Discovery and Characterization of Novel Substrate Selective Inhibitors of Human MRP1 (ABCC1)

Multidrug resistance protein 1 (MRP1/ABCC1) actively transports a variety of drugs, toxic molecules and important physiological substrates across the plasma membrane. It can confer broad-spectrum drug resistance and can decrease the bioavailability of many important drugs such as anti-cancer agents, antibiotics, antivirals, antidepressants and antiinflammatory drugs. Calcein-AM, a fluorescent reporter commonly used for studying compound interactions with MRP1 was recently used in the development of a high content imaging-based assay by our group. This assay was robust and had better sensitivity than fluorescent plate readouts. The assay identified 12 MRP1 inhibitors after screening an anticancer library of 386 compounds. Due to the multiple distinct substrate binding sites of MRP1, we sought to use different fluorescent probes to identify substrate selective inhibitors which were likely missed by the calcein-AM screening. The high content imaging-based uptake assay was modified using doxorubicin (anticancer drug) and CRO- 9 (dye) as fluorescent reporters which vary in structure and function. The doxorubicin assay, after screening the same 386 compound library identified a total of 28 MRP1 inhibitors including 16 inhibitors that have not been previously reported as inhibitors of MRP1. The CRO-9 assay identified a total of 50 MRP1 inhibitors including 19 additional inhibitors that have never been reported as inhibitors of MRP1. These 50 MRP1 inhibitors included 10 out of 12 hits identified using calcein-AM and 27 out of 28 inhibitors discovered through the doxorubicin assay. MRP1 inhibition was confirmed using flow cytometry, confocal microscopy and membrane-based transport assays. Selected drugs were evaluated for their ability to reverse resistance of MRP1-overexpressing H69AR lung cancer cells against various substrates. From the doxorubicin screening, mifepristone and doramapimod were the most effective in reversing MRP1 mediated resistance whiles celecoxib exhibited selective MRP1 inhibition. From the hits identified through the CRO- 9-based screening, LY2603618 and ZSTK474 were the most effective in reversing MRP1 mediated resistance in H69AR cells. Together, our findings signify the effectiveness and value of doxorubicin and CRO-9 based high content screening approach. Anti-cancer agents that exhibit MRP1 inhibition may be used to reverse multidrug resistance or to improve the efficacy and reduce the toxicity of various cancer chemotherapies

Calcitriol and Calcipotriol Modulate Transport Activity of ABC Transporters and Exhibit Selective Cytotoxicity in MRP1-overexpressing Cells

Efflux transporters P-glycoprotein (P-gp/ABCB1), multidrug resistance protein 1 (MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2) can affect the efficacy and toxicity of a wide variety of drugs and are implicated in multidrug resistance (MDR). Eight test compounds, recently identified from an intramolecular FRET-based high throughput screening, were characterized for their interaction with MRP1. We report that the active metabolite of vitamin D3, calcitriol, and its analog calcipotriol are selectively cytotoxic to MRP1-overexpressing cells, besides inhibiting transport function of P-gp, MRP1, and BCRP. Calcitriol and calcipotriol consistently displayed a potent inhibitory activity on MRP1-mediated doxorubicin and calcein efflux in MRP1-overexpressing H69AR and HEK293/MRP1 cells. Vesicular transport studies confirmed a strong inhibitory effect of calcitriol and calcipotriol on MRP1-mediated uptake of tritiumlabeled estradiol glucuronide and leukotriene C4. In cytotoxicity assays, MRP1-overexpressing cells exhibited hypersensitivity toward calcitriol and calcipotriol. Such collateral sensitivity, however, was not observed in HEK293/P-gp and HEK293/BCRP cells, although the vitamin D3 analogs inhibited calcein efflux in P-gp-overexpressing cells, and mitoxantrone efflux in BCRP-overexpressing cells. The selective cytotoxicity of calcitriol and calpotriol toward MRP1 over-expressing cells can be eliminated with MRP1 inhibitor MK571. Our data indicate a potential role of calcitriol and its analogs in targeting malignancies in which MRP1 expression is prominent and contributes to MDR

Development of Novel Intramolecular FRET-Based ABC Transporter Biosensors to Identify New Substrates and Modulators

Multidrug resistance protein 1 (MRP1) can efflux a wide variety of molecules including toxic chemicals, drugs, and their derivatives out of cells. Substrates of MRP1 include anti-cancer agents, antibiotics, anti-virals, anti-human immunodeficiency virus (HIV), and many other drugs. To identify novel substrates and modulators of MRP1 by exploiting intramolecular fluorescence resonance energy transfer (FRET), we genetically engineered six different two-color MRP1 proteins by changing green fluorescent protein (GFP) insertion sites, while keeping the red fluorescent protein (RFP) at the C-terminal of MRP1. Four of six recombinant proteins showed normal expression, localization, and transport activity. We quantified intramolecular FRET using ensemble fluorescence spectroscopy in response to binding of known substrate or ATP alone, substrate/ATP, and trapping of the transporter in closed conformation by vanadate. Recombinant MRP1 proteins GR-881, GR-888, and GR-905 exhibited reproducible and higher FRET changes under all tested conditions and are very promising for use as MRP1 biosensors. Furthermore, we used GR-881 to screen 40 novel anti-cancer drugs and identified 10 hits that potentially directly interact with MRP1 and could be substrates or modulators. Profiling of drug libraries for interaction with MRP1 can provide very useful information to improve the efficacy and reduce the toxicity of various therapies

The burden of drug resistance tuberculosis in Ghana; results of the First National Survey.

Resistance to Tuberculosis drugs has become a major threat to the control of tuberculosis (TB) globally. We conducted the first nation-wide drug resistance survey to investigate the level and pattern of resistance to first-line TB drugs among newly and previously treated sputum smear-positive TB cases. We also evaluated associations between potential risk factors and TB drug resistance. Using the World Health Organization (WHO) guidelines on conducting national TB surveys, we selected study participants from 33 health facilities from across the country, grouped into 29 clusters, and included them into the survey. Between April 2016 and June 2017, a total of 927 patients (859 new and 68 previously treated) were enrolled in the survey. Mycobacterium tuberculosis complex (MTBC) isolates were successfully cultured from 598 (65.5%) patient samples and underwent DST, 550 from newly diagnosed and 48 from previously treated patients. The proportion of patients who showed resistance to any of the TB drugs tested was 25.2% (95% CI; 21.8-28.9). The most frequent resistance was to Streptomycin (STR) (12.3%), followed by Isoniazid (INH) (10.4%), with Rifampicin (RIF), showing the least resistance of 2.4%. Resistance to Isoniazid and Rifampicin (multi-drug resistance) was found in 19 (3.2%; 95% CI: 1.9-4.9) isolates. Prevalence of multidrug resistance was 7 (1.3%; 95% CI: 0.5-2.6) among newly diagnosed and 12 (25.0%; 95% CI: 13.6-39.6) among previously treated patients. At both univariate and multivariate analysis, MDR-TB was positively associated with previous history of TB treatment (OR = 5.09, 95% CI: 1.75-14.75, p = 0.003); (OR = 5.41, 95% CI: 1.69-17.30, p = 0.004). The higher levels of MDR-TB and overall resistance to any TB drug among previously treated patients raises concerns about adherence to treatment. This calls for strengthening existing TB programme measures to ensure a system for adequately testing and monitoring TB drug resistance

Development of Novel Intramolecular FRET-Based ABC Transporter Biosensors to Identify New Substrates and Modulators

Multidrug resistance protein 1 (MRP1) can efflux a wide variety of molecules including toxic chemicals, drugs, and their derivatives out of cells. Substrates of MRP1 include anti-cancer agents, antibiotics, anti-virals, anti-human immunodeficiency virus (HIV), and many other drugs. To identify novel substrates and modulators of MRP1 by exploiting intramolecular fluorescence resonance energy transfer (FRET), we genetically engineered six different two-color MRP1 proteins by changing green fluorescent protein (GFP) insertion sites, while keeping the red fluorescent protein (RFP) at the C-terminal of MRP1. Four of six recombinant proteins showed normal expression, localization, and transport activity. We quantified intramolecular FRET using ensemble fluorescence spectroscopy in response to binding of known substrate or ATP alone, substrate/ATP, and trapping of the transporter in closed conformation by vanadate. Recombinant MRP1 proteins GR-881, GR-888, and GR-905 exhibited reproducible and higher FRET changes under all tested conditions and are very promising for use as MRP1 biosensors. Furthermore, we used GR-881 to screen 40 novel anti-cancer drugs and identified 10 hits that potentially directly interact with MRP1 and could be substrates or modulators. Profiling of drug libraries for interaction with MRP1 can provide very useful information to improve the efficacy and reduce the toxicity of various therapies

Tuberous sclerosis causing mutants of the TSC2 gene product affect proliferation and p27 expression

The autosomal dominant disease tuberous sclerosis (TSC) is caused by mutations in either TSC1 on chromosome 9q34, encoding hamartin, or TSC2 on chromosome 16p13.3, encoding tuberin. TSC is characterized by hamartomas that occur in many organs of a�ffected patients and these have been considered to likely result from defects in proliferation control. Although the true biochemical functions of the two TSC proteins have not been clarified, a series of independent investigations demonstrated that modulated hamartin or tuberin expression cause deregulation of proliferation/cell cycle in human, rodent and Drosophila cells. In support of tuberin acting as a tumor suppressor, ectopic over-expression of TSC2 has been shown to decrease proliferation rates of mammalian cells. Furthermore, overexpression of TSC2 has been demonstrated to trigger upregulation of the cyclin-dependent kinase inhibitor p27. We report that three di�fferent naturally occurring and TSC causing mutations within the TSC2 gene eliminate neither the anti-proliferative capacity of tuberin nor tuberin's eff�ects on p27 expression. For the first time these data provide strong evidence that deregulation of proliferation and/or upregulation of p27 are not likely to be the primary/only mechanisms of hamartoma development in TSC. These results demand reassessment of previous hypotheses of the pathogenesis of TSC