacterial actions.

Characterization of human lymphoblastoid cell lines as a novel in vitro test system to predict the immunotoxicity of xenobiotics

Evaluating immunomodulatory effects of xenobiotics is an important component of the toxicity studies. Herein we report on the establishment of a novel invitro test system for the immunotoxicity screening of xenobiotics based on human lymphoblastoid cell lines (LCLs). Four immunotoxic compounds; tributyltin chloride, cyclosporine A, benzo(a)pyrene and verapamil hydrochloride, as well as three immune-inert compounds; urethane, furosemide and mannitol were selected for characterization. The treatment of LCLs with immunosuppressive compounds resulted in reduced viability. The IC50 values determined in human LCLs were in agreement with the data obtained for human peripheral mononuclear cells. Since cytokine production reflects lymphocytes responses to external stimuli, we evaluated the functional responses of LCLs by monitoring their pro-inflammatory and immunoregulatory cytokine production. Our findings prove that LCLs allowed for reliable differentiation between immunomodulatory and immune-inert compounds. Hence, pre-treatment with immunomodulatory compounds led to a decrease in the production of pro-inflammatory TNFα, IL-6 and immunoregulatory IL-2, IL-4, IL-10 and IFNγ cytokines, when compared to untreated ionomycin/PMA stimulated cells. Moreover, testing a panel of ten LCLs derived from unrelated healthy individuals reflects inter-individual variability in response to immunomodulatory xenobiotics. In conclusion, LCLs provide a novel alternative method for the testing of the immunotoxic effects of xenobiotics.

Antimicrobial activity and cytotoxicity of some 2-amino-5-alkylidene-thiazol-4-ones.

We report a small, focused library of 30 diverse 2-amino-5-alkylidene-thiazol-4-ones that was assayed in a whole-cell antibacterial screen against a panel of several bacterial strains and a yeast. Most of the compounds exhibited modest to significant antibacterial activity against Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus, and no activity against Salmonella typhimurium and Escherichia coli. The antibacterial activity depends markedly upon substituents on the thiazol-4-one core, and the most potent compound assayed (

Selective Cytotoxicity of Amidinopiperidine Based Compounds Towards Burkitt’s Lymphoma Cells Involves Proteasome Inhibition

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