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Piperazine is a structural element present in drugs belonging to various chemical classes and used for numerous different therapeutic applications; it has been considered a privileged scaffold for drug design.The authors have searched examples of piperazine-containing compounds among drugs recently approved by the FDA and in some research fields (nicotinic receptor modulators, compounds acting against cancer, and bacterial multidrug resistance), looking in particular to the design behind the insertion of this moiety.Piperazine is widely used due to its peculiar characteristics, such as solubility, basicity, chemical reactivity, and conformational properties. This moiety has represented an important tool to modulate pharmacokinetic and pharmacodynamic properties of drugs.

Histone deacetylases (HDAC) are enzymes that regulate the concentration of acetylated histones which, in turns, interact with the bromodomain (BRD) of BET (Bromodomain and Extracellular domain) proteins to affect transcriptional activity. Simultaneous blockade of both epigenetic players has shown synergistic effects in a variety of cancer cell lines. In this paper we report the design, synthesis and activity of new dual inhibitors, obtained by adding a methyltriazole moiety to some HDAC inhibitors carrying a benzodiazepine core, which were previously developed by us. An Alphascreen FRET assay showed that the compounds were able to interact with BRD4-1 and BRD4-2 proteins, with some selectivity for the latter, while the HDAC inhibiting properties were measured by means of an immunoprecipitation assay. The antiproliferative activity was tested on C26 adenocarcinoma, SSMC2 melanoma and SHSY5Y neuroblastoma cells. Interestingly, both compounds were endowed with antihyperalgesic activity in the mouse Spared Nerve Injury (SNI) model.

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, expressed in a variety of cell types and in all tissues, control excitation and rhythm. Since their discovery in neurons and cardiac pacemaker cells, they attracted the attention of medicinal chemistry and pharmacology as novel targets to shape (patho)physiological mechanisms. To date, ivabradine represents the first-in-class drug as specific bradycardic agent in cardiac diseases; however, new applications are emerging in parallel with the demonstration of the involvement of different HCN isoforms in central and peripheral nervous system. Hence, the possibility to target specific isoforms represents an attractive development in this field; indeed, HCN1, HCN2 or HCN4 specific blockers have shown promising features in vitro and in vivo, with remarkable pharmacological differences likely depending on the diverse functional role and tissue distribution. Here, we show a recently developed compound with high potency as HCN2–HCN4 blocker; because of its unique profile, this compound may deserve further investigation.

In a continuing search of dual P-gp and hCA XII inhibitors, we synthesized and studied new

In this paper, the efficiency of the carbonic anhydrase (CA) enzyme in accelerating the hydration of CO2 is evaluated using a measurement system which consists of a vessel in which a gaseous flow of mixtures of nitrogen and CO2 is bubbled into water or water solutions containing a known quantity of CA enzyme. The pH value of the solution and the CO2 concentration at the measurement system gas exhaust are continuously monitored. The measured CO2 level allows for assessing the quantity of CO2, which, subtracted from the gaseous phase, is dissolved into the liquid phase and/or hydrated to bicarbonate. The measurement procedure consists of inducing a transient and observing and modelling the different kinetics involved in the steady-state recovery with and without CA. The main contribution of this work is exploiting dynamical system theory and chemical kinetics modelling for interpreting measurement results for characterising the activity of CA enzymes. The data for model fitting are obtained from a standard bioreactor, in principle equal to standard two-phase bioreactors described in the literature, in which two different techniques can be used to move the process itself away from the steady-state, inducing transients.

The main objective of this study was to determine whether (E)-3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2) and its structural derivative DM489 produce anti-neuropathic pain activity using the streptozotocin (STZ)- and oxaliplatin-induced neuropathic pain animal models. To assess possible mechanisms of action, the pharmacological activity of these compounds was determined at α7 and α9α10 nicotinic acetylcholine receptors (nAChRs) and CaV2.2 channels expressed alone or coexpressed with G protein-coupled GABAB receptors. The animal results indicated that a single dose of 3 mg/kg PAM-2 or DM489 decreases STZ-induced neuropathic pain in mice, and chemotherapy-induced neuropathic pain is decreased by PAM-2 (3 mg/kg) and DM489 (10 mg/kg). The observed anti-neuropathic pain activity was inhibited by the α7-selective antagonist methyllycaconitine. The coadministration of oxaliplatin with an inactive dose (1 mg/kg) of PAM-2 decreased the development of neuropathic pain after 14, but not 7, days of cotreatment. The electrophysiological results indicated that PAM-2 potentiates human (h) and rat (r) α7 nAChRs with 2-7 times higher potency than that for hCaV2.2 channel inhibition and an even greater difference compared to that for rα9α10 nAChR inhibition. These results support the notion that α7 nAChR potentiation is likely the predominant molecular mechanism underlying the observed anti-nociceptive pain activity of these compounds.

Background: The failure of anticancer chemotherapy is often due to the development of resistance to a variety of anticancer drugs. This phenomenon is called multidrug resistance (MDR) and is related to the overexpression of ABC transporters, such as P-glycoprotein, multidrug re-sistance-associated protein 1 and breast cancer resistance protein. Over the past few decades, sever-al ABC protein modulators have been discovered and studied as a possible approach to evade MDR and increase the success of anticancer chemotherapy. Nevertheless, the co-administration of pump inhibitors with cytotoxic drugs, which are substrates of the transporters, does not appear to be asso-ciated with an improvement in the therapeutic efficacy of antitumor agents. However, more recently discovered MDR reversing agents, such as the two tetrahydroisoquinoline derivatives tariquidar and elacridar, are characterized by high affinity towards the ABC proteins and by reduced negative properties. Consequently, many analogs of these two derivatives have been synthesized, with the aim of optimizing their MDR reversal properties. Objective: This review aims to describe the MDR modulators carrying the tetraidroisoquinoline scaffold reported in the literature in the period 2009-2021, highlighting the structural characteristics that confer potency and/or selectivity towards the three ABC transport proteins. Results and Conclusions: Many compounds have been synthesized in the last twelve years showing interesting properties, both in terms of potency and selectivity. Although clear structure-activity re-lationships can be drawn only by considering strictly related compounds, some of the compounds reviewed could be promising starting points for the design of new ABC protein inhibitors.

Despite the effort on developing new treatments, therapy for neuropathic pain is still a clinical challenge and combination therapy regimes of two or more drugs are often needed to improve efficacy. Accumulating evidence shows an altered expression and activity of histone acetylation enzymes in chronic pain conditions and restoration of these aberrant epigenetic modifications promotes pain-relieving activity. Recent studies showed a synergistic activity in neuropathic pain models by combination of histone deacetylases (HDACs) and bromodomain and extra-terminal domain (BET) inhibitors. On these premises, the present study investigated the pharmacological profile of new dual HDAC/BRD4 inhibitors, named SUM52 and SUM35, in the spared nerve injury (SNI) model in mice as innovative strategy to simultaneously inhibit HDACs and BETs. Intranasal administration of SUM52 and SUM35 attenuated thermal and mechanical hypersensitivity in the absence of locomotor side effects. Both dual inhibitors showed a preferential interaction with BRD4-BD2 domain, and SUM52 resulted the most active compound. SUM52 reduced microglia-mediated spinal neuroinflammation in spinal cord sections of SNI mice as showed by reduction of IBA1 immunostaining, inducible nitric oxide synthase (iNOS) expression, p65 nuclear factor-κB (NF-κB) and p38 MAPK over-phosphorylation. A robust decrease of the spinal proinflammatory cytokines content (IL-6, IL-1ß) was also observed after SUM52 treatment. Present results, showing the pain-relieving activity of HDAC/BRD4 dual inhibitors, indicate that the simultaneous modulation of BET and HDAC activity by a single molecule acting as multi-target agent might represent a promise for neuropathic pain relief.

Some 2,4-disubstituted quinazolines were synthesized and studied as multidrug resistance (MDR) reversers. The new derivatives carried the quinazoline-4-amine scaffold found in modulators of the ABC transporters involved in MDR, as the TKIs gefitinib and erlotinib. Their behaviour on the three ABC transporters, P-gp, MRP1 and BCRP, was investigated. Almost all compounds inhibited the P-gp activity in MDCK-MDR1 cells overexpressing P-gp, showing EC

The emergence of tumour recurrence and resistance limits the survival rate for most tumour-bearing patients. Only, combination therapies targeting pathways involved in the induction and in the maintenance of cancer growth and progression might potentially result in an enhanced therapeutic efficacy. Herein, we provided a prospective combination treatment that includes suberoylanilide hydroxamic acid (SAHA), a well-known inhibitor of histone deacetylases (HDACs), and SLC-0111, a novel inhibitor of carbonic anhydrase (CA) IX. We proved that HDAC inhibition with SAHA in combination with SLC-0111 affects cell viability and colony forming capability to greater extent than either treatment alone of breast, colorectal and melanoma cancer cells. At the molecular level, this therapeutic regimen resulted in a synergistically increase of histone H4 and p53 acetylation in all tested cell lines. Overall, our findings showed that SAHA and SLC-0111 can be regarded as very attractive combination providing a potential therapeutic strategy against different cancer models.

Despite the intense research on developing new therapies for neuropathic pain states, available treatments have limited efficacy and unfavorable safety profiles. Epigenetic alterations have a great influence on the development of cancer and neurological diseases, as well as neuropathic pain. Histone acetylation has prevailed as one of the well investigated epigenetic modifications in these diseases. Altered spinal activity of histone deacetylase (HDAC) and Bromo and Extra terminal domain (BET) have been described in neuropathic pain models and restoration of these aberrant epigenetic modifications showed pain-relieving activity. Over the last decades HDACs and BETs have been the focus of drug discovery studies, leading to the development of numerous small-molecule inhibitors. Clinical trials to evaluate their anticancer activity showed good efficacy but raised toxicity concerns that limited translation to the clinic. To maximize activity and minimize toxicity, these compounds can be applied in combination of sub-maximal doses to produce additive or synergistic interactions (combination therapy). Recently, of particular interest, dual BET/HDAC inhibitors (multi-target drugs) have been developed to assure simultaneous modulation of BET and HDAC activity by a single molecule. This review will summarize the most recent advances with these strategies, describing advantages and limitations of single drug treatment vs combination regimens. This review will also provide a focus on dual BET/HDAC drug discovery investigations as future therapeutic opportunity for human therapy of neuropathic pain.

The aim of this study is to determine whether type I and type II positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptors (nAChRs) induce antidepressant-like activity in mice after acute, subchronic, and chronic treatments, and to assess whether α7-PAMs inhibit neurotransmitter transporters and activate mTOR (mammalian target of rapamycin) and/or ERK (extracellular signal-regulated protein kinases) signaling. The forced swim (FST) and tail suspension (TST) test results indicated that NS-1738 (type I PAM), PNU-120596 and PAM-2 (type II PAMs) induce antidepressant-like activity after subchronic treatment, whereas PAM-2 was also active after chronic treatment. Methyllycaconitine (α7-antagonist) inhibited the observed effects, highlighting the involvement of α7 nAChRs in this process. Drug interaction studies showed synergism between PAM-2 and bupropion (antidepressant), but not between PAM-2 and DMXBA (α7-agonist). The studied PAMs showed no high affinity ( 1 µM) for the human dopamine, serotonin, and noradrenaline transporters, suggesting that transporter inhibition is not the underlying mechanism for the observed activity. To assess whether mTOR and ERK signaling pathways are involved in the activity of α7-PAMs, the phosphorylation status of key signaling nodes was determined in prefrontal cortex and hippocampus from mice chronically treated with PAM-2. In conclusion, the antidepressant-like activity of type I and type II PAMs is mediated by a mechanism involving α7 potentiation but not α7 desensitization or neurotransmitter transporter blockade, and is correlated with activation of both mTOR and ERK signaling pathways. These results support the view that α7-PAMs might be clinically used to ameliorate depression disorders .

In this paper, the efficiency of the carbonic anhydrase (CA) enzyme in accelerating the hydration of CO2 is evaluated using a measurement system which consists of a vessel in which a gaseous flow of mixtures of nitrogen and CO2 is bubbled into water or water solutions containing a known quantity of CA enzyme. The pH value of the solution and the CO2 concentration at the measurement system gas exhaust are continuously monitored. The measured CO2 level allows for assessing the quantity of CO2, which, subtracted from the gaseous phase, is dissolved into the liquid phase and/or hydrated to bicarbonate. The measurement procedure consists of inducing a transient and observing and modelling the different kinetics involved in the steady-state recovery with and without CA. The main contribution of this work is exploiting dynamical system theory and chemical kinetics modelling for interpreting measurement results for characterising the activity of CA enzymes. The data for model fitting are obtained from a standard bioreactor, in principle equal to standard two-phase bioreactors described in the literature, in which two different techniques can be used to move the process itself away from the steady-state, inducing transients.

Hyperpolarization-activated cation channels are involved, among other functions, in learning and memory, control of synaptic transmission and epileptogenesis. The importance of the HCN1 and HCN2 isoforms for brain function has been demonstrated, while the role of HCN4, the third major neuronal HCN subunit, is not known. Here we show that HCN4 is essential for oscillatory activity in the thalamocortical (TC) network. HCN4 is selectively expressed in various thalamic nuclei, excluding the thalamic reticular nucleus. HCN4-deficient TC neurons revealed a massive reduction of Ih and strongly reduced intrinsic burst firing, whereas the current was normal in cortical pyramidal neurons. In addition, evoked bursting in a thalamic slice preparation was strongly reduced in the mutant mice probes. HCN4-deficiency also significantly slowed down thalamic and cortical oscillations during active wakefulness. Taken together, these results establish that thalamic HCN4 channels are essential for the production of rhythmic intrathalamic oscillations and determine regular TC oscillatory activity during alert states.

A three-dimensional database search has been applied to design a series of endo- and exo-3-(pyridin-3-yl)bicyclo[2.2.1]heptan-2-amines as nicotinic receptor ligands. The synthesized compounds were tested in radioligand binding assay on rat cortex against [3H]-cytisine and [3H]-methyllycaconitine to measure their affinity for α4β2* and α7* nicotinic receptors. The new derivatives showed some preference for the α4β2* over the α7* subtype, with their affinity being dependent on the endo/exo isomerism and on the methylation degree of the basic nitrogen. The endo primary amines displayed the lowest Ki values on both receptor subtypes. Selected compounds (1a, 2a, 3a, and 6a) were tested on heterologously expressed α4β2, α7, and α3β2 receptors and on SHSY-5Y cells. Compounds 1a and 2a showed α4β2 antagonistic properties while behaved as full agonists on recombinant α7 and on SHSY5Y cells. On the α3β2 subtype, only the chloro derivative 2a showed full agonist activity and submicromolar potency (EC50 = 0.43 μM). T...

A new series of N,N-bis(alkanol)amine aryl diesters was synthesized and studied as dual P-glycoprotein (P-gp) and carbonic anhydrase XII inhibitors (CA XII). These hybrids should be able to synergistically overcome P-gp mediated multidrug resistance (MDR) in cancer cells. It was reported that the efflux activity of P-gp could be modulated by CA XII, as the pH reduction caused by CA XII inhibition produces a significant decrease in P-gp ATPase activity. The new compounds reported here feature both P-gp and CA XII binding moieties. These hybrids contain a N,N-bis(alkanol)amine diester scaffold found in P-glycoprotein ligands and a coumarin or benzene sulfonamide moiety to target CA XII. Many compounds displayed a dual activity against P-gp and CA XII being active in the Rhd 123 uptake test on K562/DOX cells and in the hCA XII inhibition test. On LoVo/DOX cells, that overexpress both P-gp and CA XII, some coumarin derivatives showed a high MDR reversal effect in Rhd 123 uptake and doxorubicin cytotoxicity enhancement tests. In particular, compounds 7 and 8 showed higher activity than verapamil and were more potent on LoVo/DOX than on K562/DOX cells overexpressing only P-gp. They can be considered as valuable candidates for selective P-gp/CA XII inhibition in MDR cancer cells.

The hyperpolarization-activated cyclic-nucleotide-gated (HCN) proteins are voltage-dependent ion channels, conducting both Na+and K+, blocked by millimolar concentrations of extracellular Cs+and modulated by cyclic nucleotides (mainly cAMP) that contribute crucially to the pacemaker activity in cardiac nodal cells and subsidiary pacemakers. Over the last decades, much attention has focused on HCN current, If, in non-pacemaker cardiac cells and its potential role in triggering arrhythmias. In fact, in addition to pacemakers, HCN current is constitutively present in the human atria and has long been proposed to sustain atrial arrhythmias associated to different cardiac pathologies or triggered by various modulatory signals (catecholamines, serotonin, natriuretic peptides). An atypical Ifoccurs in diseased ventricular cardiomyocytes, its amplitude being linearly related to the severity of cardiac hypertrophy. The properties of atrial and ventricular Ifand its modulation by pharmacological interventions has been object of intense study, including the synthesis and characterization of new compounds able to block preferentially HCN1, HCN2, or HCN4 isoforms. Altogether, clues emerge for opportunities of future pharmacological strategies exploiting the unique properties of this channel family: the prevalence of different HCN subtypes in organs and tissues, the possibility to target HCN gain- or loss-of-function associated with disease, the feasibility of novel isoform-selective drugs, as well as the discovery of HCN-mediated effects for old medicines.

Targeting Carbonic Anhydrases (CAs) represents a strategy to treat several diseases, from glaucoma to cancer. To widen the structure-activity relationships (SARs) of our series of piperazines endowed with potent human carbonic anhydrase (hCA) inhibition, a new series of chiral piperazines carrying a (2-hydroxyethyl) group was prepared. The Zn-binding function, the 4-sulfamoylbenzoyl moiety, was connected to one piperazine N-atom, while the other nitrogen was decorated with alkyl substituents. In analogy to the approach used for the synthesis of the previously reported series, the preparation of the new compounds started with (R)- and (S)-aspartic acid. A partial racemization occurred during the synthesis. In order to overcome this problem, other chemical strategies were investigated. The inhibitory activity of the new polar derivatives against four hCAs isoforms I, II, IV and IX using a stopped flow CO

Chemotherapy-Induced Peripheral Neuropathy (CIPN) is the most frequent adverse effect of pharmacological cancer treatments. The occurrence of neuropathy prevents the administration of fully-effective drug regimen, affects negatively the quality of life of patients, and may lead to therapy discontinuation. CIPN is currently treated with anticonvulsants, antidepressants, opioids and non-opioid analgesics, all of which are flawed by insufficient anti-hyperalgesic efficacy or addictive potential. Understandably, developing new drugs targeting CIPN-specific pathogenic mechanisms would dramatically improve efficacy and tolerability of anti-neuropathic therapies. Neuropathies are associated to aberrant excitability of DRG neurons due to the alteration in the expression or function of a variety of ion channels. In this regard, Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels are overexpressed in inflammatory and neuropathic pain states, and HCN blockers have been shown to reduce neuronal excitability and to ameliorate painful states in animal models. However, HCN channels are critical in cardiac action potential, and HCN blockers used so far in pre-clinical models do not discriminate between cardiac and non-cardiac HCN isoforms. In this work, we show an HCN current gain of function in DRG neurons from oxaliplatin-treated rats. Biochemically, we observed a downregulation of HCN2 expression and an upregulation of the HCN regulatory beta-subunit MirP1. Finally, we report the efficacy of the selective HCN1 inhibitor MEL57A in reducing hyperalgesia and allodynia in oxaliplatin-treated rats without cardiac effects. In conclusion, this study strengthens the evidence for a disease-specific role of HCN1 in CIPN, and proposes HCN1-selective inhibitors as new-generation pain medications with the desired efficacy and safety profile.

Nicotinic receptors are membrane proteins involved in several physiological processes. They are considered suitable drug targets for various CNS disorders or conditions, as shown by the large number of compounds which have entered clinical trials. In recent years, nonconventional agonists have been discovered: positive allosteric modulators, allosteric agonists, site-specific agonists and silent desensitizers are compounds able to modulate the receptor interacting at sites different from the orthodox one, or to desensitize the receptor without prior opening. While these new findings can further complicate the pharmacology of these proteins and the design and optimization of ligands, they undoubtedly offer new opportunities to find drugs for the many therapeutic indications involving nicotinic receptors.

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels are important members of the voltage-gated pore loop channels family. They show unique features: they open at hyperpolarizing potential, carry a mixed Na/K current, and are regulated by cyclic nucleotides. Four different isoforms have been cloned (HCN1-4) that can assemble to form homo- or heterotetramers, characterized by different biophysical properties. These proteins are widely distributed throughout the body and involved in different physiologic processes, the most important being the generation of spontaneous electrical activity in the heart and the regulation of synaptic transmission in the brain. Their role in heart rate, neuronal pacemaking, dendritic integration, learning and memory, and visual and pain perceptions has been extensively studied; these channels have been found also in some peripheral tissues, where their functions still need to be fully elucidated. Genetic defects and altered expression of HCN channels are linked to several pathologies, which makes these proteins attractive targets for translational research; at the moment only one drug (ivabradine), which specifically blocks the hyperpolarization-activated current, is clinically available. This review discusses current knowledge about HCN channels, starting from their biophysical properties, origin, and developmental features, to (patho)physiologic role in different tissues and pharmacological modulation, ending with their present and future relevance as drug targets.

Schizophrenic (SCH) patients show cognitive impairment in attentional performance. Positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptors (nAChRs) such as the Alzheimer’s drug galantamine (GAL) and PAM-2 are documented to have pro-cognitive properties. However, it is not well established if these properties would be lost, or may hamper antipsychotic efficacy, when given as an adjunct to an antipsychotic which is needed for managing psychotic symptoms. Using adult male Wistar rats, we here investigated the effects of: a) GAL, alone or co-administered with the antipsychotic risperidone (RISP), on acute phencyclidine (PCP)-induced deficits in the attentional set-shifting (ASST) test; b) PAM-2, alone and co-administered with RISP, in the conditioned avoidance response (CAR) test for antipsychotic activity. Acute PCP produced selective and significant SCH-like impairment in extra dimensional shift (EDS) performance, which was completely reversed by GAL. The ability of GAL to reverse PCP-induced EDS impairment was not prevented when co-administered with RISP, suggesting that the combination of GAL and low doses of RISP may be used to improve the cognitive impairment in SCH. Pretreatment with methyllycaconitine (MLA), a selective α7 nAChR antagonist, completely prevented the reversal elicited by GAL, supporting the concept that α7 nAChRs are involved in this process. On the other hand, PAM-2 alone had no effects on CAR, but enhanced, although not significantly, the antipsychotic-like effect of RISP when administered together. In conclusion, α7 PAMs, in addition to alleviate the cognitive impairments observed in SCH patients, may enhance the antipsychotic efficacy of atypical antipsychotics.

A series of derivatives were synthesized and studied with the aim to investigate the structure-activity relationships of the two P-glycoprotein (P-gp) modulators elacridar and tariquidar. Then, different aryl-substituted amides were inserted, and to explore the effects of varying the amide function, the corresponding isosteric ester derivatives and some alkylamine analogues were synthesized. The new compounds were studied to evaluate their P-gp interaction profile and selectivity toward the two other ABC transporters, multidrug-resistance-associated protein-1 (MRP-1) and breast cancer resistance protein (BCRP). Investigation of the chemical stability of the amide and ester derivatives toward spontaneous or enzymatic hydrolysis showed that these compounds were stable in phosphate-buffered saline and human plasma. This study allowed us to evaluate the selectivity of the three series on the three efflux pumps and to propose the structural requirements that define the P-gp interaction profile. We identified two P-gp substrates, a P-gp inhibitor, and three ester derivatives that were active on BCRP, which opens a new scenario in the development of ligands active toward this pump.

Although agonists and antagonists of muscarinic receptors have been known for long time, there is renewed interest in compounds (such as allosteric or bitopic ligands, or biased agonists) able to differently and selectively modulate these receptors. As a continuation of our previous research, we designed a new series of dimers of the well-known cholinergic agonist carbachol. The new compounds were tested on the five cloned human muscarinic receptors (hM1–5) expressed in CHO cells by means of equilibrium binding experiments, showing a dependence of the binding affinity on the length and position of the linker connecting the two monomers. Kinetic binding studies revealed that some of the tested compounds were able to slow the rate of NMS dissociation, suggesting allosteric behavior, also supported by docking simulations. Assessment of ERK1/2 phosphorylation on hM1, hM2 and hM3 activation showed that the new compounds are endowed with muscarinic antagonist properties. At hM2 receptors, some compounds were able to stimulate GTPγS binding but not cAMP accumulation, suggesting a biased behavior. Classification, Molecular and cellular pharmacology.

The development of multidrug resistance (MDR) is one of the major challenges to the success of chemotherapy treatment of cancer. This phenomenon is often associated with the overexpression of the ATP-binding cassette (ABC) transporters P-gp (P-glycoprotein, ABCB1), multidrug resistance-associated protein 1, ABCC1 and breast cancer resistance protein, ABCG2 (BCRP). These transporters are constitutively expressed in many tissues playing relevant protective roles by the regulation of the permeability of biological membranes, but they are also overexpressed in malignant tissues. P-gp is the first efflux transporter discovered to be involved in cancer drug resistance, and over the years, inhibitors of this pump have been disclosed to administer them in combination with chemotherapeutic agents. Three generations of inhibitors of P-gp have been examined in preclinical and clinical studies; however, these trials have largely failed to demonstrate that coadministration of pump inhibitors elicits an improvement in therapeutic efficacy of antitumor agents, although some of the latest compounds show better results. Therefore, new and innovative strategies, such as the fallback to natural products and the discover of dual activity ligands emerged as new perspectives. BCRP is the most recently ABC protein identified to be involved in multidrug resistance. It is overexpressed in several haematological and solid tumours together with P-gp, threatening the therapeutic effectiveness of different chemotherapeutic drugs. The chemistry of recently described BCRP inhibitors and dual P-gp/BCRP inhibitors, as well as their preliminary pharmacological evaluation are discussed, and the most recent advances concerning these kinds of MDR modulators are reviewed.

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels are members of the voltage-gated K+ channels family, but with unique properties. In stark contrast to close relatives, HCN channels are permeable to both Na+ and K+, and they are activated by hyperpolarization. Activation by hyperpolarization is indeed a pretty funny feature, to the point that the physiologists who first characterized HCN current in heart muscle cells named it “funny current” or If. Since then, the funny current has also been recorded from several neuronal types in both the central and peripheral nervous systems, as well as from some non-excitable cells, becoming progressively less “funny” over the years. In fact, HCN current goes now by the more serious designation of “Ih,” for “hyperpolarization-activated.” Forty years after the first current recording, it is now established that HCN channels, by virtue of their special properties and a host of modulatory mechanisms, are profoundly involved in many critical aspects of neuronal function in physiological and pathological conditions.

In a continuing search for potent P-gp-dependent multidrug-resistant (MDR) reversers we synthesized and studied a new series of N-alkanol-N-cyclohexanol amine aryl esters characterized by the presence of two linkers with different flexibility: a polymethylene chain of variable length and a cyclohexylic scaffold, that gave origin to two geometrical isomers (cis and trans). The reversal activity of the new compounds was evaluated on the K562/DOX cell line by three tests: pirarubicin uptake modulation, doxorubicin cytotoxicity enhancement (reversal fold, RF) and inhibition of P-gp-mediated rhodamine-123 (Rhd 123) efflux tests. The chemical stability of their ester function was evaluated in the experimental conditions utilized (phosphate buffer solution (PBS), bovine serum and in the presence of K562/DOX cells) and in human plasma. The new series of molecules showed very interesting MDR reversing properties; in particular compound 5b (ELF26B), characterized by trans stereochemistry and a 5-methylene chain, presented the best pharmacological profile and is stable in each tested medium. Compound 5b could be an interesting lead for the development of new potent and efficacious P-gp-dependent MDR modulators.

A series of homodimers of the well-known cholinergic agonist carbachol have been synthesized, showing the two agonist units symmetrically connected through a methylene chain of variable length. The new compounds have been tested on the five cloned muscarinic receptors (hM1-5) expressed in CHO cells by means of equilibrium binding studies, showing an increase in affinity by rising the number of methylene units up to 7 and 9. Functional experiments on guinea-pig ileum and assessment of ERK1/2 phosphorylation on hM1, hM2 and hM3 on CHO cells have shown that the new compounds are endowed with muscarinic antagonistic properties. Kinetic binding studies have revealed that some of the tested compounds are able to slow the rate of dissociation of NMS, suggesting a bitopic behavior. Docking simulations, performed on the hM1 and hM2 receptors, give a sound rationalization of the experimental data revealing how these compounds are able to interact with both orthosteric and allosteric binding sites depending on the length of their connecting chain.

In this study, a new series of N,N-bis(alkanol)amine aryl ester heterodimers was synthesized and studied. The new compounds were designed based on the structures of our previous arylamine ester derivatives endowed with high P-gp-dependent multidrug resistance reversing activity on a multidrug-resistant leukemia cell line. All new compounds were active in the pirarubicin uptake assay on the doxorubicin–resistant erythroleukemia K562 cells (K562/DOX). Compounds bearing a linker made up of 10 methylenes showed unprecedented high reversal activities regardless of the combination of aromatic moieties. Docking results obtained by an in silico study supported the data obtained by the biological tests and a study devoted to establish the chemical stability in phosphate buffer solution (PBS) and human plasma showed that only a few compounds exhibited a significant degradation in the human plasma matrix. Ten selected non-hydrolysable derivatives were able to inhibit the P-gp-mediated rhodamine-123 efflux on K562/DOX cells, and the evaluation of their apparent permeability and ATP consumption on other cell lines suggested that the compounds can behave as unambiguous or not transported substrates. The activity of these the compounds on the transport proteins breast cancer resistance protein (BCRP) and multidrug resistance associated protein 1 (MRP1) was also analyzed. All tested derivatives displayed a moderate potency on the BCRP overexpressing cells; while only four molecules showed to be effective on MRP1 overexpressing cells, highlighting a clear structural requirement for selectivity. In conclusion, we have identified a new very powerful series of compounds which represent interesting leads for the development of new potent and efficacious P-gp-dependent MDR modulators.

Stereo- and regioisomers of a series of N,N-bis(alkanol)amine aryl ester derivatives have been prepared and studied as multidrug resistance (MDR) modulators. The new compounds contain a 2-(methyl)propyl chain combined with a 3-, 5- or 7-methylenes long chain and carry different aromatic ester portions. Thus, these compounds have a methyl group on the 3-methylenes chain and represent branched homologues of previously studied derivatives. The introduction of the methyl group gives origin to a stereogenic center and consequently to (R) and (S) enantiomers. In the pirarubicin uptake assay on K562/DOX cell line these compounds showed good activity and efficacy and in many cases enantioselectivity was observed. Docking studies confirmed the influence of the stereocenter on the interaction in the P-gp pocket. The P-gp interaction mechanism and selectivity towards MRP1 and BCRP were also evaluated on MDCK transfected cells overexpressing the three transporters. Almost all these compounds inhibited both P-gp and BCRP, but only derivatives with specific structural characteristics showed MRP1 activity. Moreover, two compounds, (S)-3 and (R)-7, showed the ability to induce collateral sensitivity (CS) against MDR cells. Therefore, these two CS-promoting agents could be considered interesting leads for the development of selective cytotoxic agents for drug-resistant cells.