Sabrina Taliani

Recent advances in the development of dual topoisomerase I and II inhibitors as anticancer drugs.

DNA topoisomerases (topos) are essential enzymes that regulate the topological state of DNA during cellular processes such as replication, transcription, recombination, and chromatin remodeling. Topoisomerase I (Topo I) is a ubiquitous nuclear enzyme which catalyzes the relaxation of superhelical DNA generating a transient single strand nick in the duplex, through cycles of cleavage and religation. Topoisomerase II (Topo II) mediates the ATP-dependent induction of coordinated nicks in both strands of the DNA duplex, followed by crossing of another double strand DNA through the transiently broken duplex. Although the biological functions of Topoisomerases are important for ensuing genomic integrity, the ability to interfere with enzymes or generate enzyme-mediated damage is an effective strategy for cancer therapy and, in this connection, DNA topos (I and II) proved to be the excellent targets of clinically significant classes of anticancer drugs. Actually, specific Topo I and Topo II inhibitors reversibly trap the enzyme-DNA complexes, thus converting Topos into physiological poisons, able to produce permanent DNA damage, which triggers cell death. Given that both enzymes are good targets, it would be desirable to jointly inhibit them, but use-limiting toxicity of sequential or simultaneous combinations of topo I and II poisons include severe to life-threatening neutropenia and anemia. Furthermore, the emergence of resistance phenomena to topo I inhibitors is often accompanied by a concomitant rise in the level of topo II expression and viceversa, leading to the failure of clinical therapies. In this regard, a single compound able to inhibit both Topo I and II may present the advantage of improving antitopoisomerase activity, with reduced toxic side effects, with respect to the combination of two inhibitors. Due to the high interest in such compounds, this review represents an update of previous works dealing with the development of dual Topo I and II inhibitors as novel anti-cancer agents. The newly collected derivatives have been described focusing attention on their chemical structures and their biological profiles.

GABAA/Bz Receptor Subtypes as Targets for Selective Drugs

The gamma-aminobutyric acid type A (GABA(A)) receptors are the major inhibitory neuronal receptors in the mammalian brain. Their activation by GABA opens the intrinsic ion channel, enabling chloride flux into the cell with subsequent hyperpolarization. Several GABA(A) receptor subunit isoforms have been cloned, the major isoform containing alpha, beta, and gamma subunits, and a regional heterogeneity associated with distinct physiological effects has been suggested. As a variety of allosteric ligands can modulate GABA-gated conductance changes through binding to distinct sites, the development of subtype-selective ligands may lead to the selective treatment of GABA system-associated pathology. In particular, the best characterized binding site is the benzodiazepine site (BzR), localized at the alpha/gamma subunit interface, in which the alpha subunit is the main determinant of BzR ligand action selectivity. The alpha1-containing BzR have been proposed to be responsible for the sedative action; the alpha2 and/or the alpha3 subtypes have been suggested to mediate the anxiolytic activity and the myorelaxation effects, and the alpha5 subtype has been associated with cognition processes. The discovery of alpha-selective subtype ligands may help in the specific treatment of anxiety, sleep disorders, convulsions and memory deficits with fewer side effects. Selectivity may be achieved by two approaches: selective affinity or selective efficacy. Selective affinity needs a compound to bind with a higher affinity to one receptor subtype compared with another, whereas subtype-selective efficacy relies on a compound binding to all subtypes, but having different efficacies at various subtypes. The status of BzR ligands, subdivided on the basis of their main chemical structural features, is reviewed in relation to structure-activity relationships which determine their affinity or efficacy selectivity for a certain BzR subtype.

Arylthioamides as H2S Donors: l-Cysteine-Activated Releasing Properties and Vascular Effects in Vitro and in Vivo

A small library of arylthioamides 1-12 was easily synthesized, and their H2S-releasing properties were evaluated both in the absence or in the presence of an organic thiol such as l-cysteine. A number of arylthioamides (1-3 and 7) showed a slow and l-cysteine-dependent H2S-releasing mechanism, similar to that exhibited by the reference slow H2S-releasing agents, such as diallyl disulfide (DADS) and the phosphinodithioate derivative GYY 4137. Compound 1 strongly abolished the noradrenaline-induced vasoconstriction in isolated rat aortic rings and hyperpolarized the membranes of human vascular smooth muscle cells in a concentration-dependent fashion. Finally, a significant reduction of the systolic blood pressure of anesthetized normotensive rats was observed after its oral administration. Altogether these results highlighted the potential of arylthioamides 1-3 and 7 as H2S-donors for basic studies, and for the rational design/development of promising pharmacotherapeutic agents to treat cardiovascular diseases.

Acetic Acid Aldose Reductase Inhibitors Bearing a Five-Membered Heterocyclic Core with Potent Topical Activity in a Visual Impairment Rat Model

A number of 1,2,4-oxadiazol-5-yl-acetic acids and oxazol-4-yl-acetic acids were synthesized and tested for their ability to inhibit aldose reductase (ALR2). The oxadiazole derivatives, 7c, 7f, 7i, and 8h, 8i, proved to be the most active compounds, exhibiting inhibitory levels in the submicromolar range. In this series, the phenyl group turned out to be the preferred substitution pattern, as its lengthening to a benzyl moiety determined a general reduction of the inhibitory potency. The lead compound, 2-[3-(4-methoxyphenyl)-1,2,4-oxadiazol-5-yl]acetic acid, 7c, showed an excellent in vivo activity, proving to prevent cataract development in severely galactosemic rats when administered as an eye-drop solution in the precorneal region of the animals. Computational studies on the ALR2 inhibitors were performed to rationalize the structure-activity relationships observed and to provide the basis for further structure-guided design of novel ALR2 inhibitors.

Novel N2-substituted pyrazolo[3,4-d]pyrimidine adenosine A3 receptor antagonists: inhibition of A3-mediated human glioblastoma cell proliferation.

Adenosine induces glioma cell proliferation by means of an antiapoptotic effect, which is blocked by cotreatment with selective A(3) AR antagonists. In this study, a novel series of N(2)-substituted pyrazolo[3,4-d]pyrimidines 2a-u was developed as highly potent and selective A(3) AR antagonists. The most performing compounds were derivatives 2a (R(1) = CH(3) and R(2) = COC(6)H(5); K(i) 334, 728, and 0.60 nM at the human A(1), A(2A), and A(3) ARs, respectively) and 2b (R(1) = CH(3) and R(2) = COC(6)H(4)-4-OCH(3); K(i) 1037, 3179, and 0.18 nM at the human A(1), A(2A), and A(3) ARs, respectively), which counteracted the effect of the A(3) AR agonists Cl-IB-MECA and IB-MECA on human glioma U87MG cell proliferation. This effect was concentration-dependent, with IC(50) values comparable to A(3) AR binding affinity values of 2a and 2b, thereby suggesting that their effects were receptor-mediated. Furthermore, the antiproliferative activity of the new compounds was demonstrated to be mediated by the block of A(3) AR agonist activation of intracellular kinases ERK 1/2.

Genetic analysis of dTSPO, an outer mitochondrial membrane protein, reveals its functions in apoptosis, longevity, and Aβ42‐induced neurodegeneration

The outer mitochondrial membrane (OMM) protein, the translocator protein 18 kDa (TSPO), formerly named the peripheral benzodiazepine receptor (PBR), has been proposed to participate in the pathogenesis of neurodegenerative diseases. To clarify the TSPO function, we identified the Drosophila homolog, CG2789/dTSPO, and studied the effects of its inactivation by P‐element insertion, RNAi knockdown, and inhibition by ligands (PK11195, Ro5‐4864). Inhibition of dTSPO inhibited wing disk apoptosis in response to γ‐irradiation or H2O2 exposure, as well as extended male fly lifespan and inhibited Aβ42‐induced neurodegeneration in association with decreased caspase activation. Therefore, dTSPO is an essential mediator of apoptosis in Drosophila and plays a central role in controlling longevity and neurodegenerative disease, making it a promising drug target.

Apoptosis Therapy in Cancer: The First Single-molecule Co-activating p53 and the Translocator Protein in Glioblastoma

In the complex scenario of cancer, treatment with compounds targeting multiple cell pathways has been emerging. In Glioblastoma Multiforme (GBM), p53 and Translocator Protein (TSPO), both acting as apoptosis inducers, represent two attractive intracellular targets. On this basis, novel indolylglyoxylyldipeptides, rationally designed to activate TSPO and p53, were synthesized and biologically characterized. The new compounds were able to bind TSPO and to reactivate p53 functionality, through the dissociation from its physiological inhibitor, murine double minute 2 (MDM2). In GBM cells, the new molecules caused Δψm dissipation and inhibition of cell viability. These effects resulted significantly higher with respect to those elicited by the single target reference standards applied alone, and coherent with the synergism resulting from the simultaneous activation of TSPO and p53. Taken together, these results suggest that TSPO/MDM2 dual-target ligands could represent a new attractive multi-modal opportunity for anti-cancer strategy in GBM.

Exploiting the Pyrazolo[3,4-d]pyrimidin-4-one Ring System as a Useful Template To Obtain Potent Adenosine Deaminase Inhibitors

A number of pyrazolo[3,4-d]pyrimidin-4-ones bearing either alkyl or arylalkyl substituents in position 2 of the nucleus were synthesized and tested for their ability to inhibit adenosine deaminase (ADA) from bovine spleen. The 2-arylalkyl derivatives exhibited excellent inhibitory activity, showing Ki values in the nanomolar/subnanomolar range. The most active compound, 1-(4-((4-oxo-4,5-dihydropyrazolo[3,4-d]pyrimidin-2-yl)methyl)phenyl)-3-(4-(trifluoromethyl)phenyl)urea, 14d, was tested in rats with colitis induced by 2,4-dinitrobenzenesulfonic acid to assess its efficacy to attenuate bowel inflammation. The treatment with 14d induced a significant amelioration of both systemic and intestinal inflammatory alterations in animals with experimental colitis. Docking simulations of the synthesized compounds into the ADA catalytic site were also performed to rationalize the structure-activity relationships observed and to highlight the key pharmacophoric elements of these products, thus prospectively guiding the design of novel ADA inhibitors.

11C-ER176, a radioligand for 18-kDa translocator protein (TSPO), has adequate sensitivity to robustly image all three affinity genotypes in human brain

For PET imaging of 18-kDa translocator protein (TSPO), a biomarker of neuroinflammation, most second-generation radioligands are sensitive to the single nucleotide polymorphism rs6971; however, this is probably not the case for the prototypical agent 11C-PK11195 (11C-labeled N-butan-2-yl-1-(2-chlorophenyl)-N-methylisoquinoline-3-carboxamide), which has a relatively lower signal-to-noise ratio. We recently found that 11C-ER176 (11C-(R)-N-sec-butyl-4-(2-chlorophenyl)-N-methylquinazoline-2-carboxamide), a new analog of 11C-(R)-PK11195, showed little sensitivity to rs6971 when tested in vitro and had high specific binding in monkey brain. This study sought, first, to determine whether the sensitivity of 11C-ER176 in humans is similar to the low sensitivity measured in vitro and, second, to measure the nondisplaceable binding potential (BPND, or the ratio of specific-to-nondisplaceable uptake) of 11C-ER176 in human brain. Methods: Nine healthy volunteers—3 high-affinity binders (HABs), 3 mixed-affinity binders (MABs), and 3 low-affinity binders (LABs)—were studied with whole-body 11C-ER176 PET imaging. SUVs from 60 to 120 min after injection derived from each organ were compared between genotypes. Eight separate healthy volunteers—3 HABs, 3 MABs, and 2 LABs—underwent brain PET imaging. The 3 HABs underwent a repeated brain scan after TSPO blockade with XBD173 (N-benzyl-N-ethyl-2-(7-methyl-8-oxo-2-phenylpurin-9-yl)acetamide) to determine nondisplaceable distribution volume (VND) via Lassen occupancy plotting and thereby estimate BPND in brain. Results: Regional SUV averaged from 60 to 120 min after injection in brain and peripheral organs with high TSPO densities such as lung and spleen were greater in HABs than in LABs. On the basis of VND determined via the occupancy plot, the whole-brain BPND for LABs was estimated to be 1.4 ± 0.8, which was much lower than that for HABs (4.2 ± 1.3) but about the same as that for HABs with 11C-PBR28 ([methyl-11C]N-acetyl-N-(2-methoxybenzyl)-2-phenoxy-5-pyridinamine)) (∼1.2). Conclusion: Obvious in vivo sensitivity to rs6971 was observed in 11C-ER176 that had not been expected from in vitro studies, suggesting that the future development of any improved radioligand for TSPO should consider the possibility that in vitro properties will not be reflected in vivo. We also found that 11C-ER176 has adequately high BPND for all rs6971 genotypes. Thus, the new radioligand would likely have greater sensitivity in detecting abnormalities in patients.

Tricyclic sulfonamides incorporating benzothiopyrano[4,3-c]pyrazole and pyridothiopyrano[4,3-c]pyrazole effectively inhibit α- And β-carbonic anhydrase: X-ray crystallography and solution investigations on 15 isoforms

Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous isozymes involved in crucial physiological and pathological events, representing the targets of inhibitors with several therapeutic applications. In this connection, we report a new class of carbonic anhydrase inhibitors, based on the thiopyrano-fused pyrazole scaffold to which a pendant 4-sulfamoylphenyl moiety was attached. The new sulfonamides 3a-e were designed as constrained analogues of celecoxib and valdecoxib. The most interesting feature of sulfonamides 3 was their predominantly strong inhibition of human (h) CA I and II, as well as those of the mycobacterial β-class enzymes (Rv1284, Rv3273, and Rv3588c), whereas their inhibitory action against hCA III, IV, VA, VB, VI, VII, IX, XII, XIII, and XIV was found to be at least 2 orders of magnitude lower. X-ray crystallography and structural superposition studies made it possible to explain the very distinct inhibition profile of the tricyclic sulfonamides, different from those of celecoxib and valdecoxib.