Requires ATP Hydrolysis Research Articles

Phosphorylation of Argonaute proteins affects mRNA binding and is essential for microRNA-guided gene silencing invivo.

Argonaute proteins associate with microRNAs and are key components of gene silencing pathways. With such a pivotal role, these proteins represent ideal targets for regulatory post-translational modifications. Using quantitative mass spectrometry, we find that a C-terminal serine/threonine cluster is phosphorylated at five different residues in human and Caenorhabditis elegans In human, hyper-phosphorylation does not affect microRNA binding, localization, or cleavage activity of Ago2. However, mRNA binding is strongly affected. Strikingly, on Ago2 mutants that cannot bind microRNAs or mRNAs, the cluster remains unphosphorylated indicating a role at late stages of gene silencing. In C.elegans, the phosphorylation of the conserved cluster of ALG-1 is essential for microRNA function invivo Furthermore, a single point mutation within the cluster is sufficient to phenocopy the loss of its complete phosphorylation. Interestingly, this mutant retains its capacity to produce and bind microRNAs and represses expression when artificially tethered to an mRNA Altogether, our data suggest that the phosphorylation state of the serine/threonine cluster is important for Argonaute-mRNA interactions.

Novel DNA Binding and Regulatory Activities for σ54 (RpoN) in Salmonella enterica Serovar Typhimurium 14028s.

The variable sigma (σ) subunit of the bacterial RNA polymerase (RNAP) holoenzyme, which is responsible for promoter specificity and open complex formation, plays a strategic role in the response to environmental changes. Salmonella enterica serovar Typhimurium utilizes the housekeeping σ70 and five alternative sigma factors, including σ54 The σ54-RNAP differs from other σ-RNAP holoenzymes in that it forms a stable closed complex with the promoter and requires ATP hydrolysis by an activated cognate bacterial enhancer binding protein (bEBP) to transition to an open complex and initiate transcription. In S. Typhimurium, σ54-dependent promoters normally respond to one of 13 different bEBPs, each of which is activated under a specific growth condition. Here, we utilized a constitutively active, promiscuous bEBP to perform a genome-wide identification of σ54-RNAP DNA binding sites and the transcriptome of the σ54 regulon of S. Typhimurium. The position and context of many of the identified σ54 RNAP DNA binding sites suggest regulatory roles for σ54-RNAP that connect the σ54 regulon to regulons of other σ factors to provide a dynamic response to rapidly changing environmental conditions.IMPORTANCE The alternative sigma factor σ54 (RpoN) is required for expression of genes involved in processes with significance in agriculture, bioenergy production, bioremediation, and host-microbe interactions. The characterization of the σ54 regulon of the versatile pathogen S. Typhimurium has expanded our understanding of the scope of the σ54 regulon and how it links to other σ regulons within the complex regulatory network for gene expression in bacteria.

Rad50 ATPase activity is regulated by DNA ends and requires coordination of both active sites.

The Mre11–Rad50–Nbs1(Xrs2) (MRN/X) complex is critical for the repair and signaling of DNA double strand breaks. The catalytic core of MRN/X comprised of the Mre11 nuclease and Rad50 adenosine triphosphatase (ATPase) active sites dimerizes through association between the Rad50 ATPase catalytic domains and undergoes extensive conformational changes upon ATP binding. This ATP-bound ‘closed’ state promotes binding to DNA, tethering DNA ends and ATM activation, but prevents nucleolytic processing of DNA ends, while ATP hydrolysis is essential for Mre11 endonuclease activity at blocked DNA ends. Here we investigate the regulation of ATP hydrolysis as well as the interdependence of the two functional active sites. We find that double-stranded DNA stimulates ATP hydrolysis by hMRN over ∼20-fold in an end-dependent manner. Using catalytic site mutants to create Rad50 dimers with only one functional ATPase site, we find that both ATPase sites are required for the stimulation by DNA. MRN-mediated endonucleolytic cleavage of DNA at sites of protein adducts requires ATP hydrolysis at both sites, as does the stimulation of ATM kinase activity. These observations suggest that symmetrical engagement of the Rad50 catalytic head domains with ATP bound at both sites is important for MRN functions in eukaryotic cells.

Determination of Local pH Differences within Living Salmonella Cells by High-resolution pH Imaging Based on pH-sensitive GFP Derivative, pHluorin(M153R).

The bacterial flagellar type III protein export apparatus is composed of a transmembrane export gate complex and a cytoplasmic ATPase complex. The export apparatus requires ATP hydrolysis and the proton motive force across the cytoplasmic membrane to unfold and transport flagellar component proteins for the construction of the bacterial flagellum (Minamino, 2014). The export apparatus is a proton/protein antiporter that couples the proton flow with protein transport through the gate complex ( Minamino et al., 2011 ). A transmembrane export gate protein, FlhA, acts as an energy transducer along with the cytoplasmic ATPase complex ( Minamino et al., 2016 ). To directly measure the proton flow through the FlhA channel that is coupled with the flagellar protein export, we have developed an in vivo pH imaging system with high spatial and pH resolution ( Morimoto et al., 2016 ). Here, we describe how we measure the local pH near the export apparatus in living Salmonella cells ( Morimoto et al., 2016 ). Our approach can be applied to a wide range of living cells. Because local pH is one of the most important parameters to monitor cellular activities of living cells, our protocol would be widely used for diverse areas of life sciences.

Novel translocation intermediate allows re-evaluation of roles of ATP, proton motive force and SecG at the late stage of preprotein translocation.

Presecretory proteins such as pOmpA are translocated across the inner membrane of Escherichia coli by Sec translocase powered by ATP and proton motive force (PMF). Translocation activity has been determined by protease protection assaying invitro. We identified a new translocation intermediate at a late stage, which was protected by proteinase K (PK), but became PK sensitive upon urea extraction. At a late stage of pOmpA translocation driven by PMF in the presence of a nonhydrolyzable ATP analogue, the PK-protected materials arose, but were pulled back upon urea extraction, indicating that completion of translocation requires ATP hydrolysis. When inverted membrane vesicles prepared from secG-null strain (ΔSecG IMV) were used in the absence of PMF, the translocation intermediate was accumulated. When the ATP concentration was low in the absence of PMF, the translocation intermediate was also accumulated. Imposition of PMF in the presence of a low ATP concentration caused recovery of pOmpA translocation and resistance to urea extraction for SecG+ IMV, but not for ΔSecG IMV. Thus, analysis of the late translocation intermediate showed that two of three constituents, physiological concentration of ATP, PMF and SecG, are required for the catalytic cycle of preprotein translocation, that is, completion and subsequent initiation of translocation.

VCP/p97 Extracts Sterically Trapped Ku70/80 Rings from DNA in Double-Strand Break Repair

During DNA double-strand break (DSB) repair, the ring-shaped Ku70/80 complex becomes trapped on DNA and needs to be actively extracted, but it has remained unclear what provides the required energy. By means of reconstitution of DSB repair on beads, we demonstrate here that DNA-locked Ku rings are released by the AAA-ATPase p97. To achieve this, p97 requires ATP hydrolysis, cooperates with the Ufd1-Npl4 ubiquitin-adaptor complex, and specifically targets Ku80 that is modified by K48-linked ubiquitin chains. In U2OS cells, chemical inhibition of p97 or siRNA-mediated depletion of p97 or its adapters impairs Ku80 removal after non-homologous end joining of DSBs. Moreover, this inhibition attenuates early steps in homologous recombination, consistent with p97-driven Ku release also affecting repair pathway choice. Thus, our data answer a central question regarding regulation of Ku in DSB repair and illustrate the ability of p97 to segregate even tightly bound protein complexes for release from DNA.

Single-molecule imaging of UvrA and UvrB recruitment to DNA lesions in living Escherichia coli.

Nucleotide excision repair (NER) removes chemically diverse DNA lesions in all domains of life. In Escherichia coli, UvrA and UvrB initiate NER, although the mechanistic details of how this occurs in vivo remain to be established. Here, we use single-molecule fluorescence imaging to provide a comprehensive characterization of the lesion search, recognition and verification process in living cells. We show that NER initiation involves a two-step mechanism in which UvrA scans the genome and locates DNA damage independently of UvrB. Then UvrA recruits UvrB from solution to the lesion. These steps are coordinated by ATP binding and hydrolysis in the ‘proximal' and ‘distal' UvrA ATP-binding sites. We show that initial UvrB-independent damage recognition by UvrA requires ATPase activity in the distal site only. Subsequent UvrB recruitment requires ATP hydrolysis in the proximal site. Finally, UvrA dissociates from the lesion complex, allowing UvrB to orchestrate the downstream NER reactions.

Modulating Salmonella Typhimurium's Response to a Changing Environment through Bacterial Enhancer-Binding Proteins and the RpoN Regulon.

Transcription sigma factors direct the selective binding of RNA polymerase holoenzyme (Eσ) to specific promoters. Two families of sigma factors determine promoter specificity, the σ70 (RpoD) family and the σ54 (RpoN) family. In transcription controlled by σ54, the Eσ54-promoter closed complex requires ATP hydrolysis by an associated bacterial enhancer-binding protein (bEBP) for the transition to open complex and transcription initiation. Given the wide host range of Salmonella enterica serovar Typhimurium, it is an excellent model system for investigating the roles of RpoN and its bEBPs in modulating the lifestyle of bacteria. The genome of S. Typhimurium encodes 13 known or predicted bEBPs, each responding to a unique intracellular or extracellular signal. While the regulons of most alternative sigma factors respond to a specific environmental or developmental signal, the RpoN regulon is very diverse, controlling genes for response to nitrogen limitation, nitric oxide stress, availability of alternative carbon sources, phage shock/envelope stress, toxic levels of zinc, nucleic acid damage, and other stressors. This review explores how bEBPs respond to environmental changes encountered by S. Typhimurium during transmission/infection and influence adaptation through control of transcription of different components of the S. Typhimurium RpoN regulon.

The Walker A motif mutation recA4159 abolishes the SOS response and recombination in a recA730 mutant of Escherichia coli

In bacteria, the RecA protein forms recombinogenic filaments required for the SOS response and DNA recombination. In order to form a recombinogenic filament, wild type RecA needs to bind ATP and to interact with mediator proteins. The RecA730 protein is a mutant version of RecA with superior catalytic abilities, allowing filament formation without the help of mediator proteins. The mechanism of RecA730 filament formation is not well understood, and the question remains as to whether the RecA730 protein requires ATP binding in order to become competent for filament formation. We examined two mutants, recA730,4159 (presumed to be defective for ATP binding) and recA730,2201 (defective for ATP hydrolysis), and show that they have different properties with respect to SOS induction, conjugational recombination and double-strand break repair. We show that ATP binding is essential for all RecA730 functions, while ATP hydrolysis is required only for double-strand break repair. Our results emphasize the similarity of the SOS response and conjugational recombination, neither of which requires ATP hydrolysis by RecA730.

Trapping the Transition State of Kinesin-5 Produces a Different Multimotor Force Outcome than Inhibiting Product Release

Kinesin-5 (Eg5) is an essential mitotic motor that works in concert with multiple motors to form the bipolar spindle. Acute interest in the human kinesin-5 stems from the potential to target it for clinical therapy; many small-molecule inhibitors bind to an allosteric loop-5 (L5) pocket, inhibit ADP release, and reduce Eg5 motion to a diffusive mode that does not require ATP hydrolysis. Herein, we show that not all L5-directed drugs have the same catalytic effect nor do they have equivalent impacts upon the motor-microtubule complex. Linear free energy relationships, derived from steady state kinetics measurements of wildtype and eight L5 mutant proteins, show that monastrol is a transition state inhibitor, uniquely through an allosteric mechanism, whereas STC and ispinesib are not. Unexpectedly, in vitro “tug of war” assays between human Eg5 and human Kinesin-1 (KHC) revealed that these two categories of small chemical inhibitors also elicit dramatically different effects on Eg5 motility. Inhibitors of product release allow diffusive microtubule sliding. In contrast, trapping the transition state reveals bimodal behaviour that ultimately causes Eg5 to function as a brake that resists microtubule sliding by KHC. We conclude that ADP state inhibition of Eg5 allows the other kinesin forces on the microtubule to be unchecked while inhibition at the transition state of Eg5 hinders the entire multi-motor ensemble. It is possible that these disparate force outcomes are the result of variation in how drug inhibition at different catalytic intermediates can affect inter-head communication both within Eg5 and between motors in the ensemble. Our work highlights that not all L5 directed drugs are equal, and suggests kinesin inhibitors trapping the transition state should be further explored for their therapeutic potential.

Chaperone-assisted Post-translational Transport of Plastidic Type I Signal Peptidase 1

Type I signal peptidase (SPase I) is an integral membrane Ser/Lys protease with one or two transmembrane domains (TMDs), cleaving transport signals off translocated precursor proteins. The catalytic domain of SPase I folds to form a hydrophobic surface and inserts into the lipid bilayers at the trans-side of the membrane. In bacteria, SPase I is targeted co-translationally, and the catalytic domain remains unfolded until it reaches the periplasm. By contrast, SPases I in eukaryotes are targeted post-translationally, requiring an alternative strategy to prevent premature folding. Here we demonstrate that two distinct stromal components are involved in post-translational transport of plastidic SPase I 1 (Plsp1) from Arabidopsis thaliana, which contains a single TMD. During import into isolated chloroplasts, Plsp1 was targeted to the membrane via a soluble intermediate in an ATP hydrolysis-dependent manner. Insertion of Plsp1 into isolated chloroplast membranes, by contrast, was found to occur by two distinct mechanisms. The first mechanism requires ATP hydrolysis and the protein conducting channel cpSecY1 and was strongly enhanced by exogenously added cpSecA1. The second mechanism was independent of nucleoside triphosphates and proteinaceous components but with a high frequency of mis-orientation. This unassisted insertion was inhibited by urea and stroma extract. During import-chase assays using intact chloroplasts, Plsp1 was incorporated into a soluble 700-kDa complex that co-migrated with the Cpn60 complex before inserting into the membrane. The TMD within Plsp1 was required for the cpSecA1-dependent insertion but was dispensable for association with the 700-kDa complex and also for unassisted membrane insertion. These results indicate cooperation of Cpn60 and cpSecA1 for proper membrane insertion of Plsp1 by cpSecY1.

The Smc5/6 Complex Is an ATP-Dependent Intermolecular DNA Linker.

The structural maintenance of chromosome (SMC) protein complexes cohesin and condensin and the Smc5/6 complex (Smc5/6) are crucial for chromosome dynamics and stability. All contain essential ATPase domains, and cohesin and condensin interact with chromosomes through topological entrapment of DNA. However, how Smc5/6 binds DNA and chromosomes has remained largely unknown. Here, we show that purified Smc5/6 binds DNA through a mechanism that requires ATP hydrolysis by the complex and circular DNA to be established. This also promotes topoisomerase 2-dependent catenation of plasmids, suggesting that Smc5/6 interconnects two DNA molecules using ATP-regulated topological entrapment of DNA, similar to cohesin. We also show that a complex containing an Smc6 mutant that is defective in ATP binding fails to interact with DNA and chromosomes and leads to cell death with concomitant accumulation of DNA damage when overexpressed. Taken together, these results indicate that Smc5/6 executes its cellular functions through ATP-regulated intermolecular DNA linking.

Mode of action of closthioamide: the first member of the polythioamide class of bacterial DNA gyrase inhibitors.

The spread of MDR bacteria represents a serious threat to human society and novel antibiotic drugs, preferably from new chemical classes, are urgently needed. Closthioamide was isolated from the strictly anaerobic bacterium Clostridium cellulolyticum and belongs to a new class of natural products, the polythioamides. Here, we investigated the antimicrobial activity and mechanism of action of closthioamide. For assessing the antimicrobial activity of closthioamide, MIC values and killing kinetics were determined. To identify its target pathway, whole-cell-based assays were used including analysis of macromolecular synthesis and recording the susceptibility profile of a library of clones with down-regulated potential target genes. Subsequently, the inhibitory effect of closthioamide on the activity of isolated target enzymes, e.g. DNA gyrase and topoisomerase IV, was evaluated. Closthioamide had broad-spectrum activity against Gram-positive bacteria. Notably, closthioamide was very potent against MRSA and VRE strains. Closthioamide impaired DNA replication and inhibited DNA gyrase activity, in particular the ATPase function of gyrase and of topoisomerase IV, whereas there was little effect on the cleavage-rejoining function. Closthioamide also inhibited the relaxation activity of DNA gyrase, which does not require ATP hydrolysis, and thus may allosterically rather than directly interfere with the ATPase activity of gyrase. Cross-resistance to ciprofloxacin and novobiocin could not be detected in experimental mutants and clinical isolates. Closthioamide, a member of an unprecedented class of antibiotics, is a potent inhibitor of bacterial DNA gyrase; however, its molecular mechanism differs from that of the quinolones and aminocoumarins.

Active displacement of RecA filaments by UvrD translocase activity.

The UvrD helicase has been implicated in the disassembly of RecA nucleoprotein filaments in vivo and in vitro. We demonstrate that UvrD utilizes an active mechanism to remove RecA from the DNA. Efficient RecA removal depends on the availability of DNA binding sites for UvrD and/or the accessibility of the RecA filament ends. The removal of RecA from DNA also requires ATP hydrolysis by the UvrD helicase but not by RecA protein. The RecA-removal activity of UvrD is slowed by RecA variants with enhanced DNA-binding properties. The ATPase rate of UvrD during RecA removal is much slower than the ATPase activity of UvrD when it is functioning either as a translocase or a helicase on DNA in the absence of RecA. Thus, in this context UvrD may operate in a specialized disassembly mode.

English

ABSTRACT Pharmacodynamical studies showed that most drugs elicit their effects by acting on 3 kinds of protein molecules known as receptors, enzymes or transporters. Although their detail properties had not been explained for decades the roles of transporters in drug kinetics and dynamics has been well understood, even have been applied in the therapy. Transporters are classified into 2 major classes, the solute carriers (SLC) and ATP-binding cassette (ABC) families. SLC transporters do not possess ATP binding site property as those of ABC transporters. SLC transporters consist of 3 SLC subfamilies i.e. organic cation transporters (OCTs), organic anion transporters (OATs) and organic anion transporting polypeptides (OATPs). In contrast, ABC transporters require ATP hydrolysis to transport substrate across cell membrane. Human ABC-transporters consist of ABCA1- 13, ABCB1-11, ABCC1-12, ABCD1-4, ABCE1, ABCF1-3 and ABCG1-8 subfamily. Although the originally funtion of transporter is to transport specific physiological substrate such as nutrient, hormone, cytokines, neurotransmitters and other physiological subtances across cell membrane the specificity is not restricted to each substrate. Drugs and other xenobiotics which have structural similarity to the physiological substrates are recognized and transported by the related transporters. The competition of them on transporters therefore may lead to the occurence of drug-drug interactions (DDI) or drugphysiological substrate interaction in the drug-kinetics phase. Many transporters located in the liver, intestinal and renal epithelial cell membranes involve in the transport of endogenous substance or xenobiotics including drugs play important roles as protective barrier. Since transporters also serve as the targets of drug action it is understood that transporters play important role in the pathogenesis of diseases as well as in the drug therapy of diseases.

Actin and Dynamin Control the Fate of the Fusion Intermediate - the γ-Profile

Fusion generates an intermediate structure, an Ω-shaped membrane profile, to release vesicular contents. The subsequent fate of the intermediate structure, either Ω-profile pore closure or merge between the Ω-profile and the plasma membrane, determines whether retrieval of the Ω-profile is via fusion pore closure or classical endocytosis. Owing to difficulty of detecting Ω-profile, the mechanism controlling the Ω-profile's fate, particularly the merging process, is entirely unclear, and is often assumed an automatic process without using energy or force. By directly imaging Ω-profile in neuroendocrine chromaffin cells, we found that Ωprofile merging requires ATP hydrolysis and is mediated by actin dynamics that provides a mechanical force to shrink the Ω-profile, whereas dynamin counteracts vesicle merging by mediating Ω-profile closure, which precludes Ω-profile merging. These results reveal actin, ATP, and dynamin in control of the fate of the fusion intermediate and thus the endocytosis route for recycling of fusing vesicles.

Co- and Post-translocation Roles for HSP90 in Cholera Intoxication

Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) where the catalytic CTA1 subunit separates from the rest of the toxin. CTA1 then unfolds and passes through an ER translocon pore to reach its cytosolic target. Due to its intrinsic instability, cytosolic CTA1 must be refolded to achieve an active conformation. The cytosolic chaperone Hsp90 is involved with the ER to cytosol export of CTA1, but the mechanistic role of Hsp90 in CTA1 translocation remains unknown. Moreover, potential post-translocation roles for Hsp90 in modulating the activity of cytosolic CTA1 have not been explored. Here, we show by isotope-edited Fourier transform infrared spectroscopy that Hsp90 induces a gain-of-structure in disordered CTA1 at physiological temperature. Only the ATP-bound form of Hsp90 interacts with disordered CTA1, and refolding of CTA1 by Hsp90 is dependent upon ATP hydrolysis. In vitro reconstitution of the CTA1 translocation event likewise required ATP hydrolysis by Hsp90. Surface plasmon resonance experiments found that Hsp90 does not release CTA1, even after ATP hydrolysis and the return of CTA1 to a folded conformation. The interaction with Hsp90 allows disordered CTA1 to attain an active state, which is further enhanced by ADP-ribosylation factor 6, a host cofactor for CTA1. Our data indicate CTA1 translocation involves a process that couples the Hsp90-mediated refolding of CTA1 with CTA1 extraction from the ER. The molecular basis for toxin translocation elucidated in this study may also apply to several ADP-ribosylating toxins that move from the endosomes to the cytosol in an Hsp90-dependent process.

Geminin Inhibits a Late Step in the Formation of Human Pre-replicative Complexes

The initial step in initiation of eukaryotic DNA replication involves the assembly of pre-replicative complexes (pre-RCs) at origins of replication during the G1 phase of the cell cycle. In metazoans initiation is inhibited by the regulatory factor Geminin. We have purified the human pre-RC proteins, studied their interactions in vitro with each other and with origin DNA, and analyzed the effects of HsGeminin on formation of DNA-protein complexes. The formation of an initial complex containing the human origin recognition complex (HsORC), HsCdt1, HsCdc6, and origin DNA is cooperative, involving all possible binary interactions among the components. Maximal association of HsMCM2-7, a component of the replicative helicase, requires HsORC, HsCdc6, HsCdt1, and ATP, and is driven by interactions of HsCdt1 and HsCdc6 with multiple HsMCM2-7 subunits. Formation of stable complexes, resistant to high salt, requires ATP hydrolysis. In the absence of HsMCM proteins, HsGeminin inhibits the association of HsCdt1 with DNA or with HsORC-HsCdc6-DNA complexes. However, HsGeminin does not inhibit recruitment of HsMCM2-7 to DNA to form complexes containing all of the pre-RC proteins. In fact, HsGeminin itself is a component of such complexes, and interacts directly with the HsMcm3 and HsMcm5 subunits of HsMCM2-7, as well as with HsCdt1. Although HsGeminin does not prevent the initial formation of DNA-protein complexes containing the pre-RC proteins, it strongly inhibits the formation of stable pre-RCs that are resistant to high salt. We suggest that bound HsGeminin prevents transition of the pre-RC to a state that is competent for initiation of DNA replication.

Origin Licensing Requires ATP Binding and Hydrolysis by the MCM Replicative Helicase

SummaryLoading of the six related Minichromosome Maintenance (MCM) proteins as head-to-head double hexamers during DNA replication origin licensing is crucial for ensuring once-per-cell-cycle DNA replication in eukaryotic cells. Assembly of these prereplicative complexes (pre-RCs) requires the Origin Recognition Complex (ORC), Cdc6, and Cdt1. ORC, Cdc6, and MCM are members of the AAA+ family of ATPases, and pre-RC assembly requires ATP hydrolysis. Here we show that ORC and Cdc6 mutants defective in ATP hydrolysis are competent for origin licensing. However, ATP hydrolysis by Cdc6 is required to release nonproductive licensing intermediates. We show that ATP binding stabilizes the wild-type MCM hexamer. Moreover, by analyzing MCM containing mutant subunits, we show that ATP binding and hydrolysis by MCM are required for Cdt1 release and double hexamer formation. This work alters our view of how ATP is used by licensing factors to assemble pre-RCs.

Synthetic organotelluride compounds induce the reversal of Pdr5p mediated fluconazole resistance in Saccharomyces cerevisiae.

BackgroundResistance to fluconazole, a commonly used azole antifungal, is a challenge for the treatment of fungal infections. Resistance can be mediated by overexpression of ABC transporters, which promote drug efflux that requires ATP hydrolysis. The Pdr5p ABC transporter of Saccharomyces cerevisiae is a well-known model used to study this mechanism of antifungal resistance. The present study investigated the effects of 13 synthetic compounds on Pdr5p.ResultsAmong the tested compounds, four contained a tellurium-butane group and shared structural similarities that were absent in the other tested compounds: a lateral hydrocarbon chain and an amide group. These four compounds were capable of inhibiting Pdr5p ATPase activity by more than 90%, they demonstrated IC50 values less than 2 μM and had an uncompetitive pattern of Pdr5p ATPase activity inhibition. These organotellurides did not demonstrate cytotoxicity against human erythrocytes or S. cerevisiae mutant strains (a strain that overexpress Pdr5p and a null mutant strain) even in concentrations above 100 μM. When tested at 100 μM, they could reverse the fluconazole resistance expressed by both the S. cerevisiae mutant strain that overexpress Pdr5p and a clinical isolate of Candida albicans.ConclusionsWe have identified four organotellurides that are promising candidates for the reversal of drug resistance mediated by drug efflux pumps. These molecules will act as scaffolds for the development of more efficient and effective efflux pump inhibitors that can be used in combination therapy with available antifungals.