Nicola Micale

Subunit-specific agonist activity at NR2A, NR2B, NR2C, and NR2D containing N-methyl-D-aspartate glutamate receptors

The four N-methyl-d-aspartate (NMDA) receptor NR2 subunits (NR2A-D) have different developmental, anatomical, and functional profiles that allow them to serve different roles in normal and neuropathological situations. Identification of subunit-selective NMDA receptor agonists, antagonists, or modulators could prove to be both valuable pharmacological tools as well as potential new therapeutic agents. We evaluated the potency and efficacy of a wide range of glutamate-like compounds at NR1/NR2A, NR1/NR2B, NR1/NR2C, and NR1/NR2D receptors. Twenty-five of 53 compounds examined exhibited agonist activity at the glutamate binding site of NMDA receptors. Concentration-response relationships were determined for these agonists at each NR2 subunit. We find consistently higher potency at the NR2D subunit for a wide range of dissimilar structures, with (2S,4R)-4-methylglutamate (SYM2081) showing the greatest differential potency between NR2A- and NR2D-containing receptors (46-fold). Analysis of chimeric NR2A/D receptors suggests that enhanced agonist potency for NR2D is controlled by residues in both of the domains (Domain1 and Domain2) that compose the bilobed agonist binding domain. Molecular dynamics (MD) simulations comparing a crystallography-based hydrated NR1/NR2A model with a homology-based NR1/NR2D hydrated model of the agonist binding domains suggest that glutamate exhibits a different binding mode in NR2D compared with NR2A that accommodates a 4-methyl substitution in SYM2081. Mutagenesis of functionally divergent residues supports the conclusions drawn based on the modeling studies. Despite high homology and conserved atomic contact residues within the agonist binding pocket of NR2A and NR2D, glutamate adopts a different binding orientation that could be exploited for the development of subunit selective agonists and competitive antagonists.

Falcipain-2 inhibitors.

Malaria, particularly that one caused by Plasmodium falciparum, remains a serious health problem in Africa, South America, and many parts of Asia where it afflicts about 500 million people and is responsible for the death of more than one million children each year. The main reasons for the persistence of malaria are the emergence of resistance to common antimalarial drugs, inadequate control of mosquito vectors, and the lack of effective vaccines. Therefore, the identification and characterization of new targets for antimalarial chemotherapy are of urgent priority. This review is focused on inhibitors of falcipain‐2, a cysteine protease from P. falciparum, which represents one of the most promising targets for antimalarial drug design. Falcipain‐2 is a key enzyme in the life cycle of P. falciparum since it degrades hemoglobin, at the early trophozoite stage, and cleaves ankyrin and protein 4.1, the cytoskeletal elements vital to the stability of red cell membrane, at the schizont stage. The main classes of falcipain‐2 inhibitors are peptides or peptidomimetics bearing the most popular pharmacophores of cysteine protease inhibitors, such as vinyl sulfones, halomethyl ketones, and aldehydes. Furthermore, many other chemotypes have been identified as inhibitors of falcipain‐2, such as isoquinolines, thiosemicarbazones, and chalcones. These inhibitors represent all classes, which, to the best of our knowledge, have been disclosed in journal articles to date.

Novel Peptidomimetics Containing a Vinyl Ester Moiety as Highly Potent and Selective Falcipain-2 Inhibitors

This paper describes the synthesis and biological evaluation of a new class of peptidomimetic falcipain-2 inhibitors based on a 1,4-benzodiazepine scaffold combined with various alpha,beta-unsaturated electrophilic functions such as vinyl-ketone, -amide, -ester, and -nitrile. The profile of reactivity of this class of derivatives has been evaluated and 4c, containing a vinyl ester warhead, proved to be a highly potent and selective falcipain-2 inhibitor.

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.

Nonpeptidic Vinyl and Allyl Phosphonates as Falcipain-2 Inhibitors

Malaria remains one of the most deadly parasitic diseases, affecting 500 million people all over the world and causing more than one million deaths each year. The limitations of antimalarial chemotherapy underscore the urgent need to discover new drugs that are able to interact with new targets. Research efforts are currently focused on the design of inhibitors of malarial proteases, among which falcipain-2 (FP-2) plays a key role. FP-2 is a papain-family cysteine protease of the most virulent species of the malaria-causing parasite Plasmodium falciparum, and is required by mature schizonts for the cleavage of erythrocytic cytoskeletal proteins and by intraerythrocytic trophozoites for hemoglobin degradation, which provides free amino acids for parasite protein synthesis. Thus, selective and irreversible inhibition of FP-2 would be advantageous for the control and elimination of the parasite. Peptides and peptidomimetics containing an activated double bond, such as vinyl sulfones and vinyl esters, have been shown to be highly potent irreversible cysteine protease inhibitors. Whereas the former are active on papain-like enzymes, the latter are known in particular as inhibitors of viral proteases. Both inhibitor types interact with the target enzyme by forming a covalent bond with the thiol group of the active site cysteine. Peptidyl vinyl sulfones are stable and unreactive toward nucleophiles, and require the “catalytic machinery” of cysteine proteases for their activation. Peptidyl vinyl sulfones containing a homoPhe residue at the P1 site have been proven to be highly specific FP-2 inhibitors, with the aromatic side chain being a key structural requirement for greater selectivity toward the target enzyme. In this context we recently reported a new class of peptidomimetic FP-2 inhibitors based on a rigid benzodiazepine scaffold as a conformationally constrained form of the d-Ser-Gly fragment and on a terminal electrophilic vinyl sulfone moiety on the P1 site that reacts as classical Michael acceptor (such as compounds 1a–d, Figure 1). All the synthesized compounds showed a high level of inhibitory potency and are quite selective toward FP-2, as they weakly inhibit human cysteine proteases cathepsin B and L. In particular, compound 1b displayed potent enzymatic inhibition (k2=307000m min ) coupled with a good activity against cultured P. falciparum (IC50= 9.1 mm). Among other irreversible cysteine protease inhibitors containing a vinyl moiety conjugated to electron withdrawing groups (EWGs), vinyl phosphonates showed good inhibitory activity against cultured P. falciparum. On this basis, we designed and synthesized nonpeptidic unsaturated phosphonate structures 2 and 3 (Scheme 1) to evaluate their ability to inhibit FP-2 and to make a head-to-head

7,8-methylenedioxy-4H-2,3-benzodiazepin-4-ones as novel ampa receptor antagonists

The synthesis and anticonvulsant activity of novel 7,8-methylenedioxy-4H-2,3-benzodiazepin-4-ones 3a-e, structurally-related to GYKI 52466 1, a well-known noncompetitive AMPA-receptor antagonist, are reported. The new compounds possess marked anticonvulsant properties and, in analogy to 1, antagonize seizures induced by AMPA. In addition, when compared to the model compound 1, compounds 3 show a longer-lasting anticonvulsant activity and a lower toxicity.

Development of Novel Peptidomimetics Containing a Vinyl Sulfone Moiety as Proteasome Inhibitors

Proteasome inhibition is a topic of great interest in anticancer research. The proteolytic activity of this multicatalytic complex relies on three subunits, β1, β2 and β5, containing a caspase‐like, a trypsin‐like and a chymotrypsin‐like active site, respectively. Several studies have demonstrated that, of the three activities, the chymotrypsin‐like activity was the most necessary for cell viability and protein processing. Thus, most efforts towards the development of proteasome inhibitors have focused on the selective inhibition of the β5 subunit active site. Herein, we report the design and synthesis of a series of conformationally constrained tripeptidyl vinyl sulfones were determined to be good inhibitors of the chymotrypsin‐like activity of proteasome, with KI values in the sub‐micromolar to micromolar range. These compounds were also tested against bovine pancreatic α‐chymotrypsin and human cathepsin B and L, revealing a good selectivity for the target enzyme over these related enzymes.

Novel 2H-isoquinolin-3-ones as antiplasmodial falcipain-2 inhibitors.

A series of 1-aryl-6,7-disubstituted-2H-isoquinolin-3-ones (2-10) was synthesized and evaluated for their inhibition against Plasmodium falciparum cysteine protease falcipain-2, as well as against cultured P. falciparum strain FCBR parasites. All compounds displayed inhibitory activity against recombinant falcipain-2 and against in vitro cultured intraerythrocytic P. falciparum, with the exception of 9. The new compounds exhibited no selectivity against human cysteine proteases such as cathepsins B and L. The inhibitory activity of the synthesized compounds was also evaluated against another protozoal cysteine protease, namely rhodesain of Trypanosoma brucei rhodesiense.

Peptidomimetics containing a vinyl ketone warhead as falcipain-2 inhibitors

The design, chemical synthesis, and enzymatic activity evaluation of a set of falcipain-2 inhibitors are reported. These compounds contain a proven peptidomimetic recognition motif based on a benzo[1,4]diazepin-2-one (1,4-BDZ) framework built on a dipeptide sequence, and a Michael acceptor terminal moiety capable of deactivating the cysteine protease active site. Our goal is to modify the P(3) site of this motif in order to identify the structural requirements for the interaction with the target.

Synthesis of benzothiazole derivatives and their biological evaluation as anticancer agents

This article describes the synthesis and the biological evaluation of two sets of benzothiazole derivatives bearing at C-2 an arylamide (1a–e, 2a–e) or an arylurea (3a–d, 4a–d) moiety. Five compounds (3d and 4a–d) were selected and screened by the National Cancer Institute for the in vitro primary anticancer assay against a panel of 60 human tumor cell lines. Compounds 4a and 4c showed interesting anticancer activities, more marked for compound 4c. All compounds were also submitted to a preliminary in vitro assay as potential inhibitors of the ubiquitin-activating enzyme (E1), but they lacked significant activity.