From Batch to Continuous Flow Bioprocessing: Use of an Immobilized γ-Glutamyl Transferase from B. subtilis for the Synthesis of Biologically Active Peptide Derivatives.

γ-Glutamyl-peptides are frequently endowed with biological activities. In this work, "kokumi peptides" such as γ-glutamyl-methionine (1) and γ-glutamyl-(S)-allyl-cysteine (2), as well as the neuroprotective γ-glutamyl-taurine (3) and the antioxidant ophthalmic acid (4), were synthesized through an enzymatic transpeptidation reaction catalyzed by the γ-glutamyl transferase from Bacillus subtilis (BsGGT) using glutamine as the γ-glutamyl donor. BsGGT was covalently immobilized on glyoxyl-agarose resulting in high protein immobilization yield and activity recovery (>95%). Compounds 1-4 were obtained in moderate yields (19-40%, 5-10 g/L) with a variable purity depending on the presence of the main byproduct (γ-glutamyl-glutamine, 0-16%). To achieve process intensification and better control of side reactions, the synthesis of 2 was moved from batch to continuous flow. The specific productivity was 1.5 times higher than that in batch synthesis (13.7 µmol/min*g), but it was not accompanied by a paralleled improvement of the impurity profile.

Biocatalytic Approaches for an Efficient and Sustainable Preparation of Polyphenols and Their Derivatives.

Many sectors of industry, such as food, cosmetics, nutraceuticals, and pharmaceuticals, have increased their interest in polyphenols due to their beneficial properties. These molecules are widely found in Nature (plants) and can be obtained through direct extraction from vegetable matrices. Polyphenols introduced through the diet may be metabolized in the human body via different biotransformations leading to compounds having different bioactivities. In this context, enzyme-catalyzed reactions are the most suitable approach to produce modified polyphenols that not only can be studied for their bioactivity but also can be labeled as green, natural products. This review aims to give an overview of the potential of biocatalysis as a powerful tool for the modification of polyphenols to enhance their bioaccessibility, bioavailability, biological activity or modification of their physicochemical properties. The main polyphenol transformations occurring during their metabolism in the human body have been also presented.

The Inhibition of Cysteine Proteases Rhodesain and TbCatB: A Valuable Approach to Treat Human African Trypanosomiasis.

Human African Trypanosomiasis (HAT) is an endemic parasitic disease of sub-Saharan Africa, caused by two subspecies of protozoa belonging to Trypanosoma genus: T. brucei gambiense and T. brucei rhodesiense. In this context the inhibition of the papain-family cysteine proteases rhodesain and TbCatB has to be considered a promising strategy for HAT treatment. Rhodesain, the major cathepsin L-like cysteine protease of T. brucei rhodesiense, is a lysosomal protease essential for parasite survival. It is involved in parasite invasivity, allowing it to cross the blood-brain barrier (BBB) of the human host, causing the second lethal stage of the disease. Moreover, it plays an important role in immunoevasion, being involved in the turnover of variant surface glycoproteins of the T. brucei coat and in the degradation of immunoglobulins, avoiding a specific immune response by the host cells. On the other hand TbCatB, a cathepsin B-like cysteine protease, present in minor abundance in T. brucei, showed a key role in the degradation of host transferrin, which is necessary for iron acquisition by the parasite. In this review article we now discuss the most active peptide, peptidomimetic and non-peptide rhodesain and TbCatB inhibitors as valuable strategy to treat HAT, due also to the complementary role of the two T. brucei proteases.

Inspired by Nature: The 3-Halo-4,5-dihydroisoxazole Moiety as a Novel Molecular Warhead for the Design of Covalent Inhibitors.

Over the past few decades, there has been an increasing interest in the development of covalent enzyme inhibitors. As it was recently re-emphasized, the selective, covalent binding of a drug to the desired target can increase efficiency and lower the inhibitor concentration required to achieve a therapeutic effect. In this context, the naturally occurring antibiotic acivicin, and in particular its 3-chloro-4,5-dihydroisoxazole scaffold, has provided a wealth of inspiration to medicinal chemists and chemical biologists alike. In this Concept, to underline the great potentiality that the 3-halo-4,5-dihydroisoxazole warhead has in drug discovery, we present a number of examples, grouped by their potential biological activity and targets, in which this scaffold has been fruitfully used to develop novel biologically active compounds. Through these examples, we show that the 3-halo-4,5-dihydroisoxazole moiety represents an outstanding warhead with high potential for the design of novel covalent enzyme inhibitors.

Development of novel dipeptide-like rhodesain inhibitors containing the 3-bromoisoxazoline warhead in a constrained conformation.

Novel dipeptide-like rhodesain inhibitors containing the 3-bromoisoxazoline warhead in a constrained conformation were developed; some of them possess K(i) values in the micromolar range. We studied the structure-activity relationship of these derivatives and we performed docking studies, which allowed us to find out the key interactions established by the inhibitors with the target enzyme. Biological results indicate that the nature of the P2 and P3 substituents and their binding to the S2/S3 pockets is strictly interdependent.

Characterization of 2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl Ureido Based Inhibitors of Trypanosoma brucei FolD and Testing for Antiparasitic Activity.

The bifunctional enzyme N(5),N(10)-methylenetetrahydrofolate dehydrogenase/cyclo hydrolase (FolD) is essential for growth in Trypanosomatidae. We sought to develop inhibitors of Trypanosoma brucei FolD (TbFolD) as potential antiparasitic agents. Compound 2 was synthesized, and the molecular structure was unequivocally assigned through X-ray crystallography of the intermediate compound 3. Compound 2 showed an IC50 of 2.2 µM, against TbFolD and displayed antiparasitic activity against T. brucei (IC50 49 µM). Using compound 2, we were able to obtain the first X-ray structure of TbFolD in the presence of NADP(+) and the inhibitor, which then guided the rational design of a new series of potent TbFolD inhibitors.

Discovery of covalent inhibitors of glyceraldehyde-3-phosphate dehydrogenase, a target for the treatment of malaria.

We developed a new class of covalent inhibitors of Plasmodium falciparum glyceraldehyde-3-phosphate dehydrogenase, a validated target for the treatment of malaria, by screening a small library of 3-bromo-isoxazoline derivatives that inactivate the enzyme through a covalent, selective bond to the catalytic cysteine, as demonstrated by mass spectrometry. Substituents on the isoxazolinic ring modulated the potency up to 20-fold, predominantly due to an electrostatic effect, as assessed by computational analysis.

Synthesis and biological evaluation of papain-family cathepsin†L-like cysteine protease inhibitors containing a 1,4-benzodiazepine scaffold as antiprotozoal agents.

Novel papain-family cathepsin L-like cysteine protease inhibitors endowed with antitrypanosomal and antimalarial activity were developed, through an optimization study of previously developed inhibitors. In the present work, we studied the structure-activity relationships of these derivatives, with the aim to develop new analogues with a simplified and more synthetically accessible structure and with improved antiparasitic activity. The structure of the model compounds was significantly simplified by modifying or even eliminating the side chain appended at the C3 atom of the benzodiazepine scaffold. In addition, a simple methylene spacer of appropriate length was inserted between the benzodiazepine ring and the 3-bromoisoxazoline moiety. Several rhodesain and falcipain-2 inhibitors displaying single-digit micromolar or sub-micromolar antiparasitic activity against one or both parasites were identified, with activities that were one order of magnitude more potent than the model compounds.

Identification of a new series of amides as non-covalent proteasome inhibitors.

Proteasome inhibition has emerged as an important therapeutic strategy for the treatment of multiple myeloma (MM) and some forms of lymphoma, with potential application in other types of cancers. 20S proteasome consists of three different catalytic activities known as chymotrypsin-like (ChT-L), trypsin-like (T-L), and, post-glutamyl peptide hydrolyzing (PGPH) or caspase-like (C-L), which are located respectively on the ß5, ß2, and ß1 subunits of each heptameric ß rings. Currently a wide number of covalent proteasome inhibitors are reported in literature; however, the less widely investigated non-covalent inhibitors might be a promising alternative to employ in therapy, because of the lack of all drawbacks and side-effects related to irreversible inhibition. In the present work we identified a series of amides, two of which (1b and 1f) are good candidates to non-covalent inhibition of the chymotrypsin-like activity of the ß5 proteasome subunit. The non-covalent binding mode was corroborated by docking simulations of the most active inhibitors 1b, 1f and 2h into the yeast 20S proteasome crystal structure.

Development of rhodesain inhibitors with a 3-bromoisoxazoline warhead.

Novel rhodesain inhibitors were obtained by combining an enantiomerically pure 3-bromoisoxazoline warhead with a specific peptidomimetic recognition moiety. All derivatives behaved as inhibitors of rhodesain, with low micromolar Ki values. Their activity against the enzyme was found to be paralleled by an in vitro antitrypanosomal activity, with IC50 values in the mid-micromolar range. Notably, a preference for parasitic over human proteases, specifically cathepsins B and L, was observed.

Inhibition of rhodesain as a novel therapeutic modality for human African trypanosomiasis.

Rhodesain, a cathepsin L-like cysteine protease of T. brucei rhodesiense, is considered a potential target for the treatment of human African trypanosomiasis. Recent findings have confirmed that rhodesain, a lysosomal protease, is essential for parasite survival. Rhodesain is required by T. brucei to cross the blood-brain barrier, degrade host immunoglobulins, and turn over variant surface coat glycoproteins of T. brucei, which impair effective host immune responses. In this Perspective, we discuss the main classes of rhodesain inhibitors, including peptidic, peptidomimetic, and nonpeptidic structures, emphasizing those that have exhibited an optimal match between enzymatic affinity and trypanocidal profile and those for which preclinical investigations are currently in progress.

Synthesis and biochemical evaluation of δ(2)-isoxazoline derivatives as DNA methyltransferase 1 inhibitors.

A series of Δ(2)-isoxazoline constrained analogues of procaine/procainamide (7a-k and 8a-k) were prepared and their inhibitory activity against DNA methyltransferase 1 (DNMT1) was tested. Among them, derivative 7b is far more potent in vitro (IC(50) = 150 µM) than other non-nucleoside inhibitors and also exhibits a strong and dose-dependent antiproliferative effect against HCT116 human colon carcinoma cells. The binding mode of 7b with the enzyme was also investigated by means of a simple competition assay as well as of docking simulations conducted using the recently published crystallographic structure of human DNMT1. On the basis of the findings, we assessed that the mode of inhibition of 7b is consistent with a competition with the cofactor and propose it as a novel lead compound for the development of non-nucleoside DNMT inhibitors.

Design and synthesis of novel isoxazole-based HDAC inhibitors.

A series of isoxazole-based histone deacetylase (HDAC) inhibitors structurally related to SAHA were designed and synthesized. The isoxazole moiety was inserted in the vicinity of the Zn(2+)-binding group in order to check its participation in the coordinating process.