Emidio Camaioni

Adenosine deaminase: Functional implications and different classes of inhibitors

Adenosine deaminase (ADA) is an enzyme of the purine metabolism which catalyzes the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine, respectively. This ubiquitous enzyme has been found in a wide variety of microorganisms, plants, and invertebrates. In addition, it is present in all mammalian cells that play a central role in the differentiation and maturation of the lymphoid system. However, despite a number of studies performed to date, the physiological role played by ADA in the different tissues is not clear. Inherited ADA deficiency causes severe combined immunodeficiency desease (ADA‐SCID), in which both B‐cell and T‐cell development is impaired. ADA‐SCID has been the first disorder to be treated by gene therapy, using polyethene glycol‐modified bovine ADA (PEG‐ADA). Conversely, there are several diseases in which the level of ADA is above normal. A number of ADA inibitors have been designed and synthesized, classified as ground‐state and transition‐state inhibitors. They may be used to mimic the genetic deficiency of the enzyme, in lymphoproliferative disorders or immunosuppressive therapy (i.e., in graft rejection), to potentiate the effect of antileukemic or antiviral nucleosides, and, together with adenosine kinase, to reduce breakdown of adenosine in inflammation, hypertension, and ischemic injury.

Competitive and selective antagonism of P2Y1 receptors by N6-methyl 2'-deoxyadenosine 3',5'-bisphosphate.

The antagonist activity of N6‐methyl 2′‐deoxyadenosine 3′,5′‐bisphosphate (N6MABP) has been examined at the phospholipase C‐coupled P2Y1 receptor of turkey erythrocyte membranes. N6MABP antagonized 2MeSATP‐stimulated inositol phosphate hydrolysis with a potency approximately 20 fold greater than the previously studied parent molecule, adenosine 3′,5′‐bisphosphate. The P2Y1 receptor antagonism observed with N6MABP was competitive as revealed by Schild analysis (pKB=6.99±0.13). Whereas N6MABP was an antagonist at the human P2Y1 receptor, no antagonist effect of N6MABP was observed at the human P2Y2, human P2Y4 or rat P2Y6 receptors.

Highlights at the gate of tryptophan catabolism: a review on the mechanisms of activation and regulation of indoleamine 2,3-dioxygenase (IDO), a novel target in cancer disease

Indoleamine 2,3-dioxygenase (IDO) catalyzes the first and rate-limiting step of Kynurenine pathway along the major route of Tryptophan catabolism. The scientific interest in the enzyme has been growing since the observations of the involvement of IDO in the mechanisms of immune tolerance and in the mechanisms of tumor immuno-editing process. In view of this latter observation, in particular, preclinical studies of small molecule inhibitors of the enzyme have indicated the feasibility to thwart the immuno-editing process and to enhance the efficacy of current chemotherapeutic agents, supporting the notion that IDO is a novel target in cancer disease.This review covers the structural and conformational aspects of substrate recognition by IDO, including the catalytic mechanism and the so-far puzzling mechanisms of enzyme activation. Furthermore, we discuss the recent advances of medicinal chemistry in the field of IDO inhibitors.

New substituted 9-alkylpurines as adenosine receptor ligands

In the present study an investigation of the structure-activity relationships in 9-ethylpurine derivatives, aimed at preparing A1, A2A, A2B, and A3 selective adenosine receptor antagonists, was undertaken. Our synthetic approach was to introduce various substituents (amino, alkoxy and alkynyl groups) into the 2-, 6-, or 8-positions of the purine ring. The starting compounds for each series of derivatives were respectively: 2-iodo-9-ethyladenine (9), obtained from 2-amino-6-chloropurine (5); 9-ethyl-6-iodo-9H-purine (11), 8-bromo-9-ethyl-adenine (3) and 8-bromo-9-ethyl-6-iodo-9H-purine (13), obtained from 9-ethyl-adenine (2). The synthesized compounds were tested in in vitro radioligand binding assays at A1, A2A, and A3 human adenosine receptor subtypes. Due to the lack of a suitable radioligand the affinity of the 9-ethyladenine derivatives at A2B adenosine receptors was determined in adenylyl cyclase experiments. In general, the series of 9-ethylpurine derivatives exhibited a similar pharmacological profile at A1 and A2A receptors whereas some differences were found for the A3 and the A2B subtypes. 8-Bromo-9-ethyladenine (3) showed higher affinity for all receptors in comparison to the parent compound 2, and the highest affinity in the series for the A2A and A2B subtypes (Ki = 0.052 and 0.84 microM, respectively). Analyzing the different substituents, a phenethoxy group in 2-position (10a) gave the highest A2A versus A2B selectivity (near 400-fold), whereas a phenethylamino group in 2- and 6-position (10b and 12b, respectively) improved the affinity at A2B receptors, compared to the parent compound 2. The presence of a hexynyl substituent in 8-position led to a compound with good affinity at the A3 receptor (4d, Ki = 0.62 microM), whereas (ar)alkynyl groups are detrimental for the potency at the A2B subtype. These differences give raise to the hope that further modifications will result in the development of currently unavailable leads with good affinity and selectivity for A2B adenosine receptors.

Poly(ADP-ribose) Catabolism Triggers AMP-dependent Mitochondrial Energy Failure

Upon massive DNA damage, hyperactivation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP)-1 causes severe depletion of intracellular NAD and ATP pools as well as mitochondrial dysfunction. Thus far, the molecular mechanisms contributing to PARP-1-dependent impairment of mitochondrial functioning have not been identified. We found that degradation of the PARP-1 product poly(ADP-ribose) through the concerted actions of poly(ADP-ribose) glycohydrolase and NUDIX (nucleoside diphosphate-X) hydrolases leads to accumulation of AMP. The latter, in turn, inhibits the ADP/ATP translocator, prompting mitochondrial energy failure. For the first time, our findings identify NUDIX hydrolases as key enzymes involved in energy derangement during PARP-1 hyperactivity. Also, these data disclose unanticipated AMP-dependent impairment of mitochondrial exchange of adenine nucleotides, which can be of relevance to organelle functioning and disease pathogenesis.

PARP inhibitors: polypharmacology versus selective inhibition

Inhibition of ADP‐ribosyltransferases with diphtheria toxin homology (ARTD), widely known as the poly(ADP‐ribose) polymerase (PARP) family, is a strategy under development for treatment of various conditions, including cancers and ischemia. Here, we give a brief summary of ARTD enzyme functions and the implications for their potential as therapeutic targets. We present an overview of the PARP inhibitors that have been used in clinical trials. Finally, we summarize recent insights from structural biology, and discuss the molecular aspects of PARP inhibitors in terms of broad‐range versus selective inhibition of ARTD family enzymes.

On the Way to Selective PARP-2 Inhibitors. Design, Synthesis, and Preliminary Evaluation of a Series of Isoquinolinone Derivatives

PARP‐1 and PARP‐2 are members of the family of poly(ADP‐ribose)polymerases, which are involved in the maintenance of genomic integrity under conditions of genotoxic stimuli. The different roles of the two isoforms under pathophysiological conditions have not yet been fully clarified, and this is partially due to the lack of selective inhibitors. We report herein the synthesis and preliminary pharmacological evaluation of a large series of isoquinolinone derivatives as PARP‐1/PARP‐2 inhibitors. Among them, we identified the 5‐benzoyloxyisoquinolin‐1(2 H)‐one derivative as the most selective PARP‐2 inhibitor reported so far, with a PARP‐2/PARP‐1 selectivity index greater than 60.

Synthesis and Structure-Activity Relationships of Pyridoxal-6-arylazo-5'-phosphate and Phosphonate Derivatives as P2 Receptor Antagonists.

Novel analogs of the P2 receptor antagonist pyridoxal‐5′‐phosphate‐6‐phenylazo‐2′,4′‐disulfonate (PPADS) were synthesized. Modifications were made through functional group substitution on the sulfophenyl ring and at the phosphate moiety through the inclusion of phosphonates, demonstrating that a phosphate linkage is not required for P2 receptor antagonism. Substituted 6‐phenylazo and 6‐naphthylazo derivatives were also evaluated. Among the 6‐phenylazo derivatives, 5′‐methyl, ethyl, propyl, vinyl, and allyl phosphonates were included. The compounds were tested as antagonists at turkey erythrocyte and guinea‐pig taenia coli P2Y1 receptors, in guinea‐pig vas deferens and bladder P2X1 receptors, and in ion flux experiments by using recombinant rat P2X2 receptors expressed in Xenopus oocytes. Competitive binding assay at human P2X1 receptors in differentiated HL‐60 cell membranes was carried out by using [35S]ATP‐γ‐S. A 2′‐chloro‐5′‐sulfo analog of PPADS (C14H12O9N3ClPSNa), a vinyl phosphonate derivative (C15H12O11N3PS2Na3), and a naphthylazo derivative (C18H14O12N3PS2Na2), were particularly potent in binding to human P2X1 receptors. The potencies of phosphate derivatives at P2Y1 receptors were generally similar to PPADS itself, except for the p‐carboxyphenylazo phosphate derivative C15H13O8N3PNa and its m‐chloro analog C15H12O8N3ClPNa, which were selective for P2X vs. P2Y1 receptors. C15H12O8N3ClPNa was very potent at rat P2X2 receptors with an IC50 value of 0.82 μM. Among the phosphonate derivatives, [4‐formyl‐3‐hydroxy‐2‐methyl‐6‐(2‐chloro‐5‐sulfonylphenylazo)‐pyrid‐5‐yl]methylphosphonic acid (C14H12O8N3ClPSNa) showed high potency at P2Y1 receptors with an IC50 of 7.23 μM. The corresponding 2,5‐disulfonylphenyl derivative was nearly inactive at turkey erythrocyte P2Y1 receptors, whereas at recombinant P2X2 receptors had an IC50 value of 1.1 μM. An ethyl phosphonate derivative (C15H15O11N3PS2Na3), whereas inactive at turkey erythrocyte P2Y1 receptors, was particularly potent at recombinant P2X2 receptors. Drug Dev. Res. 45:52–66, 1998. Published 1998 Wiley‐Liss, Inc.

Long‐lasting neuroprotection and neurological improvement in stroke models with new, potent and brain permeable inhibitors of poly(ADP‐ribose) polymerase

BACKGROUND AND PURPOSES Thienyl‐isoquinolone (TIQ‐A) is a relatively potent PARP inhibitor able to reduce post‐ischaemic neuronal death in vitro. Here we have studied, in different stroke models in vivo, the neuroprotective properties of DAMTIQ and HYDAMTIQ, two TIQ‐A derivatives able to reach the brain and to inhibit PARP‐1 and PARP‐2.

Characterization of potent ligands at human recombinant adenosine receptors

The four adenosine receptor subtypes have been stably transfected into Chinese hamster ovary (CHO) cells allowing for comparative studies in a similar cellular background, using radioligand binding studies (A1, A2A, A3) or adenylyl cyclase activity assays (A2B). We are currently using the transfected CHO cells for extensive screening of nucleosides and purine derivatives of our library. Screening of a number of 2‐alkynyl analogs of 5′‐N‐ethylcarboxamidoadenosine (NECA) indicated that introduction of particular substituents, such as the racemic 2‐phenylhydroxypropynyl group, led to a highly potent, nonselective agonist at A1, A2A, and A3 subtypes (PHPNECA, Ki in the low nanomolar range at the three subtypes). In the A2B functional assay, it has been found that PHPNECA (EC50 A2B = 0.88 μM) is threefold more potent than NECA. This article is the first report in which the introduction of a bulky group in the 2‐position of NECA led to a compound that is active as an agonist at the human A2B subtype. On the other hand, the presence of a phenyl ring conjugated to the triple bond as in phenylethynylNECA (PENECA) enhanced selectivity for the A3 subtype. In the purine series (potential antagonists), 8‐bromo‐9‐ethyladenine (8‐BEA) showed good affinity toward all adenosine receptor subtypes (Ki A1 = 0.28 μM, Ki A2A = 0.052 μM, Ki A2B = 0.84 μM, Ki A3 = 27.8 μM). On the other hand, the introduction of alkynyl chains in the 8‐position resulted in an increased affinity at the A3 receptor (8‐hexynyl‐9‐ethyladenine, 8‐HEEA, Ki A3 = 0.62 μM and 8‐phenylethynyl‐9‐ethyladenine, 8‐PEEA, Ki A3 = 0.086 μM). Drug Dev. Res. 45:176–181, 1998.