Arylamino Esters As P‐Glycoprotein Modulators: SAR Studies to Establish Requirements for Potency and Selectivity

A set of basic aryl‐group‐containing compounds was synthesized with the aim of developing potent and selective P‐glycoprotein (P‐gp) modulators that are able to reverse multidrug resistance (MDR). The natures of the spacer (dicyclohexylamine or dialkylamine) and the aryl moieties were modified to investigate selectivity and the mechanism of P‐gp interaction. The inhibitory activities of the compounds toward P‐gp, multidrug resistance‐associated protein 1 (MRP1), and breast cancer resistance protein (BCRP), the most relevant ATP binding cassette (ABC) transporters for MDR, were evaluated. The mechanism of P‐gp interaction for each compound was investigated with three biological assays: apparent permeability (P_app) determination (B→A/A→B) in Caco‐2 cell monolayers, ATP cell depletion, and inhibition of Calcein‐AM transport in MDCK‐MDR1 cells. These assays allowed us to estimate the selectivity of the compounds for the three efflux pumps and to identify the structural requirements that define the P‐gp‐interaction profile. All dicyclohexylamine derivatives were found to be P‐gp substrates, whereas one dialkylamine derivative was shown to be a P‐gp inhibitor. The good MRP1 activity of one cis/cis isomer highlighted this as a lead candidate for the development of MRP1 ligands.     

Flavonoid Dimers as Bivalent Modulators for P‐Glycoprotein‐Based Multidrug Resistance: Structure–Activity Relationships

Bivalent modulators of P‐glycoprotein : A small library of flavonoid homodimers and heterodimers was synthesized, and their in vitro activity in reversing paclitaxel resistance was evaluated along with structure–activity relationships. SAR trends indicate that flavonoid dimers with nonpolar, hydrophobic, less bulky substituents generally show more potent reversing activity. This will help guide future efforts in the search for more potent compounds.

     

From Quinoline to Quinazoline-Based S. aureus NorA Efflux Pump Inhibitors by Coupling a Focused Scaffold Hopping Approach and a Pharmacophore Search

Antibiotic resistance breakers, such as efflux pump inhibitors (EPIs), represent a powerful alternative to the development of new antimicrobials. Recently, by using previously described EPIs, we developed pharmacophore models able to identify inhibitors of NorA, the most studied efflux pump of Staphylococcus aureus. Herein we report the pharmacophore-based virtual screening of a library of new potential NorA EPIs generated by an in-silico scaffold hopping approach of the quinoline core. After chemical synthesis and biological evaluation of the best virtual hits, we found the quinazoline core as the best performing scaffold. Accordingly, we designed and synthesized a series of functionalized 2-arylquinazolines, which were further evaluated as NorA EPIs. Four of them exhibited a strong synergism with ciprofloxacin and a good inhibition of ethidium bromide efflux on resistant S. aureus strains coupled with low cytotoxicity against human cell lines, thus highlighting a promising safety profile.     

6,7‐Dimethoxy‐2‐{2‐[4‐(1H‐1,2,3‐triazol‐1‐yl)phenyl]ethyl}‐1,2,3,4‐tetrahydroisoquinolines as Superior Reversal Agents for P‐Glycoprotein‐Mediated Multidrug Resistance

P‐glycoprotein (P‐gp)‐mediated multidrug resistance (MDR) is a major obstacle for successful cancer chemotherapy. Based on our previous study, 17 novel compounds with the 6,7‐dimethoxy‐2‐{2‐[4‐(1H‐1,2,3‐triazol‐1‐yl)phenyl]ethyl}‐1,2,3,4‐tetrahydroisoquinoline scaffold were designed and synthesized. Among them, 2‐[(1‐{4‐[2‐(6,7‐dimethoxy‐3,4‐dihydroisoquinolin‐2(1H)‐yl)ethyl]phenyl}‐1H‐1,2,3‐triazol‐4‐yl)methoxy]‐N‐(p‐tolyl)benzamide (compound 7 h ) was identified as a potent modulator of P‐gp‐mediated MDR, with high potency (EC₅₀=127.5±9.1 nM ), low cytotoxicity (TI>784.3), and long duration (>24 h) in reversing doxorubicin (DOX) resistance in K562/A02 cells. Compound 7 h also enhanced the effects of other MDR‐related cytotoxic agents (paclitaxel, vinblastine, and daunorubicin), increased the accumulation of DOX and blocked P‐gp‐mediated rhodamine 123 efflux function in K562/A02 MDR cells. Moreover, 7 h did not have any effect on cytochrome (CYP3A4) activity. These results indicate that 7 h is a relatively safe modulator of P‐gp‐mediated MDR that has good potential for further development.     

A Potent and Selective P‐gp Modulator for Altering Multidrug Resistance Due to Pump Overexpression

P‐glycoprotein (P‐gp) is a membrane protein responsible for the active transport of several endogenous and exogenous substances. It constitutes a defense mechanism and, at the same time, it severely compromises the success rate of antitumor chemotherapy. In this study a small library of alkyl/oxyalkyl derivatives of MC70 [4′‐(6,7‐dimethoxy‐3,4‐dihydro‐1H‐isoquinolin‐2‐ylmethyl)biphenyl‐4‐ol], a well‐known P‐gp inhibitor, was synthesized through straightforward functionalization of the phenolic group present in the structure of MC70. All compounds were characterized for their effect on P‐gp, proving capable of blocking P‐gp‐mediated calcein‐AM efflux with micromolar potency, following their ability to act as high‐affinity substrates of this transporter. Excitingly, compound 4 [6,7‐dimethoxy‐2‐((4′‐butoxybiphen‐4‐yl)methyl)‐1,2,3,4‐tetrahydroisoquinoline] exhibited low nanomolar potency (5.2 nm ) and had a peculiar activity profile, acting both as a positive allosteric modulator and as a substrate of the transporter. A new and more efficient synthesis of MC70 is also described.     

Interactions of the Multidrug Resistance Modulators Tariquidar and Elacridar and their Analogues with P‐glycoprotein

Tariquidar and elacridar are among the most potent inhibitors of the multidrug resistance transporter P‐glycoprotein (P‐gp), but how they interact with the protein is yet unknown. In this work, we describe a possible way in which these inhibitors interact with P‐gp. We rely on structure–activity relationship analysis of a small group of tariquidar and elacridar analogues that was purposefully selected, designed, and tested. Structural modifications of the compounds relate to the presence or absence of functional groups in the tariquidar and elacridar scaffolds. The activity of the compounds was evaluated by their effects on the accumulation of P‐gp substrates rhodamine 123 and Hoechst 33342 in resistant tumor cells. The data allow estimation of the ability of the compounds to interact with the experimentally proposed R‐ and H‐sites to which rhodamine 123 and Hoechst 33342 bind, respectively. Using an inward‐facing homology model of human P‐gp based on the crystallographic structure of mouse P‐gp, we demonstrate that these binding sites may overlap with the binding sites of the QZ59 ligands co‐crystallized with mouse P‐gp. Based on this SAR analysis, and using flexible alignment and docking, we propose possible binding modes for tariquidar and elacridar. Our results suggest the possibility for the studied compounds to bind to sites that coincide or overlap with the binding sites of rhodamine 123 and Hoechst 33342. These results contribute to further understanding of structure–function relationships of P‐gp and can help in the design of selective and potent P‐gp inhibitors with potential clinical use.     

Design,Synthesis, and Biological Evaluation of (S)‐Valine Thiazole‐Derived Cyclic and Noncyclic Peptidomimetic Oligomers as Modulators of Human P‐Glycoprotein (ABCB1)

Multidrug resistance caused by ATP binding cassette transporter P‐glycoprotein (P‐gp) through extrusion of anticancer drugs from the cells is a major cause of failure in cancer chemotherapy. Previously, selenazole‐containing cyclic peptides were reported as P‐gp inhibitors and were also used for co‐crystallization with mouse P‐gp, which has 87 % homology to human P‐gp. It has been reported that human P‐gp can simultaneously accommodate two to three moderately sized molecules at the drug binding pocket. Our in silico analysis, based on the homology model of human P‐gp, spurred our efforts to investigate the optimal size of (S)‐valine‐derived thiazole units that can be accommodated at the drug binding pocket. Towards this goal, we synthesized varying lengths of linear and cyclic derivatives of (S)‐valine‐derived thiazole units to investigate the optimal size, lipophilicity, and structural form (linear or cyclic) of valine‐derived thiazole peptides that can be accommodated in the P‐gp binding pocket and affects its activity, previously an unexplored concept. Among these oligomers, lipophilic linear ( 13 ) and cyclic trimer ( 17 ) derivatives of QZ59S‐SSS were found to be the most and equally potent inhibitors of human P‐gp (IC₅₀=1.5 μM ). As the cyclic trimer and linear trimer compounds are equipotent, future studies should focus on noncyclic counterparts of cyclic peptides maintaining linear trimer length. A binding model of the linear trimer 13 within the drug binding site on the homology model of human P‐gp represents an opportunity for future optimization, specifically replacing valine and thiazole groups in the noncyclic form.     

1‐(1H‐Indol‐3‐yl)ethanamine Derivatives as Potent Staphylococcus aureus NorA Efflux Pump Inhibitors

The synthesis of 37 1‐(1H‐indol‐3‐yl)ethanamine derivatives, including 12 new compounds, was achieved through a series of simple and efficient chemical modifications. These indole derivatives displayed modest or no intrinsic anti‐staphylococcal activity. By contrast, several of the compounds restored, in a concentration‐dependent manner, the antibacterial activity of ciprofloxacin against Staphylococcus aureus strains that were resistant to fluoroquinolones due to overexpression of the NorA efflux pump. Structure–activity relationships studies revealed that the indolic aldonitrones halogenated at position 5 of the indole core were the most efficient inhibitors of the S. aureus NorA efflux pump. Among the compounds, (Z)‐N‐benzylidene‐2‐(tert‐butoxycarbonylamino)‐1‐(5‐iodo‐1H‐indol‐3‐yl)ethanamine oxide led to a fourfold decrease of the ciprofloxacin minimum inhibitory concentration against the SA‐1199B strain when used at a concentration of 0.5 mg L ^?1. To the best of our knowledge, this activity is the highest reported to date for an indolic NorA inhibitor. In addition, a new antibacterial compound, tert‐butyl (2‐(3‐hydroxyureido)‐2‐(1H‐indol‐3‐yl)ethyl)carbamate, which is not toxic for human cells, was also found.     

Specific Inhibitors of the Breast Cancer Resistance Protein (BCRP)

A new class of specific breast cancer resistance protein (BCRP) inhibitors was identified, showing no inhibition of the ATP binding cassette (ABC) transporters P‐gp and MRP1. Some of these modulators inhibit BCRP with high potency; they are only slightly less potent than Ko143 and could serve as promising lead structures for the design of novel effective BCRP inhibitors. These inhibitors are structurally related to tariquidar (XR9576) and belong to a library of multidrug‐resistance modulators synthesized by our research group. The absence of the tetrahydroisoquinoline substructure appears to play a crucial role for specificity; we found that the presence of this substructure is not essential for interaction with BCRP. To determine the type of interaction between pheophorbide A and compounds with and without the tetrahydroisoquinoline substructure, various substrate pheophorbide A concentrations were used in enzyme kinetics assays. The resulting data show that these compounds share a noncompetitive‐type interaction with pheophorbide A. Experiments with imatinib and pheophorbide A revealed a mixed‐type interaction. The combination of imatinib and compounds with and without the tetrahydroisoquinoline substructure resulted in a positive cooperative effect, indicating that imatinib engages a binding site distinct from that of the new compounds on one side and distinct from that of pheophorbide A on the other side as well. The results of this study suggest that the category of BCRP‐specific inhibitors, which includes only fumitremorgin C, Ko143 and analogues, and novobiocin needs to be extended by this new class of inhibitors, which possess three key characteristics: specificity, potency, and low toxicity.     

Multi‐Drug‐Resistance‐Reverting Agents: 2‐Aryloxazole and 2‐Arylthiazole Derivatives as Potent BCRP or MRP1 Inhibitors

The 2‐aryloxazole and 2‐arylthiazole scaffolds were used for generating compounds that we characterized for their inhibitory activity toward ATP binding cassette transporters involved in multi‐drug resistance, such as BCRP and MRP1, by using tumor cell lines overexpressing each transporter. These SAR studies are a significant step toward improving the inhibitory potency against P‐glycoprotein, BCRP, and MRP1.

     

Enzyme Inhibitors: The Best Strategy to Tackle Superbug NDM-1 and Its Variants

Multidrug bacterial resistance endangers clinically effective antimicrobial therapy and continues to cause major public health problems, which have been upgraded to unprecedented levels in recent years, worldwide. β-Lactam antibiotics have become an important weapon to fight against pathogen infections due to their broad spectrum. Unfortunately, the emergence of antibiotic resistance genes (ARGs) has severely astricted the application of β-lactam antibiotics. Of these, New Delhi metallo-β-lactamase-1 (NDM-1) represents the most disturbing development due to its substrate promiscuity, the appearance of variants, and transferability. Given the clinical correlation of β-lactam antibiotics and NDM-1-mediated resistance, the discovery, and development of combination drugs, including NDM-1 inhibitors, for NDM-1 bacterial infections, seems particularly attractive and urgent. This review summarizes the research related to the development and optimization of effective NDM-1 inhibitors. The detailed generalization of crystal structure, enzyme activity center and catalytic mechanism, variants and global distribution, mechanism of action of existing inhibitors, and the development of scaffolds provides a reference for finding potential clinically effective NDM-1 inhibitors against drug-resistant bacteria.     

A New Class of 1‐Aryl‐5,6‐dihydropyrrolo[2,1‐a]isoquinoline Derivatives as Reversers of P‐Glycoprotein‐Mediated Multidrug Resistance in Tumor Cells

A number of aza‐heterocyclic compounds, which share the 5,6‐dihydropyrrolo[2,1‐a]isoquinoline (DHPIQ) scaffold with members of the lamellarin alkaloid family, were synthesized and evaluated for their ability to reverse in vitro multidrug resistance in cancer cells through inhibition of P‐glycoprotein (P‐gp) and/or multidrug‐resistance‐associated protein 1. Most of the investigated DHPIQ compounds proved to be selective P‐gp modulators, and the most potent modulator, 8,9‐diethoxy‐1‐(3,4‐diethoxyphenyl)‐3‐(furan‐2‐yl)‐5,6‐dihydropyrrolo[2,1‐a]isoquinoline‐2‐carbaldehyde, attained sub‐micromolar inhibitory potency (IC₅₀: 0.19 μm ). Schiff bases prepared by the condensation of some 1‐aryl‐DHPIQ aldehydes with p‐aminophenol also proved to be of some interest, and one of them, 4‐((1‐(4‐fluorophenyl)‐5,6‐dihydro‐8,9‐dimethoxypyrrolo[2,1‐a]isoquinolin‐2‐yl)methyleneamino)phenol, had an IC₅₀ value of 1.01 μm . In drug combination assays in multidrug‐resistant cells, some DHPIQ compounds, at nontoxic concentrations, significantly increased the cytotoxicity of doxorubicin in a concentration‐dependent manner. Studies of structure–activity relationships and investigation of the chemical stability of Schiff bases provided physicochemical information useful for molecular optimization of lamellarin‐like cytotoxic drugs active toward chemoresistant tumors as well as nontoxic reversers of P‐gp‐mediated multidrug resistance in tumor cells.     

Activators of P‐glycoprotein: Structure–Activity Relationships and Investigation of their Mode of Action

P‐glycoprotein (P‐gp), a 170 kDa plasma membrane protein, is one of the most relevant ABC transporters involved in the development of multidrug resistance (MDR). Understanding its mechanism of transport as well as its interactions with various substrates are basic requirements for the development of adequate therapeutic approaches to overcome this kind of resistance against a broad spectrum of structurally unrelated cytostatic drugs. P‐gp modulators (activators) that exert various effects on the intracellular accumulation of distinct P‐gp substrates are useful tools for investigating the interactions between multiple drug binding sites of this transport protein. In this study, a series of 27 different imidazobenzothiazoles and imidazobenzimidazoles structurally related to the known P‐gp activators QB102 and QB11 was designed, and their modulating properties were investigated. Most of them were able to stimulate P‐gp‐mediated efflux of daunorubicin and rhodamine 123 in a concentration‐dependent manner, but some compounds also displayed weak inhibitory effects. Additionally, P‐gp‐mediated efflux of vinblastine and colchicine was inhibited by several compounds. Therefore, we concluded that the novel compounds bind to the H site of P‐gp and activate the efflux of specific substrates of the R site in a positive cooperative manner, whereas binding of H‐type substrates is inhibited competitively. This hypothesis is confirmed by the observation that the modulators do not influence hydrolysis of ATP or its affinity toward P‐gp.     

Design of gem‐Difluoro‐bis‐Tetrahydrofuran as P2 Ligand for HIV‐1 Protease Inhibitors to Improve Brain Penetration: Synthesis,X‐ray Studies,and Biological Evaluation

The structure‐based design, synthesis, biological evaluation, and X‐ray structural studies of fluorine‐containing HIV‐1 protease inhibitors are described. The synthesis of both enantiomers of the gem‐difluoro‐bis‐THF ligands was carried out in a stereoselective manner using a Reformatskii–Claisen reaction as the key step. Optically active ligands were converted into protease inhibitors. Two of these inhibitors, (3R,3aS,6aS)‐4,4‐difluorohexahydrofuro[2,3‐b]furan‐3‐yl(2S,3R)‐3‐hydroxy‐4‐((N‐isobutyl‐4‐methoxyphenyl)sulfonamido)‐1‐phenylbutan‐2‐yl) carbamate ( 3 ) and (3R,3aS,6aS)‐4,4‐difluorohexahydrofuro[2,3‐b]furan‐3‐yl(2S,3R)‐3‐hydroxy‐4‐((N‐isobutyl‐4‐aminophenyl)sulfonamido)phenylbutan‐2‐yl) carbamate ( 4 ), exhibited HIV‐1 protease inhibitory K_i values in the picomolar range. Both 3 and 4 showed very potent antiviral activity, with respective EC₅₀ values of 0.8 and 3.1 nM against the laboratory strain HIV‐1_LAI. The two inhibitors exhibited better lipophilicity profiles than darunavir, and also showed much improved blood–brain barrier permeability in an in vitro model. A high‐resolution X‐ray structure of inhibitor 4 in complex with HIV‐1 protease was determined, revealing that the fluorinated ligand makes extensive interactions with the S2 subsite of HIV‐1 protease, including hydrogen bonding interactions with the protease backbone atoms. Moreover, both fluorine atoms on the bis‐THF ligand formed strong interactions with the flap Gly 48 carbonyl oxygen atom.     

Derivatives of Trimethoxybenzoic Acid and Gallic Acid as Potential Efflux Pump Inhibitors: In Silico and In Vitro Studies

The overexpression of efflux pumps is one of the strategies used by bacteria to resist antibiotics and could be targeted to circumvent the antibiotic crisis. In this work, a series of trimethoxybenzoic acid derivatives previously described as antifouling compounds was explored for potential antimicrobial activity and efflux pump (EP) inhibition. First, docking studies on the acridine resistance proteins A and B coupled to the outer membrane channel TolC (AcrAB-TolC) efflux system and a homology model of the quinolone resistance protein NorA EP were performed on 11 potential bioactive trimethoxybenzoic acid and gallic acid derivatives. The synthesis of one new trimethoxybenzoic acid derivative (derivative 13) was accomplished. To investigate the potential of this series of 11 derivatives as antimicrobial agents, and in reverting drug resistance, the minimum inhibitory concentration was determined on several strains (bacteria and fungi), and synergy with antibiotics and EP inhibition were investigated. Derivative 10 showed antibacterial activity against the studied strains, derivatives 5 and 6 showed the ability to inhibit EPs in the acrA gene inactivated mutant Salmonella enterica serovar Typhimurium SL1344, and 6 also inhibited EPs in Staphylococcus aureus 272123. Structure-activity relationships highlighted trimethoxybenzoic acid as important for EP inhibitory activity. Although further studies are necessary, these results show the potential of simple trimethoxybenzoic acid derivatives as a source of feasible EP inhibitors.     

A Combination Strategy to Inhibit Pim‐1: Synergism between Noncompetitive and ATP‐Competitive Inhibitors

Pim‐1 is a serine/threonine kinase critically involved in the initiation and progression of various types of cancer, especially leukemia, lymphomas and solid tumors such as prostate, pancreas and colon, and is considered a potential drug target against these malignancies. In an effort to discover new potent Pim‐1 inhibitors, a previously identified ATP‐competitive indolyl‐pyrrolone scaffold was expanded to derive structure–activity relationship data. A virtual screening campaign was also performed, which led to the discovery of additional ATP‐competitive inhibitors as well as a series of 2‐aminothiazole derivatives, which are noncompetitive with respect to both ATP and peptide substrate. This mechanism of action, which resembles allosteric inhibition, has not previously been characterized for Pim‐1. Notably, further evaluation of the 2‐aminothiazoles indicated a synergistic inhibitory effect in enzymatic assays when tested in combination with ATP‐competitive inhibitors. A synergistic effect in the inhibition of cell proliferation by ATP‐competitive and ATP‐noncompetitive compounds was also observed in prostate cancer cell lines (PC3), where all Pim‐1 inhibitors tested in showed synergism with the known anticancer agent, paclitaxel. These results further establish Pim‐1 as a target in cancer therapy, and highlight the potential of these agents for use as adjuvant agents in the treatment of cancer diseases in which Pim‐1 is associated with chemotherapeutic resistance.     

Tepotinib Inhibits Several Drug Efflux Transporters and Biotransformation Enzymes: The Role in Drug-Drug Interactions and Targeting Cytostatic Resistance In Vitro and Ex Vivo

Tepotinib is a novel tyrosine kinase inhibitor recently approved for the treatment of non-small cell lung cancer (NSCLC). In this study, we evaluated the tepotinib’s potential to perpetrate pharmacokinetic drug interactions and modulate multidrug resistance (MDR). Accumulation studies showed that tepotinib potently inhibits ABCB1 and ABCG2 efflux transporters, which was confirmed by molecular docking. In addition, tepotinib inhibited several recombinant cytochrome P450 (CYP) isoforms with varying potency. In subsequent drug combination experiments, tepotinib synergistically reversed daunorubicin and mitoxantrone resistance in cells with ABCB1 and ABCG2 overexpression, respectively. Remarkably, MDR-modulatory properties were confirmed in ex vivo explants derived from NSCLC patients. Furthermore, we demonstrated that anticancer effect of tepotinib is not influenced by the presence of ABC transporters associated with MDR, although monolayer transport assays designated it as ABCB1 substrate. Finally, tested drug was observed to have negligible effect on the expression of clinically relevant drug efflux transporters and CYP enzymes. In conclusion, our findings provide complex overview on the tepotinib’s drug interaction profile and suggest a promising novel therapeutic strategy for future clinical investigations.     

Multispecificity of drug transporters: probing inhibitor selectivity for the human drug efflux transporters ABCB1 and ABCG2

Multidrug resistance transporters P-glycoprotein/ABCB1 and ABCG2 limit the effect of a large number of cytostatic and cytotoxic drugs by energy-dependent efflux. In experimental models, pump inhibitors reestablish sensitivity towards these drugs. Both transporters demonstrate remarkably broad and partly overlapping substrate specificity. Propafenone analogues are inhibitors of a large number of drug efflux pumps including P-glycoprotein and ABCG2 as well as the microbial pumps. Here the two human ABC transporters ABCB1 and ABCG2 have been studied with respect to interaction with this class of compounds. Data indicate that within the same chemical scaffold, selectivity indices span three orders of magnitude. Compounds with the highest selectivity indices contain a non-ionizable nitrogen atom. Qualitative and quantitative pharmacophore models indicate that hydrophobicity, the number of rotatable bonds, and the number of H-bond acceptors are key features for both activity and selectivity.     

PET Radiotracers for Imaging P‐glycoprotein: The Challenge for Early Diagnosis in AD

PET radiotracer development to target in vivo P‐glycoprotein (P‐gp) could be an important strategy for the early diagnosis of neurodegenerative disorders such as Alzheimer’s disease (AD). Indeed, as a dysfunction of P‐gp is responsible for the accumulation of β‐amyloid plaques (a hallmark of AD) in brain parenchyma, P‐gp is the cause of AD onset. P‐gp substrates and inhibitors are useful for imaging the activity or expression of this protein, respectively; herein we discuss the in vivo evaluation of some ¹¹C radiotracers with P‐gp‐inhibitory activity, such as [¹¹C]MC18 and [¹¹C]MC113, as well as P‐gp substrates [¹¹C]MC266 and [¹¹C]MC80. Moreover, the radiosynthesis of all these P‐gp probes is reported.