Indole Derivative Interacts with Estrogen Receptor Beta and Inhibits Human Ovarian Cancer Cell Growth.

Ovarian cancer remains the leading cause of mortality among gynecological tumors. Estrogen receptor beta (ERß) expression has been suggested to act as a tumor suppressor in epithelial ovarian cancer by reducing both tumor growth and metastasis. ERß expression abnormalities represent a critical step in the development and progression of ovarian cancer: for these reasons, its re-expression by genetic engineering, as well as the use of targeted ERß therapies, still constitute an important therapeutic approach. 3-{[2-chloro-1-(4-chlorobenzyl)-5-methoxy-6-methyl-1H-indol-3-yl]methylene}-5-hydroxy-6-methyl-1,3-dihydro-2H-indol-2-one, referred to here as compound 3, has been shown to have cytostatic as well cytotoxic effects on various hormone-dependent cancer cell lines. However, the mechanism of its anti-carcinogenic activity is not well understood. Here, we offer a possible explanation of such an effect in the human ovarian cancer cell line IGROV1. Chromatin binding protein assay and liquid chromatography mass spectrometry were exploited to localize and quantify compound 3 in cells. Molecular docking was used to prove compound 3 binding to ERß. Mass spectrometry-based approaches were used to analyze histone post-translational modifications. Finally, gene expression analyses revealed a set of genes regulated by the ERß/3 complex, namely CCND1, MYC, CDKN2A, and ESR2, providing possible molecular mechanisms that underline the observed antiproliferative effects.

Unveiling the Biochemistry of the Epigenetic Regulator SMYD3.

SET and MYND domain-containing protein 3 (SMYD3) is a lysine methyltransferase that plays a central role in a variety of cancer diseases, exerting its pro-oncogenic activity by methylation of key proteins, of both nuclear and cytoplasmic nature. However, the role of SMYD3 in the initiation and progression of cancer is not yet fully understood and further biochemical characterization is required to support the discovery of therapeutics targeting this enzyme. We have therefore developed robust protocols for production, handling, and crystallization of SMYD3 and biophysical and biochemical assays for clarification of SMYD3 biochemistry and identification of useful lead compounds. Specifically, a time-resolved biosensor assay was developed for kinetic characterization of SMYD3 interactions. Functional differences in SMYD3 interactions with its natural small molecule ligands SAM and SAH were revealed, with SAM forming a very stable complex. A variety of peptides mimicking putative substrates of SMYD3 were explored in order to expose structural features important for recognition. The interaction between SMYD3 and some peptides was influenced by SAM. A nonradioactive SMYD3 activity assay using liquid chromatography-mass spectrometry (LC-MS) analysis explored substrate features of importance also for methylation. Methylation was notable only toward MAP kinase kinase kinase 2 (MAP3K2_K260)-mimicking peptides, although binary and tertiary complexes were detected also with other peptides. The analysis supported a random bi-bi mechanistic model for SMYD3 methyltransferase catalysis. Our work unveiled complexities in SMYD3 biochemistry and resulted in procedures suitable for further studies and identification of novel starting points for design of effective and specific leads for this potential oncology target.

Structural basis for the magnesium-dependent activation of transketolase from Chlamydomonas reinhardtii.

BACKGROUND: In photosynthetic organisms, transketolase (TK) is involved in the Calvin-Benson cycle and participates to the regeneration of ribulose-5-phosphate. Previous studies demonstrated that TK catalysis is strictly dependent on thiamine pyrophosphate (TPP) and divalent ions such as Mg2+. METHODS: TK from the unicellular green alga Chlamydomonas reinhardtii (CrTK) was recombinantly produced and purified to homogeneity. Biochemical properties of the CrTK enzyme were delineated by activity assays and its structural features determined by CD analysis and X-ray crystallography. RESULTS: CrTK is homodimeric and its catalysis depends on the reconstitution of the holo-enzyme in the presence of both TPP and Mg2+. Activity measurements and CD analysis revealed that the formation of fully active holo-CrTK is Mg2+-dependent and proceeds with a slow kinetics. The 3D-structure of CrTK without cofactors (CrTKapo) shows that two portions of the active site are flexible and disordered while they adopt an ordered conformation in the holo-form. Oxidative treatments revealed that Mg2+ participates in the redox control of CrTK by changing its propensity to be inactivated by oxidation. Indeed, the activity of holo-form is unaffected by oxidation whereas CrTK in the apo-form or reconstituted with the sole TPP show a strong sensitivity to oxidative inactivation. CONCLUSION: These evidences indicate that Mg2+ is fundamental to allow gradual conformational arrangements suited for optimal catalysis. Moreover, Mg2+ is involved in the control of redox sensitivity of CrTK. GENERAL SIGNIFICANCE: The importance of Mg2+ in the functionality and redox sensitivity of CrTK is correlated to light-dependent fluctuations of Mg2+ in chloroplasts.

Absolute Configuration Assignment of Chiral Resorcin[4]arenes from ECD Spectra.

Racemates of five chiral resorcin[4]arenes, four tetra-O-substituted and one hepta-O-substituted, have been resolved by enantioselective HPLC, and their ECD spectra have been recorded online by stopped-flow measurements. The absolute configuration has been assigned by comparison of the experimental ECD spectra with DFT and semiempirical calculations. For the four tetra-O-substituted resorcin[4]arenes, the ECD exciton couplet at longer wavelength depends on the chirality induced in the arene scaffold by the substituents rather than on the precise nature of the substituents themselves. Accordingly, the exciton chirality model with excitons localized on the arene scaffold, here generalized to Cn symmetry, accurately describes the relationship between stereochemistry and chiroptical properties for this couplet, while its application at shorter wavelengths is unsafe. For the significantly larger hepta-O-substituted system the assignment particularly benefits from the use of the semiempirical ZINDO method.

Surface plasmon resonance and isothermal titration calorimetry to monitor the Ni(II)-dependent binding of Helicobacter pylori NikR to DNA.

NikR is a transcription factor that regulates the expression of Ni(II)-dependent enzymes and other proteins involved in nickel trafficking. In the human pathogenic bacterium Helicobacter pylori, NikR (HpNikR) controls, among others, the expression of the Ni(II) enzyme urease by binding the double-strand DNA (dsDNA) operator region of the urease promoter (OP ureA ) in a Ni(II)-dependent mode. This article describes the complementary use of surface plasmon resonance (SPR) spectroscopy and isothermal titration calorimetry (ITC) to carry out a mechanistic characterization of the HpNikR-OP ureA interaction. An active surface was prepared by affinity capture of OP ureA and validated for the recognition process in the SPR experiments. Subsequently, the Ni(II)-dependent affinity of the transcription factor for its operator region was assessed through kinetic evaluation of the binding process at variable Ni(II) concentrations. The kinetic data are consistent with a two-step binding mode involving an initial encounter between the two interactants, followed by a conformational rearrangement of the HpNikR-OP ureA complex, leading to high affinity binding. This conformational change is only observed in the presence of the full set of four Ni(II) ions bound to the protein. The SPR assay developed and validated in this study constitutes a suitable method to screen potential drug lead candidates acting as inhibitors of this protein-dsDNA interaction. Graphical Abstract Pictorial representation of the interaction between HpNikR, flowing in solution, and the OP ureA urease promoter immobilized on the sensor chip surface.

A SMYD3 Small-Molecule Inhibitor Impairing Cancer Cell Growth.

SMYD3 is a histone lysine methyltransferase that plays an important role in transcriptional activation as a member of an RNA polymerase complex, and its oncogenic role has been described in different cancer types. We studied the expression and activity of SMYD3 in a preclinical model of colorectal cancer (CRC) and found that it is strongly upregulated throughout tumorigenesis both at the mRNA and protein level. Our results also showed that RNAi-mediated SMYD3 ablation impairs CRC cell proliferation indicating that SMYD3 is required for proper cancer cell growth. These data, together with the importance of lysine methyltransferases as a target for drug discovery, prompted us to carry out a virtual screening to identify new SMYD3 inhibitors by testing several candidate small molecules. Here we report that one of these compounds (BCI-121) induces a significant reduction in SMYD3 activity both in vitro and in CRC cells, as suggested by the analysis of global H3K4me2/3 and H4K5me levels. Of note, the extent of cell growth inhibition by BCI-121 was similar to that observed upon SMYD3 genetic ablation. Most of the results described above were obtained in CRC; however, when we extended our observations to tumor cell lines of different origin, we found that SMYD3 inhibitors are also effective in other cancer types, such as lung, pancreatic, prostate, and ovarian. These results represent the proof of principle that SMYD3 is a druggable target and suggest that new compounds capable of inhibiting its activity may prove useful as novel therapeutic agents in cancer treatment.

Posttranscriptional changes of serum albumin: clinical and prognostic significance in hospitalized patients with cirrhosis.

UNLABELLED: Beside the regulation of fluid distribution, human serum albumin (HSA) carries other activities, such as binding, transport, and detoxification of many molecules. In patients with cirrhosis, HSA exhibits posttranscriptional alterations that likely affect its functions. This study aimed at identifying the structural HSA alterations occurring in cirrhosis and determining their relationship with specific clinical complications and patient survival. One hundred sixty-eight patients with cirrhosis, 35 with stable conditions and 133 hospitalized for acute clinical complications, and 94 healthy controls were enrolled. Posttranscriptional HSA molecular changes were identified and quantified by using a high-performance liquid chromatography/electrospray ionization mass spectrometry technique. Clinical and biochemical parameters were also recorded and hospitalized patients were followed for up to 1 year. Seven HSA isoforms carrying one or more posttranscriptional changes were identified. Altered HSA isoforms were significantly more represented in patients than in healthy controls. Conversely, the native, unchanged HSA isoform was significantly reduced in cirrhosis. Native HSA and most altered isoforms correlated with both Child-Pugh and Model for End-Stage Liver Disease scores. In hospitalized patients, oxidized and N-terminal truncated isoforms were independently associated with ascites, renal impairment, and bacterial infection. Finally, the native HSA and cysteinylated/N-terminal truncated isoforms were predictors of 1-year survival, with greater prognostic accuracy than total serum albumin concentration. CONCLUSIONS: Extensive posttranscriptional changes of HSA, involving several molecular sites and increasing in parallel with disease severity, occur in patients with cirrhosis. Altered isoforms are independently associated with specific clinical complications, whereas the residual, native HSA isoform independently predicts patient survival. These findings support the concept of the "effective albumin concentration," which implies that the global HSA function is related not only to its serum concentration, but also to the preservation of its structural integrity.

Ischaemia-modified albumin: a marker of bacterial infection in hospitalized patients with cirrhosis.

BACKGROUND & AIMS: Patients with cirrhosis present structural changes of human serum albumin (HSA) affecting non-oncotic functions. Ischaemia-modified albumin (IMA), which reflects the capacity to bind cobalt, has been associated to patient mortality during acute-on-chronic liver failure. This study aimed to assess whether circulating IMA is elevated in advanced cirrhosis and its relationship with severity of cirrhosis and specific complications. METHODS: A total of 127 cirrhotic patients hospitalized for an acute complication of the disease and 44 healthy controls were enrolled. Plasma IMA and IMA to albumin ratio (IMAr) were measured with a cobalt-binding assay. HSA isoforms carrying post-transcriptional molecular changes were assessed with HPLC-ESI-MS. The effect of endotoxemia on IMA was evaluated in rats with CCl4 -cirrhosis. RESULTS: IMA/IMAr is significantly higher in cirrhotic patients than in controls, but no correlations were found with prognostic scores. IMA did not correlate with the altered HSA isoforms. Ascites, renal impairment and hepatic encephalopathy did not influence IMA/IMAr levels. In contrast, IMA/IMAr is significantly higher in infected than non-infected patients. ROC curves showed that IMA/IMAr had similar discriminating performances for bacterial infection as C-reactive protein (CRP). Moreover, CRP and IMA were independently associated with bacterial infection. Consistently, endotoxin injection significantly increased IMA in cirrhotic, but not in healthy rats. CONCLUSIONS: IMA is elevated in patients with advanced cirrhosis. The IMA level does not correlate with disease severity scores, but it is specifically associated to bacterial infection, showing a discriminating performance similar to CRP. Further investigations to assess IMA as a novel diagnostic test for bacterial infection are advocated.

Stopped-Flow Enantioselective HPLC-CD Analysis and TD-DFT Stereochemical Characterization of Methyl Trans-3-(3,4-Dimethoxyphenyl)Glycidate.

Caffeic acid-derived polyethers are a class of natural products isolated from the root extracts of comfrey and bugloss, which are endowed with intriguing pharmacological properties as anticancer agents. The synthesis of new polyether derivatives is achieved through ring-opening polymerization of chiral 2,3-disubstituted oxiranes, whose absolute configurations define the overall stereochemistry of the produced polymer. The absolute stereochemistry of one of these building blocks, methyl trans-3-(3,4-dimethoxy-phenyl)glycidate (3), was therefore characterized by the combination of enantioselective high-performance liquid chromatography (HPLC), electronic circular dichroism (ECD) spectroscopy, and time-dependent density functional theory (TD-DFT) calculations. Initial efforts aiming at the isolation of enantiomers by means of a standard preparative HPLC protocol followed by offline ECD analysis failed due to unexpected degradation of the samples after collection. The stopped-flow HPLC-CD approach, by which the ECD spectra of enantiomers are measured online with the HPLC system, was applied to overcome this issue and allowed a fast, reliable, and chemical-saving analysis, while avoiding the risks of sample degradation during the collection and processing of enantiomeric fractions. Subsequent TD-DFT calculations identified ( as the first eluted enantiomeric fraction on the Lux Cellulose-2 column, therefore achieving a full stereochemical characterization of the chiral oxirane under investigation.

Short-range solvation effects on chiroptical properties: a time-dependent density functional theory and ab initio molecular dynamics computational case study on austdiol.

The description of solvation effects on the chiroptical properties of chiral molecules is still a difficult challenge in the field of computational spectroscopy; this issue is critical in stereochemical characterization, since a reliable assessment of absolute configuration requires high accuracy. The present case study reports the huge effect of solvation on the chiroptical properties of austdiol, a fungal metabolite of known stereochemistry. Standard protocols based on time-dependent density functional theory calculations failed to reproduce its experimental chiroptical properties in methanol. When short-range solvation effects are explicitly considered by means of ab initio molecular dynamics, the correlation between calculated and experimental data is greatly improved because of a better description of the chiral environment around the ketone chromophore, showing that the modeling of subtle solvent-induced perturbations may require the most accurate computational methods.

Mycoleptones A-C and polyketides from the endophyte Mycoleptodiscus indicus.

Three new azaphilones with an unusual methylene bridge, named mycoleptones A, B, and C (2, 4, and 5), were isolated from cultures of Mycoleptodiscus indicus, a fungus associated with the South American medicinal plant Borreria verticillata. Additionally, four known polyketides, austdiol (1), eugenitin (3), 6-methoxyeugenin (6), and 9-hydroxyeugenin (7), were also isolated. The structural characterization of compounds was carried out by nuclear magnetic resonance spectroscopy, high-resolution mass spectrometry, electronic circular dichroism spectroscopy, time-dependent density functional theory calculations, and X-ray crystallography. Compounds 1-9 were weakly active when tested in antileishmanial and cytotoxicity assays.

Chiral triazole fungicide difenoconazole: absolute stereochemistry, stereoselective bioactivity, aquatic toxicity, and environmental behavior in vegetables and soil.

In this study, the systemic assessments of the stereoisomers of triazole fungicide difenoconazole are reported for the first time, including absolute stereochemistry, stereoselective bioactivity toward pathogens (Alternaria sonali, Fulvia fulva, Botrytis cinerea, and Rhizoctonia solani), and toxicity toward aquatic organisms (Scenedesmus obliquus, Daphnia magna, and Danio rerio). Moreover, the stereoselective degradation of difenoconazole in vegetables (cucumber, Cucumis sativus and tomato, Lycopersicon esculentum) under field conditions and in soil under laboratory-controlled conditions (aerobic and anaerobic) was investigated. There were 1.33-24.2-fold and 1.04-6.78-fold differences in bioactivity and toxicity, respectively. Investigations on the stereoselective degradation of difenoconazole in vegetables showed that the highest-toxic and lowest-bioactive (2S,4S)-stereoisomer displays a different enrichment behavior in different plant species. Under aerobic or anaerobic conditions, (2R,4R)- and (2R,4S)-difenoconazole were preferentially degraded in the soil. Moreover, difenoconazole was configurationally stable in the test soil matrices. On the basis of biological activity, ecotoxicity, and environmental behavior, it is likely that the use of pure (2R,4S)-difenoconazole instead of the commercial stereoisomer mix may help to increase the bioactivity and reduce environmental pollution.

Surface plasmon resonance, fluorescence, and circular dichroism studies for the characterization of the binding of BACE-1 inhibitors.

The mechanism of action underlying ß-secretase 1 (BACE-1) inhibition was characterized by a surface plasmon resonance (SPR) method using primary amino groups to immobilize OM99-2, a well-known highly potent peptidic BACE-1 inhibitor, on the carboxyl groups of the dextran layer of a sensor chip. The diluted BACE-1 was mixed with buffer or the test compound and the mixture was flushed through the chip. BACE-1 binding to the immobilized peptide inhibitor was quantified. This SPR method was used to identify BACE-1 inhibitor binding sites and the mechanism of action (competitive/noncompetitive) and to validate findings of fluorescence resonance energy transfer (FRET) inhibition studies. To support this, a multimethodological approach (circular dichroism and fluorescence spectroscopy) was applied in parallel to FRET inhibition studies to characterize the binding modes of peptidic and nonpeptidic BACE-1 inhibitors. Circular dichroism spectroscopy served to correlate the conformation of BACE-1 with enzymatic activity and to monitor secondary structure changes upon ligand binding. In a complementary approach, direct fluorescence spectroscopy was used to characterize different BACE-1 inhibitor binding sites. The influence of pH and inhibitors on BACE-1 secondary structure was also elucidated. This multimethodological approach was applied to identify binding modes of bis(7)-tacrine and myricetin in comparison with well-known peptidic inhibitors.

Conformational flexibility and absolute stereochemistry of (3R)-3-hydroxy-4-aryl-ß-lactams investigated by chiroptical properties and TD-DFT calculations.

The effect of conformational flexibility on the chiroptical properties of a series of synthetic (3R)-3-hydroxy-4-aryl-ß-lactams of known stereochemistry (1-6) was investigated by means of electronic circular dichroism (ECD) measurements and time-dependent density functional theory (TD-DFT) calculations. The application of the ß-lactam sector rules allowed a correct stereochemical characterization of these compounds, with the exception of a thienyl-substituted derivative (cis-). TD-DFT calculations yielded accurate predictions of experimental ECD spectra and [α](D) values, allowing us to assign the correct absolute configuration to all the investigated compounds. A detailed analysis of the ß-lactam ring equilibrium geometry on optimized conformers identified regular patterns for the arrangement of atoms around the amide chromophore, confirming the validity of the ß-lactam sector rules. However, relevant variations in theoretical chiroptical properties were found for compounds bearing a heterocyclic substituent at C4 or a phenyl substituent at C3, whose conformers deviate from these regular geometric patterns. This behavior explains the failure of the ß-lactam sector rules in cis-. This study showed the importance of conformational flexibility for the determination of chiroptical properties and highlighted the strengths and weaknesses of the different methods for the stereochemical characterization of chiral molecules in solution.

Analysis of carvedilol enantiomers in human plasma using chiral stationary phase column and liquid chromatography with tandem mass spectrometry.

Carvedilol is an antihypertensive drug available as a racemic mixture. (-)-(S)-carvedilol is responsible for the nonselective ß-blocker activity but both enantiomers present similar activity on α(1)-adrenergic receptor. To our knowledge, this is the first study of carvedilol enantiomers in human plasma using a chiral stationary phase column and liquid chromatography with tandem mass spectrometry. The method involves plasma extraction with diisopropyl ether using metoprolol as internal standard and direct separation of the carvedilol enantiomers on a Chirobiotic T® (Teicoplanin) column. Protonated ions [M + H](+) and their respective ion products were monitored at transitions of 407 > 100 for the carvedilol enantiomers and 268 > 116 for the internal standard. The quantification limit was 0.2 ng ml(-1) for both enantiomers in plasma. The method was applied to study enantioselectivity in the pharmacokinetics of carvedilol administered as a single dose of 25 mg to a hypertensive patient. The results showed a higher plasma concentration of (+)-(R)-carvedilol (AUC(0-∞) 205.52 vs. 82.61 (ng h) ml(-1)), with an enantiomer ratio of 2.48.

Reversible human serum albumin binding of lipocrine: a circular dichroism study.

Lipocrine has been selected as an effective candidate for in vivo investigation because of its multiple biological properties, namely inhibition of AChE and BChE activities, inhibition of AChE-induced Aß aggregation, and ability to protect cells against reactive oxygen species. To evaluate the possibility for lipocrine to become a lead and to be developed as a multipotent drug for the treatment of Alzheimer's disease, ADMET (absorption, distribution, metabolism, excretion, and toxicity) parameters need to be determined. Among ADMET parameters, distribution plays a key role in determining the lead drugability, and the drug binding to plasma proteins greatly influences the drug distribution. Here, the human serum albumin (HSA) binding of lipocrine has been studied by circular dichroism (CD) spectroscopy. The reversible binding of lipocrine is stereoselective as shown by the well-defined induced CD spectrum in its binding to HSA. The intensity of the CD signal changes upon changing the [drug]/[HSA] molar ratio, showing a different behavior for a [drug]/[HSA] up to 2/1 or over this molar ratio, suggesting a binding to multiple sites. Competition experiments show that lipocrine interacts significantly with all the main binding sites on the serum carrier. A direct competition has been monitored for site II and bilirubin-binding site, whereas a noncooperative binding should better describe the displacement observed at site I. Rac-lipocrine and its enantiomers are characterized by two different binding modes. Almost the same induced CD spectra were obtained for both (R)- and (S)-lipocrine complexed to HSA, suggesting a similar stereochemistry for the bound enantiomers.

Harnessing the Role of HDAC6 in Idiopathic Pulmonary Fibrosis: Design, Synthesis, Structural Analysis, and Biological Evaluation of Potent Inhibitors.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by a progressive-fibrosing phenotype. IPF has been associated with aberrant HDAC activities confirmed by our immunohistochemistry studies on HDAC6 overexpression in IPF lung tissues. We herein developed a series of novel hHDAC6 inhibitors, having low inhibitory potency over hHDAC1 and hHDAC8, as potential pharmacological tools for IPF treatment. Their inhibitory potency was combined with low in vitro and in vivo toxicity. Structural analysis of 6h and structure-activity relationship studies contributed to the optimization of the binding mode of the new molecules. The best-performing analogues were tested for their efficacy in inhibiting fibrotic sphere formation and cell viability, proving their capability in reverting the IPF phenotype. The efficacy of analogue 6h was also determined in a validated human lung model of TGF-ß1-dependent fibrogenesis. The results highlighted in this manuscript may pave the way for the identification of first-in-class molecules for the treatment of IPF.

Circular dichroism in drug discovery and development: an abridged review.

Chirality plays a fundamental role in determining the pharmacodynamic and pharmacokinetic properties of drugs, and contributes significantly to our understanding of the mechanisms that lie behind biorecognition phenomena. Circular dichroism spectroscopy is the technique of choice for determining the stereochemistry of chiral drugs and proteins, and for monitoring and characterizing molecular recognition phenomena in solution. The role of chirality in our understanding of recognition phenomena at the molecular level is discussed here via several selected systems of interest in the drug discovery and development area. The examples were selected in order to underline the utility of circular dichroism in emerging studies of protein-protein interactions in biological context. In particular, the following aspects are discussed here: the relationship between stereochemistry and pharmacological activity--stereochemical characterization of new leads and drugs; stereoselective binding of leads and drugs to target proteins--the binding of drugs to serum albumins; conformational transitions of peptides and proteins of physiological relevance, and the stereochemical characterization of therapeutic peptides.

HSA binding of HIV protease inhibitors: a high-performance affinity chromatography study.

The binding of HIV protease inhibitors, drugs important for anti-HIV chemotherapy, to HSA was examined by high-performance affinity chromatography. Frontal analysis was first used to determine the amount of anchored protein and the binding capacity for selected markers on this column. Zonal elution experiments then ranked the HSA bound fraction of the examined compounds. Information on the binding region was obtained by competitive zonal elution experiments using probe compounds with known sites on HSA. An allosteric competition between HIV protease inhibitors (PIs) and valproate (a probe for the bilirubin site) was detected, consistent with a noncooperative binding mechanism. No significant competition was observed between the examined compounds and salicylate or ibuprofen, probes for sites I and II, respectively. The observations were confirmed by circular dichroism spectroscopy, based on the change in the induced circular dichroism signals of selected markers for the main binding sites of HSA when ritonavir was added as the competitor. These results were in good agreement with previous literature reports and provide more details on how PIs are transported in plasma and how they may compete with other drugs in the body.