Discovery of Bis-Imidazoline Derivatives as New CXCR4 Ligands.

The chemokine receptor CXCR4 and its ligand CXCL12 regulate leukocyte trafficking, homeostasis and functions and are potential therapeutic targets in many diseases such as HIV-1 infection and cancers. Here, we identified new CXCR4 ligands in the CERMN chemical library using a FRET-based high-throughput screening assay. These are bis-imidazoline compounds comprising two imidazole rings linked by an alkyl chain. The molecules displace CXCL12 binding with submicromolar potencies, similarly to AMD3100, the only marketed CXCR4 ligand. They also inhibit anti-CXCR4 mAb 12G5 binding, CXCL12-mediated chemotaxis and HIV-1 infection. Further studies with newly synthesized derivatives pointed out to a role of alkyl chain length on the bis-imidazoline properties, with molecules with an even number of carbons equal to 8, 10 or 12 being the most potent. Interestingly, these differ in the functions of CXCR4 that they influence. Site-directed mutagenesis and molecular docking predict that the alkyl chain folds in such a way that the two imidazole groups become lodged in the transmembrane binding cavity of CXCR4. Results also suggest that the alkyl chain length influences how the imidazole rings positions in the cavity. These results may provide a basis for the design of new CXCR4 antagonists targeting specific functions of the receptor.

A New Thienopyrimidinone Chemotype Shows Multistage Activity against Plasmodium falciparum, Including Artemisinin-Resistant Parasites.

Human malaria infection begins with a one-time asymptomatic liver stage followed by a cyclic symptomatic blood stage. For decades, the research for novel antimalarials focused on the high-throughput screening of molecules that only targeted the asexual blood stages. In a search for new effective compounds presenting a triple action against erythrocytic and liver stages in addition to the ability to block the transmission of the disease via the mosquito vector, 2-amino-thienopyrimidinone derivatives were synthesized and tested for their antimalarial activity. One molecule, named gamhepathiopine (denoted as "M1" herein), was active at submicromolar concentrations against both erythrocytic (50% effective concentration [EC50] = 0.045 µM) and liver (EC50 = 0.45 µM) forms of Plasmodium falciparum. Furthermore, gamhepathiopine efficiently blocked the development of the sporogonic cycle in the mosquito vector by inhibiting the exflagellation step. Moreover, M1 was active against artemisinin-resistant forms (EC50 = 0.227 µM), especially at the quiescent stage. Nevertheless, in mice, M1 showed modest activity due to its rapid metabolization by P450 cytochromes into inactive derivatives, calling for the development of new parent compounds with improved metabolic stability and longer half-lives. These results highlight the thienopyrimidinone scaffold as a novel antiplasmodial chemotype of great interest to search for new drug candidates displaying multistage activity and an original mechanism of action with the potential to be used in combination therapies for malaria elimination in the context of artemisinin resistance. IMPORTANCE This work reports a new chemical structure that (i) displays activity against the human malaria parasite Plasmodium falciparum at 3 stages of the parasitic cycle (blood stage, hepatic stage, and sexual stages), (ii) remains active against parasites that are resistant to the first-line treatment recommended by the World Health Organization (WHO) for the treatment of severe malaria (artemisinins), and (iii) reduces transmission of the parasite to the mosquito vector in a mouse model. This new molecule family could open the way to the conception of novel antimalarial drugs with an original multistage mechanism of action to fight against Plasmodium drug resistance and block interhuman transmission of malaria.

Benzothienoquinazolinones as new multi-target scaffolds: Dual inhibition of human Topoisomerase I and tubulin polymerization.

3-(Alkyl(dialkyl)amino)benzothieno[2,3-f]quinazolin-1(2H)-ones (4-9) have been designed using Ellipticine structure as a model, replacing the carbazole core and the pyridine ring with a dibenzothiophene and a pyrimidine moiety, respectively. New benzothienoquinazolinones (4-9) have been synthesized by a simple one-pot reaction employing 3-aminodibenzothiophene as starting material. The benzothienoquinazolinones obtained (4-9), were evaluated for their anticancer activity against two breast cancer cell lines, MDA-MB-231 and MCF-7. The results revealed that compounds 4 and 7 presented a good antitumor activity toward the triple negative MDA-MB-231, without cytotoxicity against non-tumoral cells. Furthermore, the compounds 4 and 7 can be considered important molecular multi-target tools for their dual inhibition of different cellular proteins, i.e. Tubulin and human Topoisomerase I, involved in relevant cellular processes, as predicted by in silico studies and demonstrated by in vitro assays (for compound 4).

Structure-guided design of pyridoclax derivatives based on Noxa / Mcl-1 interaction mode.

Protein-protein interactions are attractive targets because they control numerous cellular processes. In oncology, apoptosis regulating Bcl-2 family proteins are of particular interest. Apoptotic cell death is controlled via PPIs between the anti-apoptotic proteins hydrophobic groove and the pro-apoptotic proteins BH3 domain. In ovarian carcinoma, it has been previously demonstrated that Bcl-xL and Mcl-1 cooperate to protect tumor cells against apoptosis. Moreover, Mcl-1 is a key regulator of cancer cell survival and is a known resistance factor to Bcl-2/Bcl-xL pharmacological inhibitors making it an attractive therapeutic target. Here, using a structure-guided design from the oligopyridine lead Pyridoclax based on Noxa/Mcl-1 interaction we identified a new derivative, active at lower concentration as compared to Pyridoclax. This new derivative selectively binds to the Mcl-1 hydrophobic groove and releases Bak and Bim from Mcl-1 to induce cell death and sensitize cancer cells to Bcl-2/Bcl-xL targeting strategies.

Toward Understanding Mcl-1 Promiscuous and Specific Binding Mode.

Mcl-1, which is an anti-apoptotic member of the Bcl-2 protein family, is overexpressed in various cancers and promotes the aberrant survival of tumor cells. To inhibit Mcl-1, and initiate apoptosis, an interaction between BH3-only proteins and Mcl-1 anti-apoptotic protein is necessary. These protein-protein interactions exhibit some selectivity: Mcl-1 binds specifically to Noxa, whereas Bim and Puma bind strongly to all anti-apoptotic proteins. Even if the three-dimensional (3D) structures of several Mcl-1/BH3-only complexes have been solved, the BH3-only binding specificity to Mcl-1 is still not completely understood. In this study, molecular dynamics simulations were used to elucidate the molecular basis of the interactions with Mcl-1. Our results corroborate the importance of four conserved hydrophobic residues and a conserved aspartic acid on BH3-only as a common binding pattern. Furthermore, our results highlight the contribution of the fifth hydrophobic residue in the C-terminal part and a negatively charged patch in the N-terminal of BH3-only peptides as important for their fixation to Mcl-1. We hypothesize that this negatively charged patch will be an Mcl-1 specific binding pattern.

Tau protein aggregation in Alzheimer's disease: An attractive target for the development of novel therapeutic agents.

Alzheimer's Disease (AD) is a neurodegenerative brain disorder in which many biological dysfunctions are involved. Among them, two main types of lesions were discovered and widely studied: the amyloid plaques and the neurofibrillary tangles (NFTs). These two lesions are caused by the dysfunction and the accumulation of two proteins which are, respectively, the beta-amyloid peptide and the tau protein. The process that leads these two proteins to aggregate is complex and is the subject of current studies. After a brief description of the aggregation mechanisms, we will provide an overview of new therapeutic agents targeting the different dysfunctions and toxic species found during aggregation.

Multifaceted properties of 1,4-dimethylcarbazoles: Focus on trimethoxybenzamide and trimethoxyphenylurea derivatives as novel human topoisomerase II inhibitors.

Natural or synthetic carbazole derivatives have recently attracted the attention of the scientific world because of their multiple biological activity, leading to an increase of designed, synthesized and studied analogues. In this paper, four 1,4-dimethylcarbazole derivatives, analogues of Ellipticine, have been investigated for their ability to block cancer cells growth, with low effects on the proliferation of normal cells. DNA topoisomerases inhibition assays, docking simulations, stability studies and effects on a membrane model are reported. Particularly, compounds 2 and 3 have been found thermally stable and able to inhibit, strongly and selectively, the human DNA topoisomerase II. These properties confer a good and broad antitumoral activity in vitro, with very low cytotoxic effect on the proliferation of normal cell lines and without damaging, in contrast with Ellipticine, the cell membrane model. The presented outcomes set the most active compounds as good candidates for pre-clinical studies useful in cancer treatment.

3-(Dipropylamino)-5-hydroxybenzofuro[2,3-f]quinazolin-1(2H)-one (DPA-HBFQ-1) plays an inhibitory role on breast cancer cell growth and progression.

A series of unknown 3-(alkyl(dialkyl)amino)benzofuro[2,3-f]quinazolin-1(2H)-ones 4-17 has been synthesized as new ellipticine analogs, in which the carbazole moiety and the pyridine ring were replaced by a dibenzofuran residue and a pyrimidine ring, respectively. The synthesis of these benzofuroquinazolinones 4-17 was performed in a simple one-pot reaction using 3-aminodibenzofuran or its 2-methoxy derivative, as starting materials. From 3-(dipropylamino)-5-methoxybenzofuro[2,3-f] quinazolin-1(2H)-one (13), we prepared 3-(dipropylamino)-5-hydroxybenzofuro[2,3-f]quinazolin-1(2H)-one (18), referred to as DPA-HBFQ-1. The cytotoxic activities of all the synthesized compounds, tested in different human breast cancer cell lines, revealed that DPA-HBFQ-1 was the most active compound. In particular, the latter was able to inhibit anchorage-dependent and -independent cell growth and to induce apoptosis in estrogen receptor alpha (ERα)-positive and -negative breast cancer cells. It did not affect proliferation and apoptotic responses in MCF-10A normal breast epithelial cells. The observed effects have been ascribed to an enhanced p21(Cip1/WAF1) expression in a p53-dependent manner of tumor suppressor and to a selective inhibition of human topoisomerase II. In addition, DPA-HBFQ-1 exerted growth inhibitory effects also in other cancer cell lines, even though with a lower cytotoxic activity. Our results indicate DPA-HBFQ-1 as a good candidate to be useful as cancer therapeutic agent, particularly for breast cancer.

The Toxicity of a Novel Antifungal Compound Is Modulated by Endoplasmic Reticulum-Associated Protein Degradation Components.

In a search for new antifungal compounds, we screened a library of 4,454 chemicals for toxicity against the human fungal pathogen Aspergillus fumigatus. We identified sr7575, a molecule that inhibits growth of the evolutionary distant fungi A. fumigatus, Cryptococcus neoformans, Candida albicans, and Saccharomyces cerevisiae but lacks acute toxicity for mammalian cells. To gain insight into the mode of inhibition, sr7575 was screened against 4,885 S. cerevisiae mutants from the systematic collection of haploid deletion strains and 977 barcoded haploid DAmP (decreased abundance by mRNA perturbation) strains in which the function of essential genes was perturbed by the introduction of a drug resistance cassette downstream of the coding sequence region. Comparisons with previously published chemogenomic screens revealed that the set of mutants conferring sensitivity to sr7575 was strikingly narrow, affecting components of the endoplasmic reticulum-associated protein degradation (ERAD) stress response and the ER membrane protein complex (EMC). ERAD-deficient mutants were hypersensitive to sr7575 in both S. cerevisiae and A. fumigatus, indicating a conserved mechanism of growth inhibition between yeast and filamentous fungi. Although the unfolded protein response (UPR) is linked to ERAD regulation, sr7575 did not trigger the UPR in A. fumigatus and UPR mutants showed no enhanced sensitivity to the compound. The data from this chemogenomic analysis demonstrate that sr7575 exerts its antifungal activity by disrupting ER protein quality control in a manner that requires ERAD intervention but bypasses the need for the canonical UPR. ER protein quality control is thus a specific vulnerability of fungal organisms that might be exploited for antifungal drug development.

Antileishmanial pharmacomodulation in 8-nitroquinolin-2(1H)-one series.

An antileishmanial pharmacomodulation at position 4 of 8-nitroquinolin-2(1H)-one was conducted by using the Sonogashira and Suzuki-Miyaura coupling reactions. A series of 25 derivatives was tested in vitro on the promastigote stage of Leishmania donovani along with an in vitro cytotoxicity evaluation on the human HepG2 cell line. Only the derivatives bearing a phenyl moiety at position 4 of the quinoline ring displayed interesting biologic profile, when the phenyl moiety was substituted at the para position by a Br or Cl atom, or by a CF3 group. Among them, molecules 17 and 19 were the most selective and were then tested in vitro on the intracellular amastigote stage of both L. donovani and Leishmania infantum, in parallel with complementary in vitro cytotoxicity assays on the macrophage cell lines THP-1 and J774A.1. Molecule 19 showed no activity on the amastigote stages of the parasites and some cytotoxicity on the J774A.1 cell line while molecule 17, less cytotoxic than 19, showed anti-amastigote activity in L. infantum, being 3 times less active than miltefosine but more active and selective than pentamidine. Nevertheless, hit-molecule 17 did not appear as selective as the parent compound.

Discovery of new thienopyrimidinone derivatives displaying antimalarial properties toward both erythrocytic and hepatic stages of Plasmodium.

A preliminary in vitro screening of compounds belonging to various chemical families from our library revealed the thieno[3,2-d]pyrimidin-4(3H)-one scaffold displayed a promising profile against Plasmodium falciparum. Then, 120 new derivatives were synthesized and evaluated in vitro; compared to drug references, 40 showed good activity toward chloroquine sensitive (IC50 35-344 nM) and resistant (IC50 45-800 nM) P. falciparum strains. They were neither cytotoxic (CC50 15-50 µM) toward HepG2 and CHO cells, nor mutagenic. Structure-activity relationships were defined. The lead-compound also appeared active against the Plasmodium liver stages (Plasmodium yoelii IC50 = 35 nM) and a preliminary in vivo evaluation indicated the in vitro activity was preserved (45% reduction in parasitemia compared to untreated infected mice). A mechanistic study demonstrated these molecules do not involve any of the pathways described for commercial drugs and exert a specific activity on the ring and trophozoite stages.

Boronic species as promising inhibitors of the Staphylococcus aureus NorA efflux pump: study of 6-substituted pyridine-3-boronic acid derivatives.

In response to the extensive use of antibiotics, bacteria have evolved numerous mechanisms of defense against antimicrobial agents. Among them, extrusion of the antimicrobial agents outside the bacterial cell through efflux pumps is a major cause of concern. At first limited to one or few structurally-related antibiotics, bacterial resistance have then progressed towards cross-resistance between different classes of antibiotics, leading to multidrug-resistant microorganisms. Emergence of these pathogens requires development of novel therapeutic strategies and inhibition of efflux pumps appears to be a promising strategy that could restore the potency of existing antibiotics. NorA is the most studied chromosomal efflux pump of Staphylococcus aureus; it is known to be implied in resistance of Methicillin-resistant S. aureus (MRSA) strains against a wide range of unrelated substrates, including hydrophilic fluoroquinolones. Starting from 6-benzyloxypyridine-3-boronic acid I that we previously identified as a potential inhibitor of the NorA efflux pump against the NorA-overexpressing S. aureus 1199B strain (SA1199B), we describe here the synthesis and biological evaluation of a series of 6-(aryl)alkoxypyridine-3-boronic acids. 6-(3-Phenylpropoxy)pyridine-3-boronic acid 3i and 6-(4-phenylbutoxy)pyridine-3-boronic acid 3j were found to potentiate ciprofloxacin activity by a 4-fold increase compared to the parent compound I. In addition, it has been shown that both compounds promote Ethidium Bromide (EtBr) accumulation in SA1199B, thus corroborating their potential mode of action as NorA inhibitors.

(6-bromo-1,4-dimethyl-9H-carbazol-3-yl-methylene)-hydrazine (carbhydraz) acts as a GPER agonist in breast cancer cells.

Estrogens control a wide number of aspects of human physiology and play a key role in multiple diseases, including cancer. Estrogens act by binding to and activating the cognate receptor (ER), however numerous studies have revealed that the G protein-coupled receptor named GPR30/GPER mediates also estrogen signals. As ER and GPER share the ability to bind to same compounds, the use of GPER-selective ligands has allowed a better understanding of the biological responses mediated by GPER. In the present study, we designed and synthesized two novel carbazole derivatives and then investigated their ability to interact with and activate the GPER-mediated transduction pathway in breast cancer cells. Both compounds did not activate the classical ER in MCF7 cells, whereas one of the two compounds synthesized triggered through GPER the rapid ERK activation in ER-negative SkBr3 cells, demonstrating a good affinity for GPER in docking studies. The characterization of this novel selective GPER agonist could represent a potential useful tool to provide further insights into the physiopathological role exerted by GPER.

Looking for new antileishmanial derivatives in 8-nitroquinolin-2(1H)-one series.

From a recently identified antileishmanial pharmacophore, a structure-activity relationship study was conducted by introducing various aminated, phenoxy or thiophenoxy moieties at position 4 of the 8-nitroquinolin-2(1H)-one scaffold, using SNAr reactions. Thus a series of 47 derivatives was synthesized and evaluated in vitro on the promastigote stage of Leishmania donovani. In parallel, the cytotoxicity of the active molecules was tested on the human HepG2 cell line. The results we obtained showed that the introduction of a substituent at position 4 of the antileishmanial pharmacophore can either lead to inactive or active derivatives, depending on the nature of the substituent. Aminated moieties appear as very unfavorable toward antileishmanial activity, while phenoxy or thiophenoxy moieties were shown to maintain the in vitro antileishmanial profile, especially when the phenyl ring of these moieties was substituted at the para or ortho position by a halogen atom (except fluorine), a trifluoromethyl group or a methyl group. Most of these derivatives showed a lack of solubility in the culture media which hindered the in vitro determination of both their cytotoxicity and activity against the intracellular amastigoste stage of L. donovani.

Tampering with cell division by using small-molecule inhibitors of CDK-CKS protein interactions.

Cyclin-dependent kinases (CDKs) control many cellular processes and are considered important therapeutic targets. Large collections of inhibitors targeting CDK active sites have been discovered, but their use in chemical biology or drug development has been often hampered by their general lack of specificity. An alternative approach to develop more specific inhibitors is targeting protein interactions involving CDKs. CKS proteins interact with some CDKs and play important roles in cell division. We discovered two small-molecule inhibitors of CDK-CKS interactions. They bind to CDK2, do not inhibit its enzymatic activity, inhibit the proliferation of tumor cell lines, induce an increase in G1 and/or S-phase cell populations, and cause a decrease in CDK2, cyclin A, and p27(Kip1) levels. These molecules should help decipher the complex contributions of CDK-CKS complexes in the regulation of cell division, and they might present an interesting therapeutic potential.

First evidence that oligopyridines, α-helix foldamers, inhibit Mcl-1 and sensitize ovarian carcinoma cells to Bcl-xL-targeting strategies.

Apoptosis control defects such as the deregulation of Bcl-2 family member expression are frequently involved in chemoresistance. In ovarian carcinoma, we previously demonstrated that Bcl-xL and Mcl-1 cooperate to protect cancer cells against apoptosis and their concomitant inhibition leads to massive apoptosis even in the absence of chemotherapy. Whereas Bcl-xL inhibitors are now available, Mcl-1 inhibition, required to sensitize cells to Bcl-xL-targeting strategies, remains problematic. In this context, we designed and synthesized oligopyridines potentially targeting the Mcl-1 hydrophobic pocket, evaluated their capacity to inhibit Mcl-1 in live cells, and implemented a functional screening assay to evaluate their ability to sensitize ovarian carcinoma cells to Bcl-xL-targeting strategies. We established structure-activity relationships and focused our attention on MR29072, named Pyridoclax. Surface plasmon resonance assay demonstrated that pyridoclax directly binds to Mcl-1. Without cytotoxic activity when administered as a single agent, pyridoclax induced apoptosis in combination with Bcl-xL-targeting siRNA or with ABT-737 in ovarian, lung, and mesothelioma cancer cells.

Synthesis and in vitro evaluation of 4-trichloromethylpyrrolo[1,2-a]quinoxalines as new antiplasmodial agents.

Thanks to a preliminary in vitro screening of several CCl3-substituted-nitrogen containing heterocycles belonging to our chemical library, the 2-trichloromethylquinoxaline scaffold appeared to be of potential interest for developing new antiplasmodial agents. Then, combining these experimental results to the antimalarial properties reported for various pyrrolo[1,2-a]quinoxaline derivatives, an original series of fifteen 7-substituted-4-trichoromethylpyrrolo[1,2-a]quinoxalines was synthesized in a 4 to 5 reaction steps pathway. All molecules were evaluated in vitro toward both their antiplasmodial activity on the K1 multi-resistant Plasmodium falciparum strain and their cytotoxicity on the HepG2 human cell line. Thus, 3 hit molecules were identified, displaying IC50 values in the micromolar range and low cytotoxicity values, reaching good selectivity indexes, in comparison with the reference drugs chloroquine and doxycycline. Structure-activity relationship studies showed that the pyrrolo[1,2-a]quinoxaline scaffold can support selective antiplasmodial activity when substituted at position 4 by a CCl3 group. However, substitution at position 7 of the same scaffold is neither beneficial for cytotoxicity nor favourable for the solubility in the biological media.

Dual histamine H3R/serotonin 5-HT4R ligands with antiamnesic properties: pharmacophore-based virtual screening and polypharmacology.

In recent years, preclinical and clinical studies have generated considerable interest in the development of histamine H3 receptor (H3R) antagonists as novel treatment for degenerative disorders associated with impaired cholinergic function. To identify novel scaffolds for H3R antagonism, a common feature-based pharmacophore model was developed and used to screen the 17,194 compounds of the CERMN (Centre d'Etudes et de Recherche sur le Médicament de Normandie) chemical library. Out of 268 virtual hits which have been gathered in 34 clusters, we were particularly interested in tricyclic derivatives also exhibiting a potent 5HT4R affinity. Benzo[h][1,6]naphthyridine derivatives showed the highest H3R affinity, and compound 17 (H3R Ki = 41.6 nM; 5-HT4R Ki = 208 nM) completely reversed the amnesiant effect of scopolamine at 3 mg/kg in a spatial working memory experiment. For the first time we demonstrated the feasibility to combine H3R and 5-HT4R activities in a single molecule, raising the exciting possibility that dual H3R antagonist/5HT4R agonist have potential for the treatment of neurodegenerative diseases such as Alzheimer's disease.

Small-animal single-photon emission computed tomographic imaging of the brain serotoninergic systems in wild-type and mdr1a knockout rats.

The pharmacokinetic properties of radiotracers are crucial for successful in vivo single-photon emission computed tomographic (SPECT) imaging. Our goal was to determine if MDR1A-deficient animals could allow better SPECT imaging outcomes than wild-type (WT) animals for a selection of serotoninergic radioligands. Thus, we compared the performances of 123I-p-MPPI, 123I-R91150, 123I-SB207710, and 123I-ADAM radioligands, for imaging of their respective targets (5-hydroxytryptamine [5-HT]1A, 5-HT2A, 5-HT4, and serotonin transporter [SERT]), in WT and Mdr1a knockout (KO) rats. With 123I-SB207710, virtually no SPECT signal was recorded in the brain of WT or KO animals. For 123I-p-MPPI, low nondisplaceable binding potentials (BPND, mean ± SD) were observed in WT (0.49 ± 0.25) and KO (0.89 ± 0.52) animals. For 123I-ADAM, modest imaging contrast was observed in WT (1.27 ± 0.02) and KO (1.31 ± 0.09) animals. For 123I-R91150, the BPND were significantly higher in Mdr1a KO (3.98 ± 0.65) animals compared to WT animals (1.22 ± 0.26). The pharmacokinetics of 123I-SB207710 and 123I-p-MPPI do not make them ideal tracers for preclinical SPECT neuroimaging. 123I-ADAM showed adequate brain uptake regardless of Mdr1a expression and appeared suitable for preclinical SPECT neuroimaging in both animal strains. The use of Mdr1a KO animals significantly improved the brain penetration of 123I-R91150, making this animal strain an interesting option when considering SPECT neuroimaging of 5-HT2A receptors in rat.

First identification of boronic species as novel potential inhibitors of the Staphylococcus aureus NorA efflux pump.

Overexpression of efflux pumps is an important mechanism of bacterial resistance that results in the extrusion of antimicrobial agents outside the bacterial cell. Inhibition of such pumps appears to be a promising strategy that could restore the potency of existing antibiotics. The NorA efflux pump of Staphylococcus aureus confers resistance to a wide range of unrelated substrates, such as hydrophilic fluoroquinolones, leading to a multidrug-resistance phenotype. In this work, approximately 150 heterocyclic boronic species were evaluated for their activity against susceptible and resistant strains of S. aureus. Twenty-four hit compounds, although inactive when tested alone, were found to potentiate ciprofloxacin activity by a 4-fold increase at concentrations ranging from 0.5 to 8 µg/mL against S. aureus 1199B, which overexpresses NorA. Boron-free analogues showed no biological activity, thus revealing that the boron atom is crucial for biological activity. This work describes the first reported efflux pump inhibitory activity of boronic acid derivatives.