Unlocking the Immunomodulatory Potential of Rosmarinic Acid Isolated from Punica granatum L. Using Bioactivity-Guided Approach: In Silico, In Vitro, and In Vivo Approaches.

BACKGROUND: Punica granatum L. is well-known for its multifaceted therapeutic potential, including anti-inflammatory and immunomodulatory activities. AIM: This study aimed to characterize an immunomodulatory compound isolated from Punica granatum L. using a bioactivity-guided approach. METHODS: Chromatographic techniques were adopted for isolation and purification of secondary metabolites. In silico, in vitro, and in vivo methods were performed to characterize the therapeutic potential of the isolated compound. RESULTS: Using preparative thin-layer chromatography, rosmarinic acid was isolated from F4 (column chromatography product obtained from a butanolic fraction of the extract). The impact of rosmarinic acid was assessed in rats using the neutrophil adhesion test, DTH response, and phagocytic index. In immunized rats, rosmarinic acid demonstrated significant immunomodulatory potential. Computational experiments, like molecular docking and molecular dynamics, were also conducted against two targeted receptors, Cereblon (PDB ID: 8AOQ) and human CD22 (PDB ID: 5VKM). Computational studies suggested that an increase in phagocytic index by rosmarinic acid could be attributed to inhibiting Cereblon and CD22. Pharmacokinetics and toxicity prediction also suggested the drug-likeness of rosmarinic acid. CONCLUSION: Rosmarinic acid is a potential candidate, but extensive research needs to be done to translate this molecule from bench to bedside.

Identification of synthetically tractable MERS-CoV main protease inhibitors using structure-based virtual screening and molecular dynamics potential of mean force (PMF) calculations.

The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a potentially lethal infection that presents a substantial threat to health, especially in Middle East nations. Given that no FDA-approved specific therapy for MERS infection exists, designing and discovering a potent antiviral therapy for MERS-CoV is crucial. One pivotal strategy for inhibiting MERS replication is to focus on the viral main protease (Mpro). In this study, we identify potential novel Mpro inhibitors employing structure-based virtual screening of our recently reported Ugi reaction-derived library (URDL) consisting of cherry-picked molecules from the literature. The key features of the URDL library include synthetic tractability (1-2 pot synthesis) of the molecules scaffold and unexplored chemical space. The hits were ranked based on the docking score, MM-GBSA free energy of binding, and the interaction pattern with the active site residues. A molecular dynamics (MD) simulation study was performed for the first two top-ranked compounds to analyze the stability and free binding energy based on the molecular mechanics Poisson-Boltzmann surface area. The potential mean force calculated from the steered molecular dynamics (SMD) simulations of the hits indicates improved H-bond potential, enhanced conformational stability, and binding affinity toward the target, compared to the cocrystallized ligand. The discovered hits represent novel synthetically tractable scaffolds as potential MERS-CoV Mpro inhibitors.Communicated by Ramaswamy H. Sarma.

Crystal structures of non-uracil ring fragments in complex with Mycobacterium tuberculosis uracil DNA glycosylase (MtUng) as a starting point for novel inhibitor design: A case study with the barbituric acid fragment.

Uracil DNA glycosylase (UDG or Ung) is a key enzyme involved in uracil excision from the DNA as a repair mechanism. Designing Ung inhibitors is thus a promising strategy to treat different cancers and infectious diseases. The uracil ring and its derivatives have been shown to inhibit Mycobacterium tuberculosis Ung (MtUng), resulting from specific and strong binding with the uracil-binding pocket (UBP). To design novel MtUng inhibitors, we screened several non-uracil ring fragments hypothesised to occupy MtUng UBP due to their high similarity to the uracil structural motif. These efforts have resulted in the discovery of novel MtUng ring inhibitors. Here we report the co-crystallised poses of these fragments, confirming their binding within the UBP, thus providing a robust structural framework for the design of novel lead compounds. We selected the barbituric acid (BA) ring as a case study for further derivatisation and SAR analysis. The modelling studies predicted the BA ring of the designed analogues to interact with the MtUng UBP much like the uracil ring. The synthesised compounds were screened in vitro using radioactivity and a fluorescence-based assay. These studies led to a novel BA-based MtUng inhibitor 18a (IC50 = 300 µM) displaying ∼24-fold potency over the uracil ring.

Basic Nitrogen (BaN) Is a Key Property of Antimalarial Chemical Space.

Most antimalarials are based on basic N-heterocycles and possess amine functionalities. Despite this, the role of basic nitrogen (BaN) in antimalarial drug design has not been studied systematically. Our cheminformatics analysis indicates that BaN is an important feature of antimalarial space. We show that potent research antiplasmodials (RAP) and advanced-stage antimalarials (ASAMs) consistently show a higher BaN count (#BaN) compared to oral drugs. Similarly, BaN is often a vital feature of the hits obtained from phenotypic screenings despite the use of varied assay conditions. The literature review demonstrates that in several unrelated scaffolds, the addition of BaN results in enhanced antiplasmodial activity. In addition, potent antiplasmodials and HTS hits are bulky, lipophilic, and less polar and have a high aromatic ring count (#AR). This characterization of antimalarial space may be used to collate a focused compound collection to achieve higher hit rates in HTS, as shown retrospectively in this perspective.

Curation and cheminformatics analysis of a Ugi-reaction derived library (URDL) of synthetically tractable small molecules for virtual screening application.

Virtual screening (VS) is an important approach in drug discovery and relies on the availability of a virtual library of synthetically tractable molecules. Ugi reaction (UR) represents an important multi-component reaction (MCR) that reliably produces a peptidomimetic scaffold. Recent literature shows that a tactically assembled Ugi adduct can be subjected to further chemical modifications to yield a variety of rings and scaffolds, thus, renewing the interest in this old reaction. Given the reliability and efficiency of UR, we collated an UR derived library (URDL) of small molecules (total = 5773) for VS. The synthesis of the majority of URDL molecules may be carried out in 1-2 pots in a time and cost-effective manner. The detailed analysis of the average property and chemical space of URDL was also carried out using the open-source Datawarrior program. The comparison with FDA-approved oral drugs and inhibitors of protein-protein interactions (iPPIs) suggests URDL molecules are 'clean', drug-like, and conform to a structurally distinct space from the other two categories. The average physicochemical properties of compounds in the URDL library lie closer to iPPI molecules than oral drugs thus suggesting that the URDL resource can be applied to discover novel iPPI molecules. The URDL molecules consist of diverse ring systems, many of which have not been exploited yet for drug design. Thus, URDL represents a small virtual library of drug-like molecules with unexplored chemical space designed for VS. The structures of all molecules of URDL, oral drugs, and iPPI compounds are being made freely accessible as supplementary information for broader application.

Identification of a new and diverse set of Mycobacterium tuberculosis uracil-DNA glycosylase (MtUng) inhibitors using structure-based virtual screening: Experimental validation and molecular dynamics studies.

Mycobacterium tuberculosis uracil-DNA glycosylase (MtUng), a key DNA repair enzyme, represents an attractive target for the design of new antimycobacterial agents. However, only a limited number of weak MtUng inhibitors are reported, primarily based on the uracil ring, and hence, lack diversity. We report the first structure-based virtual screening (SBVS) using three separate libraries consisting of uracil and non-uracil small molecules, together with the FDA-approved drugs. Twenty diverse virtual hits with the highest predicted binding were procured and screened using a fluorescence-based assay to evaluate their potential to inhibit MtUng. Several of these molecules were found to inhibit MtUng activity at low mM and µM levels, comparable to or better than several other reported Ung inhibitors. Thus, these molecules represent a diverse set of scaffolds for developing next-generation MtUng inhibitors. The most active uracil-based compound 5 (IC50 = 0.14 mM) was found to be âˆ¼ 15-fold more potent than the positive control, uracil. The binding stability and conformation of compound 5 in complex with the enzyme were further confirmed using molecular dynamics simulation.

Quantitative Proteomics Reveals that Hsp90 Inhibition Dynamically Regulates Global Protein Synthesis in Leishmania mexicana.

Heat shock protein 90 (Hsp90) is a conserved molecular chaperone responsible for the folding and maturation of nascent proteins. Hsp90 is regarded as a master regulator of protein homeostasis in the cell, and its inhibition affects the functions of a large array of client proteins. The classical Hsp90 inhibitor tanespimycin has shown potent antileishmanial activity. Despite the increasing importance of Hsp90 inhibition in the development of antileishmanial agents, the global effects of these inhibitors on the parasite proteome remain unknown. By combining tanespimycin treatment with bioorthogonal noncanonical amino acid tagging (BONCAT) metabolic labeling and isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic mass spectrometry, for the first time, we robustly profiled the relative changes in the synthesis of hundreds of parasite proteins as functions of dose and duration of the inhibitor treatment. We showed that Hsp90 inhibition dynamically regulates nascent protein synthesis in Leishmania mexicana, with many chaperones and virulence factors showing inhibitor concentration- and treatment duration-dependent changes in relative expression. Many ribosomal proteins showed a downregulation upon severe Hsp90 inhibition, providing the first protein-level evidence that Hsp90 inhibition affects the protein synthesis capacity of the ribosome in this organism. We also provide an unbiased target validation of tanespimycin in L. mexicana using live parasite photoaffinity labeling with a novel chemical probe and quantitative proteomic mass spectrometry. We showed that the classical Hsp90 inhibitor not only engages with its presumed target, Hsp83-1, in L. mexicana promastigotes but also affects multiple proteins involved in protein synthesis and quality control in the parasite. This study defines the Leishmania parasites' response to Hsp90 inhibition at the level of nascent global protein synthesis and provides a rich resource for future studies on Leishmania spp. biology and antileishmanial drug development.IMPORTANCE Leishmania spp. are the causative agents of leishmaniasis, a poverty-related disease, which is endemic in >90 countries worldwide, affecting approximately 12 million people, with an estimated 700,000 to 1 million new cases and around 70,000 deaths annually. Inhibitors of the chaperone protein Hsp90 have shown promising antileishmanial activity. However, further development of the Hsp90 inhibitors as antileishmanials is hampered by a lack of direct information of their downstream effects on the parasite proteome. Using a combination of mass spectrometry-based quantitative proteomics and chemical and metabolic labeling, we provide the first protein-level evidence that Hsp90 inhibition affects global protein synthesis in Leishmania We also provide the precise relative quantitative changes in the expressions of hundreds of affected proteins as functions of both the concentration and duration of the inhibitor treatment. We find that Leishmania regulates its ribosomal proteins under Hsp90 inhibition while a set of virulence factors and chaperones are preferentially synthesized.

Use of a molecular beacon based fluorescent method for assaying uracil DNA glycosylase (Ung) activity and inhibitor screening.

Uracil DNA glycosylases are an important class of enzymes that hydrolyze the N-glycosidic bond between the uracil base and the deoxyribose sugar to initiate uracil excision repair. Uracil may arise in DNA either because of its direct incorporation (against A in the template) or because of cytosine deamination. Mycobacteria with G, C rich genomes are inherently at high risk of cytosine deamination. Uracil DNA glycosylase activity is thus important for the survival of mycobacteria. A limitation in evaluating the druggability of this enzyme, however, is the absence of a rapid assay to evaluate catalytic activity that can be scaled for medium to high-throughput screening of inhibitors. Here we report a fluorescence-based method to assay uracil DNA glycosylase activity. A hairpin DNA oligomer with a fluorophore at its 5' end and a quencher at its 3' ends was designed incorporating five consecutive U:A base pairs immediately after the first base pair (5' C:G 3') at the top of the hairpin stem. Enzyme assays performed using this fluorescent substrate were seen to be highly sensitive thus enabling investigation of the real time kinetics of uracil excision. Here we present data that demonstrate the feasibility of using this assay to screen for inhibitors of Mycobacterium tuberculosis uracil DNA glycosylase. We note that this assay is suitable for high-throughput screening of compound libraries for uracil DNA glycosylase inhibitors.

Physicochemical Profiling and Comparison of Research Antiplasmodials and Advanced Stage Antimalarials with Oral Drugs.

To understand the property space of antimalarials, we collated a large dataset of research antiplasmodial (RAP) molecules with known in vitro potencies and advanced stage antimalarials (ASAMs) with established oral bioavailability. While RAP molecules are "non-druglike", ASAM molecules display properties closer to Lipinski's and Veber's thresholds. Comparison within the different potency groups of RAP molecules indicates that the in vitro potency is positively correlated to the molecular weight, the calculated octanol-water partition coefficient (clog P), aromatic ring counts (#Ar), and hydrogen bond acceptors. Despite both categories being bioavailable, the ASAM molecules are relatively larger and more lipophilic, have a lower polar surface area, and possess a higher count of heteroaromatic rings than oral drugs. Also, antimalarials are found to have a higher proportion of aromatic (#ArN) and basic nitrogen (#BaN) counts, features implicitly used in the design of antimalarial molecules but not well studied hitherto. We also propose using descriptors scaled by the sum of #ArN and #BaN (SBAN) to define an antimalarial property space. Together, these results may have important applications in the identification and optimization of future antimalarials.

Non-hydroxamate inhibitors of 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR): A critical review and future perspective.

1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) catalyzes the second step of the non-mevalonate (or MEP) pathway that functions in several organisms and plants for the synthesis of isoprenoids. DXR is essential for the survival of multiple pathogenic bacteria/parasites, including those that cause tuberculosis and malaria in humans. DXR function is inhibited by fosmidomycin (1), a natural product, which forms a chelate with the active site divalent metal (Mg2+/Mn2+) through its hydroxamate metal-binding group (MBG). Most of the potent DXR inhibitors are structurally similar to 1 and retain hydroxamate despite the unfavourable pharmacokinetic and toxicity profile of the latter. We provide our perspective on the lack of non-hydroxamate DXR inhibitors. We also highlight the fundamental flaws in the design of MBG in these molecules, primarily responsible for their failure to inhibit DXR. We also suggest that for designing next-generation non-hydroxamate DXR inhibitors, approaches followed for other metalloenzymes targets may be exploited.

Tubulin inhibitory activity of a novel colchicine-binding compounds based on a dinaphthospiropyranran scaffold.

Spiropyrans have been investigated for their thermo- and photochromic characteristics, but their biotherapeutic properties have not been addressed. We report anti-proliferative properties of a novel dinaphthospiropyran analogue (1). The compound 1 was synthesized by a simple and expedient method using a one-pot acid-catalyzed aldol condensation of 2-hydroxy-1-naphthaldehyde with 4-piperidone followed by an acetalization reaction. Compound 1 was submitted to anticancer drug screen in the National Cancer Institute's panel of 60 human tumor cell lines. The average concentration of 1 to inhibit 50% cell growth was 5.4 ± 0.23 µM. All cell lines responded at almost the same concentration, suggesting that the action of 1 is not selective for cancer of origin. COMPARE analysis of dose-response data revealed interaction with tubulin as the possible mechanism of action of 1. At molecular level, 1 induced tubulin reorganization in colon cancer HCT-116 cells. Under cell-free conditions, the efficacy of 1 to inhibit tubulin polymerization was comparable to that of paclitaxel and vinblastine. Molecular docking showed that compound 1 binds to the colchicine-binding site of tubulin. We conclude that dinaphthospiropyrans present a novel scaffold for the development of tubulin inhibitors.

Identification of antimalarial leads with dual falcipain-2 and falcipain-3 inhibitory activity.

Falcipains (FPs), cysteine proteases in the malarial parasite, are emerging as the promising antimalarial drug targets. In order to identify novel FP inhibitors, we generated a pharmacophore derived from the reported co-crystal structures of inhibitors of Plasmodium falciparum Falcipain-3 to screen the ZINC library. Further, the filters were applied for dock score, drug-like characters, and clustering of similar structures. Sixteen molecules were purchased and subject to in vitro enzyme (FP-2 and FP-3) inhibition assays. Two compounds showed in vitro inhibition of FP-2 and FP-3 at low µM concentration. The selectivity of the inhibitors can be explained based on the predicted interactions of the molecule in the active site. Further, the inhibitors were evaluated in a functional assay and were found to induce morphological changes in line with their mode of action arresting Plasmodium development. Compound 15 was most potent inhibitor identified in this study.

Thienopyrimidinone Based Sirtuin-2 (SIRT2)-Selective Inhibitors Bind in the Ligand Induced Selectivity Pocket.

Sirtuins (SIRTs) are NAD-dependent deacylases, known to be involved in a variety of pathophysiological processes and thus remain promising therapeutic targets for further validation. Previously, we reported a novel thienopyrimidinone SIRT2 inhibitor with good potency and excellent selectivity for SIRT2. Herein, we report an extensive SAR study of this chemical series and identify the key pharmacophoric elements and physiochemical properties that underpin the excellent activity observed. New analogues have been identified with submicromolar SIRT2 inhibtory activity and good to excellent SIRT2 subtype-selectivity. Importantly, we report a cocrystal structure of one of our compounds (29c) bound to SIRT2. This reveals our series to induce the formation of a previously reported selectivity pocket but to bind in an inverted fashion to what might be intuitively expected. We believe these findings will contribute significantly to an understanding of the mechanism of action of SIRT2 inhibitors and to the identification of refined, second generation inhibitors.

Histone lysine methyltransferase structure activity relationships that allow for segregation of G9a inhibition and anti-Plasmodium activity.

Plasmodium falciparum HKMTs (PfHKMTs) play a key role in controlling Plasmodium gene expression and represent exciting new anti-malarial epigenetic targets. Using an inhibitor series derived from the diaminoquinazoline HKMT inhibitory chemotype, we have previously identified compounds with highly promising antimalarial activity, including irreversible asexual cycle blood stage-independent cytotoxic activity at nM concentrations, oral efficacy in in vivo models of disease, and the unprecedented ability to reactivate dormant liver stage parasites (hypnozoites). However, future development of this series will need to address host versus parasite selectivity, where inhibitory activity against human G9a is removed from the lead compounds, while maintaining potent anti-Plasmodium activity. Herein, we report an extensive study of the SAR of this series against both G9a and P. falciparum. We have identified key SAR features which demonstrate that high parasite vs. G9a selectivity can be achieved by selecting appropriate substituents at position 2, 4 and 7 of the quinazoline ring. We have also, in turn, discovered that potent G9a inhibitors can be identified by employing a 6-carbon 'Nle mimic' at position 7. Together, this data suggests that while broadly similar, the G9a and potential PfHKMT target(s) binding pockets and/or binding modes of the diaminoquinazoline analogues exhibit clear and exploitable differences. Based on this, we believe this scaffold to have clear potential for development into a novel anti-malarial therapeutic.

Histone methyltransferase inhibitors are orally bioavailable, fast-acting molecules with activity against different species causing malaria in humans.

Current antimalarials are under continuous threat due to the relentless development of drug resistance by malaria parasites. We previously reported promising in vitro parasite-killing activity with the histone methyltransferase inhibitor BIX-01294 and its analogue TM2-115. Here, we further characterize these diaminoquinazolines for in vitro and in vivo efficacy and pharmacokinetic properties to prioritize and direct compound development. BIX-01294 and TM2-115 displayed potent in vitro activity, with 50% inhibitory concentrations (IC50s) of <50 nM against drug-sensitive laboratory strains and multidrug-resistant field isolates, including artemisinin-refractory Plasmodium falciparum isolates. Activities against ex vivo clinical isolates of both P. falciparum and Plasmodium vivax were similar, with potencies of 300 to 400 nM. Sexual-stage gametocyte inhibition occurs at micromolar levels; however, mature gametocyte progression to gamete formation is inhibited at submicromolar concentrations. Parasite reduction ratio analysis confirms a high asexual-stage rate of killing. Both compounds examined displayed oral efficacy in in vivo mouse models of Plasmodium berghei and P. falciparum infection. The discovery of a rapid and broadly acting antimalarial compound class targeting blood stage infection, including transmission stage parasites, and effective against multiple malaria-causing species reveals the diaminoquinazoline scaffold to be a very promising lead for development into greatly needed novel therapies to control malaria.

Development of diaminoquinazoline histone lysine methyltransferase inhibitors as potent blood-stage antimalarial compounds.

Modulating epigenetic mechanisms in malarial parasites is an emerging avenue for the discovery of novel antimalarial drugs. Previously we demonstrated the potent in vitro and in vivo antimalarial activity of (1-benzyl-4-piperidyl)[6,7-dimethoxy-2-(4-methyl-1,4-diazepin-1-yl)-4-quinazolinyl]amine (BIX01294; 1), a known human G9a inhibitor, together with its dose-dependent effects on histone methylation in the malarial parasite. This work describes our initial medicinal chemistry efforts to optimise the diaminoquinazoline chemotype for antimalarial activity. A variety of analogues were designed by substituting the 2 and 4 positions of the quinazoline core, and these molecules were tested against Plasmodium falciparum (3D7 strain). Several analogues with IC50 values as low as 18.5 nM and with low mammalian cell toxicity (HepG2) were identified. Certain pharmacophoric features required for antimalarial activity were found to be analogous to the previously published SAR of these analogues for G9a inhibition, thereby suggesting potential similarities between the malarial and human HKMT targets of this chemotype. Physiochemical, in vitro activity, and in vitro metabolism studies were also performed for a select set of potent analogues to evaluate their potential as antimalarial leads.

Identification of 2,4-diamino-6,7-dimethoxyquinoline derivatives as G9a inhibitors†Electronic supplementary information (ESI) available. See DOI: 10.1039/c4md00274a.

G9a is a histone lysine methyltransferase (HKMT) involved in epigenetic regulation via the installation of histone methylation marks. 6,7-Dimethoxyquinazoline analogues, such as BIX-01294, are established as potent, substrate competitive inhibitors of G9a. With an objective to identify novel chemotypes for substrate competitive inhibitors of G9a, we have designed and synthesised a range of heterocyclic scaffolds, and investigated their ability to inhibit G9a. These studies have led to improved understanding of the key pharmacophoric features of BIX-01294 and the identification of a new core quinoline inhibitory scaffold, which retains excellent potency and high selectivity. Molecular docking was carried out to explain the observed in vitro data.

On the histone lysine methyltransferase activity of fungal metabolite chaetocin.

Histone lysine methyltransferases (HKMTs) are an important class of targets for epigenetic therapy. 1 (chaetocin), an epidithiodiketopiperazine (ETP) natural product, has been reported to be a specific inhibitor of the SU(VAR)3-9 class of HKMTs. We have studied the inhibition of the HKMT G9a by 1 and functionally related analogues. Our results reveal that only the structurally unique ETP core is required for inhibition, and such inhibition is time-dependent and irreversible (in the absence of DTT), ultimately resulting in protein denaturation. Mass spectrometric data provide a molecular basis for this effect, demonstrating covalent adduct formation between 1 and the protein. This provides a potential rationale for the selectivity observed in the inhibition of a variety of HKMTs by 1 in vitro and has implications for the activity of ETPs against these important epigenetic targets.

2-Substituted 4,5-dihydrothiazole-4-carboxylic acids are novel inhibitors of metallo-ß-lactamases.

Bacterial resistance to ß-lactam antibiotics caused by class B metallo-ß-lactamases (MBL), especially for certain hospital-acquired, Gram-negative pathogens, poses a significant threat to public health. We report several 2-substituted 4,5-dihydrothiazole-4-carboxylic acids to be novel MBL inhibitors. Structure activity relationship (SAR) and molecular modeling studies were performed and implications for further inhibitor design are discussed.

Apicoplast isoprenoid precursor synthesis and the molecular basis of fosmidomycin resistance in Toxoplasma gondii.

Apicomplexa are important pathogens that include the causative agents of malaria, toxoplasmosis, and cryptosporidiosis. Apicomplexan parasites contain a relict chloroplast, the apicoplast. The apicoplast is indispensable and an attractive drug target. The apicoplast is home to a 1-deoxy-D-xylulose-5-phosphate (DOXP) pathway for the synthesis of isoprenoid precursors. This pathway is believed to be the most conserved function of the apicoplast, and fosmidomycin, a specific inhibitor of the pathway, is an effective antimalarial. Surprisingly, fosmidomycin has no effect on most other apicomplexans. Using Toxoplasma gondii, we establish that the pathway is essential in parasites that are highly fosmidomycin resistant. We define the molecular basis of resistance and susceptibility, experimentally testing various host and parasite contributions in T. gondii and Plasmodium. We demonstrate that in T. gondii the parasite plasma membrane is a critical barrier to drug uptake. In strong support of this hypothesis, we engineer de novo drug-sensitive T. gondii parasites by heterologous expression of a bacterial transporter protein. Mice infected with these transgenic parasites can now be cured from a lethal challenge with fosmidomycin. We propose that the varied extent of metabolite exchange between host and parasite is a crucial determinator of drug susceptibility and a predictor of future resistance.