In vivo evaluation of Clostridioides difficile enoyl-ACP reductase II (FabK) inhibition by phenylimidazole unveils a promising narrow-spectrum antimicrobial strategy.

Clostridioides difficile infection (CDI) is a leading cause of hospital-acquired diarrhea, which often stems from disruption of the gut microbiota by broad-spectrum antibiotics. The increasing prevalence of antibiotic-resistant C. difficile strains, combined with disappointing clinical trial results for recent antibiotic candidates, underscores the urgent need for novel CDI antibiotics. To this end, we investigated C. difficile enoyl ACP reductase (CdFabK), a crucial enzyme in de novo fatty acid synthesis, as a drug target for microbiome-sparing antibiotics. To test this concept, we evaluated the efficacy and in vivo spectrum of activity of the phenylimidazole analog 296, which is validated to inhibit intracellular CdFabK. Against major CDI-associated ribotypes 296 had an Minimum inhibitory concentration (MIC90) of 2 µg/mL, which was comparable to vancomycin (1 µg/mL), a standard of care antibiotic. In addition, 296 achieved high colonic concentrations and displayed dosed-dependent efficacy in mice with colitis CDI. Mice that were given 296 retained colonization resistance to C. difficile and had microbiomes that resembled the untreated mice. Conversely, both vancomycin and fidaxomicin induced significant changes to mice microbiomes, in a manner consistent with prior reports. CdFabK, therefore, represents a potential target for microbiome-sparing CDI antibiotics, with phenylimidazoles providing a good chemical starting point for designing such agents.

Synthesis and evaluation of phenylimidazole FabK inhibitors as new Anti-C. Difficile agents.

Previously, 1-((4-(4-bromophenyl)-1H-imidazol-2-yl)methyl)-3-(5-(pyridin-2-ylthio)thiazol-2-yl)urea bearing a p-bromine substitution was shown to possess selective inhibitory activity against the Clostridioides difficile enoyl-acyl carrier protein (ACP) reductase II enzyme, FabK. Inhibition of CdFabK by this compound translated to promising antibacterial activity in the low micromolar range. In these studies, we sought to expand our knowledge of the SAR of the phenylimidazole CdFabK inhibitor series while improving the potency of the compounds. Three main series of compounds were synthesized and evaluated based on: 1) pyridine head group modifications including the replacement with a benzothiazole moiety, 2) linker explorations, and 3) phenylimidazole tail group modifications. Overall, improvement in the CdFabK inhibition was achieved, while maintaining the whole cell antibacterial activity. Specifically, compounds 1-((4-(4-bromophenyl)-1H-imidazol-2-yl)methyl)-3-(5-((3-(trifluoromethyl)pyridin-2-yl)thio)thiazol-2-yl)urea, 1-((4-(4-bromophenyl)-1H-imidazol-2-yl)methyl)-3-(6-(trifluoromethyl)benzo[d]thiazol-2-yl)urea, and 1-((4-(4-bromophenyl)-1H-imidazol-2-yl)methyl)-3-(6-chlorobenzo[d]thiazol-2-yl)urea showed CdFabK inhibition (IC50 = 0.10 to 0.24 µM), a 5 to 10-fold improvement in biochemical activity relative to 1-((4-(4-bromophenyl)-1H-imidazol-2-yl)methyl)-3-(5-(pyridin-2-ylthio)thiazol-2-yl)urea, with anti-C. difficile activity ranging from 1.56 to 6.25 µg/mL. Detailed analysis of the expanded SAR, supported by computational analysis, is presented.

Recent Advances in Macrocyclic Drugs and Microwave-Assisted and/or Solid-Supported Synthesis of Macrocycles.

Macrocycles represent attractive candidates in organic synthesis and drug discovery. Since 2014, nineteen macrocyclic drugs, including three radiopharmaceuticals, have been approved by FDA for the treatment of bacterial and viral infections, cancer, obesity, immunosuppression, etc. As such, new synthetic methodologies and high throughput chemistry (e.g., microwave-assisted and/or solid-phase synthesis) to access various macrocycle entities have attracted great interest in this chemical space. This article serves as an update on our previous review related to macrocyclic drugs and new synthetic strategies toward macrocycles (Molecules, 2013, 18, 6230). In this work, I first reviewed recent FDA-approved macrocyclic drugs since 2014, followed by new advances in macrocycle synthesis using high throughput chemistry, including microwave-assisted and/or solid-supported macrocyclization strategies. Examples and highlights of macrocyclization include macrolactonization and macrolactamization, transition-metal catalyzed olefin ring-closure metathesis, intramolecular C-C and C-heteroatom cross-coupling, copper- or ruthenium-catalyzed azide-alkyne cycloaddition, intramolecular SNAr or SN2 nucleophilic substitution, condensation reaction, and multi-component reaction-mediated macrocyclization, and covering the literature since 2010.

Recent Reports of Solid-Phase Cyclohexapeptide Synthesis and Applications.

Macrocyclic peptides are privileged scaffolds for drug development and constitute a significant portion of macrocyclic drugs on the market today in fields spanning from infectious disease to oncology. Developing orally bioavailable peptide-based drugs remains a challenging task; however, macrocyclization of linear peptides can be an effective strategy to improve membrane permeability, proteolytic stability, oral bioavailability, and overall drug-like characteristics for this class. Significant advances in solid-phase peptide synthesis (SPPS) have enabled the efficient construction of macrocyclic peptide and peptidomimetic libraries with macrolactamization being performed on-resin or in solution phase. The primary goal of this review is to summarize solid-phase cyclohexapeptide synthesis using the on-resin and solution-phase macrocyclization methodologies published since 2013. We also highlight their broad applications ranging from natural product total synthesis, synthetic methodology development, and medicinal chemistry, to drug development and analyses of conformational and physiochemical properties.

Structure-activity relationships and docking studies of synthetic 2-arylindole derivatives determined with aromatase and quinone reductase 1.

In our ongoing effort of discovering anticancer and chemopreventive agents, a series of 2-arylindole derivatives were synthesized and evaluated toward aromatase and quinone reductase 1 (QR1). Biological evaluation revealed that several compounds (e.g., 2d, IC50 = 1.61 µM; 21, IC50 = 3.05 µM; and 27, IC50 = 3.34 µM) showed aromatase inhibitory activity with half maximal inhibitory concentration (IC50) values in the low micromolar concentrations. With regard to the QR1 induction activity, 11 exhibited the highest QR1 induction ratio (IR) with a low concentration to double activity (CD) value (IR = 8.34, CD = 2.75 µM), while 7 showed the most potent CD value of 1.12 µM. A dual acting compound 24 showed aromatase inhibition (IC50 = 9.00 µM) as well as QR1 induction (CD = 5.76 µM) activities. Computational docking studies using CDOCKER (Discovery Studio 3.5) provided insight in regard to the potential binding modes of 2-arylindoles within the aromatase active site. Predominantly, the 2-arylindoles preferred binding with the 2-aryl group toward a small hydrophobic pocket within the active site. The C-5 electron withdrawing group on indole was predicted to have an important role and formed a hydrogen bond with Ser478 (OH). Alternatively, meta-pyridyl analogs may orient with the pyridyl 3'-nitrogen coordinating with the heme group.

Solid-phase synthesis of cyclic hexapeptides wollamides A, B and desotamide B.

Solid-phase synthesis of antibacterial cyclohexapeptides including wollamides A, B and desotamide B has been developed. Briefly, the protected linear hexapeptides were assembled on 2-chlorotrityl chloride resin using standard Fmoc chemistry and diisopropylcarbodiimide/hydroxybenzotriazole coupling reagents, cleaved off-resin with hexafluoroisopropanol/dichloromethane to keep side-chain protecting groups intact, and cyclized in solution. Final global removal of all protecting groups using a cocktail of trifluoroacetic acid/triisopropylsilane/dichloromethane afforded the desired cyclic hexapeptides, which were characterized by 1H, 13C NMR, and HRMS. Subsequent investigation of macrocyclization parameters such as terminal residues, coupling reagents, and cyclization concentration revealed the optimized conditions for the synthesis of this class of cyclic hexapeptides.

Synthesis and antibacterial evaluation of macrocyclic diarylheptanoid derivatives.

Bacterial infections, caused by Mycobacterium tuberculosis and other problematic bacterial pathogens, continue to pose a significant threat to global public health. As such, new chemotype antibacterial agents are desperately needed to fuel and strengthen the antibacterial drug discovery and development pipeline. As part of our antibacterial research program to develop natural product-inspired new antibacterial agents, here we report synthesis, antibacterial evaluation, and structure-activity relationship studies of an extended chemical library of macrocyclic diarylheptanoids with diverse amine, amide, urea, and sulfonamide functionalities. Results of this study have produced macrocyclic geranylamine and 4-fluorophenethylamine substituted derivatives, exhibiting moderate to good activity against M. tuberculosis and selected Gram-positive bacterial pathogens.

Determination of the absolute configuration of chaetoviridins and other bioactive azaphilones from the endophytic fungus Chaetomium globosum.

Chemical investigation of an endophytic fungus Chaetomium globosum isolated from leaves of Wikstroemia uva-ursi led to the isolation of two new azaphilones, chaetoviridins J and K (1 and 3), along with five known derivatives (2 and 4-7). The structures of azaphilones were determined by NMR, X-ray diffraction, Mosher's method, and CD analysis. The isolated compounds were evaluated for their cancer chemopreventive-potential based on their abilities to inhibit tumor necrosis factor alpha (TNF-α)-induced nuclear factor-kappa B (NF-κB). Compounds 4, 5, 7, and synthetic 8 and 9 inhibit nitric oxide (NO) production with IC50 values in the range of 0.3-5.8 µM. Compounds 4, 5, and 9 also displayed (TNF-α)-induced NF-κB activity with IC50 values in the range of 0.9-5.1 µM.

Inhibitory effect of a callophycin A derivative on iNOS expression via inhibition of Akt in lipopolysaccharide-stimulated RAW 264.7 cells.

In previous studies, (R)-2-isobutyl 3-methyl 3,4-dihydro-1H-pyrido[3,4-b]indole-2,3(9H)-dicarboxylate (1), a callophycin A derivative, was found to strongly inhibit nitrite production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells, while (R)- or (S)-callophycin A showed only weak inhibition. We currently report additional studies to define the mechanisms underlying the inhibitory action of 1. Expression of inducible nitric oxide synthase (iNOS) was reduced at both protein and mRNA levels. Major upstream signaling molecules and transcription factors regulating iNOS expression were examined, but it was found that 1 did not affect the phosphorylated and total protein levels of p38 mitogen-activated protein kinase (p38 MAPK), Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2), and signal transducer and activator of transcription 1 (STAT1), nor did it mediate the degradation of the inhibitor of nuclear factor-κB α-isoform (IκBα). However, starting at early time points, 1 consistently inhibited the phosphorylation of protein kinase B/Akt at serine 473. In addition, 1 suppressed the protein expression of octamer-binding transcription factor-2 (Oct-2) and the expression of microRNA 155 (miR-155). In sum, compound 1 inhibits LPS-induced nitrite production by a unique and complex mechanism. Reduction of iNOS expression is accompanied by inhibition of Akt activation, Oct-2 protein expression, and miR-155 expression.

Herbicidin congeners, undecose nucleosides from an organic extract of Streptomyces sp. L-9-10.

Four new undecose nucleosides (herbicidin congeners), three known herbicidins, and 9-(ß-d-arabinofuranosyl)hypoxanthine (Ara-H) were isolated from the organic extract of a fermentation culture of Streptomyces sp. L-9-10 using proton NMR-guided fractionation. Their structures were elucidated on the basis of comprehensive 1D and 2D NMR and mass spectrometry analyses. These structures included 2'-O-demethylherbicidin F (1), 9'-deoxy-8',8'-dihydroxyherbicidin B (2), 9'-deoxy-8'-oxoherbicidin B (2a), and the 8'-epimer of herbicidin B (3). This is the first detailed assignment of proton and carbon chemical shifts for herbicidins A, B, and F. The isolated compounds were evaluated for cancer chemopreventive potential based on inhibition of tumor necrosis factor alpha (TNF-α)-induced nuclear factor-kappa B (NF-κB) activity.

Evaluation of Fusobacterium nucleatum Enoyl-ACP Reductase (FabK) as a Narrow-Spectrum Drug Target.

Fusobacterium nucleatum, a pathobiont inhabiting the oral cavity, contributes to opportunistic diseases, such as periodontal diseases and gastrointestinal cancers, which involve microbiota imbalance. Broad-spectrum antimicrobial agents, while effective against F. nucleatum infections, can exacerbate dysbiosis. This necessitates the discovery of more targeted narrow-spectrum antimicrobial agents. We therefore investigated the potential for the fusobacterial enoyl-ACP reductase II (ENR II) isoenzyme FnFabK (C4N14_ 04250) as a narrow-spectrum drug target. ENRs catalyze the rate-limiting step in the bacterial fatty acid synthesis pathway. Bioinformatics revealed that of the four distinct bacterial ENR isoforms, F. nucleatum specifically encodes FnFabK. Genetic studies revealed that fabK was indispensable for F. nucleatum growth, as the gene could not be deleted, and silencing of its mRNA inhibited growth under the test conditions. Remarkably, exogenous fatty acids failed to rescue growth inhibition caused by the silencing of fabK. Screening of synthetic phenylimidazole analogues of a known FabK inhibitor identified an inhibitor (i.e., 681) of FnFabK enzymatic activity and F. nucleatum growth, with an IC50 of 2.1 µM (1.0 µg/mL) and a MIC of 0.4 µg/mL, respectively. Exogenous fatty acids did not attenuate the activity of 681 against F. nucleatum. Furthermore, FnFabK was confirmed as the intracellular target of 681 based on the overexpression of FnFabK shifting MICs and 681-resistant mutants having amino acid substitutions in FnFabK or mutations in other genetic loci affecting fatty acid biosynthesis. 681 had minimal activity against a range of commensal flora, and it was less active against streptococci in physiologic fatty acids. Taken together, FnFabK is an essential enzyme that is amenable to drug targeting for the discovery and development of narrow-spectrum antimicrobial agents.

Macrocyclic drugs and synthetic methodologies toward macrocycles.

Macrocyclic scaffolds are commonly found in bioactive natural products and pharmaceutical molecules. So far, a large number of macrocyclic natural products have been isolated and synthesized. The construction of macrocycles is generally considered as a crucial and challenging step in the synthesis of macrocyclic natural products. Over the last several decades, numerous efforts have been undertaken toward the synthesis of complex naturally occurring macrocycles and great progresses have been made to advance the field of total synthesis. The commonly used synthetic methodologies toward macrocyclization include macrolactonization, macrolactamization, transition metal-catalyzed cross coupling, ring-closing metathesis, and click reaction, among others. Selected recent examples of macrocyclic synthesis of natural products and druglike macrocycles with significant biological relevance are highlighted in each class. The primary goal of this review is to summarize currently used macrocyclic drugs, highlight the therapeutic potential of this underexplored drug class and outline the general synthetic methodologies for the synthesis of macrocycles.

Advancing Pharmacy Education by Moving From Sequenced "Integration" to True Curricular Integration.

OBJECTIVES: Traditional pharmacy education focuses on teaching content, which is affectionately known as "silos". Each topic area or discipline includes a course or an individual class session designed to impart knowledge, skills, or abilities needed for the student pharmacist to become a practice-ready, team-ready pharmacist. With expanding content and educational standards, there have been calls to simplify and streamline content. Truly "integrated" curricula (sequenced, coordinated, and collaboratively taught) where silos are removed to foster student integrative learning and build connections across disciplines (foundational, clinical, and social or administrative sciences) could be one such approach. Thus, the objectives of this integrative review are to provide recommendations for decreasing curriculum overload by moving to truly integrated curricula, explore integrated approaches, discuss challenges and barriers, and propose next steps for creating integrated curricula that decrease content overload. FINDINGS: Although there are different approaches to curricular integration, most curricular integration occurs through sequenced courses or integrated cases. In order to truly streamline content and foster connections across disciplines, integration must move beyond simply sequencing of content to content that includes all the disciplines taught seamlessly. When taught together, curricular integration offers the opportunity to cover medication classes quickly and efficiently with multiple opportunities for reinforcement. SUMMARY: There remains limited data and examples of these types of true integration approaches. Thus, it is important for the Academy to determine if the integration of content improves curricular outcomes, positively affects students' learning, and addresses curriculum overload by increasing efficiency and streamlining curricula.

In vivo evaluation of Clostridioides difficile enoyl-ACP reductase II (FabK) Inhibition by phenylimidazole unveils a promising narrow-spectrum antimicrobial strategy.

Clostridioides difficile infection (CDI) is a leading cause of hospital-acquired diarrhea, which often stem from disruption of the gut microbiota by broad-spectrum antibiotics. The increasing prevalence of antibiotic-resistant C. difficile strains, combined with disappointing clinical trials results for recent antibiotic candidates, underscore the urgent need for novel CDI antibiotics. To this end, we investigated C. difficile enoyl ACP reductase (CdFabK), a crucial enzyme in de novo fatty acid synthesis, as a drug target for microbiome-sparing antibiotics. To test this concept, we evaluated the efficacy and in vivo spectrum of activity of the phenylimidazole analog 296, which is validated to inhibit intracellular CdFabK. Against major CDI-associated ribotypes 296 had an MIC90 of 2 µg/ml, which was comparable to vancomycin (1 µg/ml), a standard of care antibiotic. In addition, 296 achieved high colonic concentrations and displayed dosed-dependent efficacy in mice with colitis CDI. Mice that were given 296 retained colonization resistance to C. difficile and had microbiomes that resembled the untreated mice. Conversely, both vancomycin and fidaxomicin induced significant changes to mice microbiomes, in a manner consistent with prior reports. CdFabK therefore represents a potential target for microbiome-sparing CDI antibiotics, with phenylimidazoles providing a good chemical starting point for designing such agents.

Synthesis of 2-arylindole derivatives and evaluation as nitric oxide synthase and NFκB inhibitors.

Development of small molecule drug-like inhibitors blocking both nitric oxide synthase and NFκB could offer a synergistic therapeutic approach in the prevention and treatment of inflammation and cancer. During the course of evaluating the biological potential of a commercial compound library, 2-phenylindole (1) displayed inhibitory activity against nitrite production and NFκB with IC(50) values of 38.1 ± 1.8 and 25.4 ± 2.1 µM, respectively. Based on this lead, synthesis and systematic optimization have been undertaken in an effort to find novel and more potent nitric oxide synthase and NFκB inhibitors with antiinflammatory and cancer preventive potential. First, chemical derivatizations of 1 and 2-phenylindole-3-carboxaldehyde (4) were performed to generate a panel of N-alkylated indoles and 3-oxime derivatives 2­7. Second, a series of diversified 2-arylindole derivatives (10) were synthesized from an array of substituted 2-iodoanilines (8) and terminal alkynes (9) by applying a one-pot palladium catalyzed Sonogashira-type alkynylation and base-assisted cycloaddition. Subsequent biological evaluations revealed 3-carboxaldehyde oxime and cyano substituted 2-phenylindoles 5 and 7 exhibited the strongest nitrite inhibitory activities (IC(50) = 4.4 ± 0.5 and 4.8 ± 0.4 µM, respectively); as well as NFκB inhibition (IC(50) = 6.9 ± 0.8 and 8.5 ± 2.0 µM, respectively). In addition, the 6'-MeO-naphthalen-2'-yl indole derivative 10at displayed excellent inhibitory activity against NFκB with an IC(50) value of 0.6 ± 0.2 µM.

Microwave-assisted synthesis of macrocycles via intramolecular and/or bimolecular Ullmann coupling.

Microwave-assisted synthesis of macrocyclic diaryl ethers via intramolecular and/or bimolecular Ullmann coupling is described. Using the optimized conditions, a panel of macrocycles, with different substitution patterns, ring sizes, and linkers, has been successfully synthesized using microwave irradiation. To the best of our knowledge, this work represents the first examples of the microwave-assisted synthesis of macrocyclic diaryl ethers via intramolecular and/or bimolecular Ullmann coupling.

Design, synthesis, and biological evaluation of callophycin A and analogues as potential chemopreventive and anticancer agents.

Callophycin A was originally isolated from the red algae Callophycus oppositifolius and shown to mediate anticancer and cytotoxic effects. In our collaborative effort to identify potential chemopreventive and anticancer agents with enhanced potency and selectivity, we employed a tetrahydro-ß-carboline-based template inspired by callophycin A for production of a chemical library. Utilizing a parallel synthetic approach, 50 various functionalized tetrahydro-ß-carboline derivatives were prepared and assessed for activities related to cancer chemoprevention and cancer treatment: induction of quinone reductase 1 (QR1) and inhibition of aromatase, nitric oxide (NO) production, tumor necrosis factor (TNF)-α-induced NFκB activity, and MCF7 breast cancer cell proliferation. Biological results showed that the n-pentyl urea S-isomer 6a was the strongest inducer of QR1 with an induction ratio (IR) value of 4.9 at 50 µM [the concentration to double the activity (CD)=3.8 µM] and its corresponding R-isomer 6f had an IR value of 4.3 (CD=0.2 µM). The isobutyl carbamate derivative 3d with R stereochemistry demonstrated the most potent inhibitory activity of NFκB, with the half maximal inhibitory concentration (IC(50)) value of 4.8 µM, and also showed over 60% inhibition at 50 µM of NO production (IC(50)=2.8 µM). The R-isomer urea derivative 6j, having an appended adamantyl group, exhibited the most potent MCF7 cell proliferation inhibitory activity (IC(50)=14.7 µM). The S-isomer 12a of callophycin A showed the most potent activity in aromatase inhibition (IC(50)=10.5 µM).

The structure-activity relationship of urea derivatives as anti-tuberculosis agents.

The treatment of tuberculosis is becoming more difficult due to the ever increasing prevalence of drug resistance. Thus, it is imperative that novel anti-tuberculosis agents, with unique mechanisms of action, be discovered and developed. The direct anti-tubercular testing of a small compound library led to discovery of adamantyl urea hit compound 1. In this study, the hit was followed up through the synthesis of an optimization library. This library was generated by systematically replacing each section of the molecule with a similar moiety until a clear structure-activity relationship was obtained with respect to anti-tubercular activity. The best compounds in this series contained a 1-adamantyl-3-phenyl urea core and had potent activity against Mycobacterium tuberculosis plus an acceptable therapeutic index. It was noted that the compounds identified and the pharmacophore developed is consistent with inhibitors of epoxide hydrolase family of enzymes. Consequently, the compounds were tested for inhibition of representative epoxide hydrolases: M. tuberculosis EphB and EphE; and human soluble epoxide hydrolase. Many of the optimized inhibitors showed both potent EphB and EphE inhibition suggesting the antitubercular activity is through inhibition of multiple epoxide hydrolase enzymes. The inhibitors also showed potent inhibition of humans soluble epoxide hydrolase, but limited cytotoxicity suggesting that future studies must be towards increasing the selectivity of epoxide hydrolase inhibition towards the M. tuberculosis enzymes.

Total synthesis and structural revision of engelhardione.

The total synthesis of the macrocyclic natural product engelhardione is reported. This effort led to the structural revision of the published structure of engelhardione to that of pterocarine. The revision reflects the change of the substitution pattern of one phenyl ether ring from the meta to the para position. To confirm, pterocarine (2) and its close regioisomer 3 were subsequently synthesized for comparison. Moreover, to the best of our knowledge, our synthesis of 1 represents the first example of a 14-membered macrocyclic diarylheptanoid with a meta-meta substitution pattern at the diphenyl ether moiety.

Macrocycle-Antibiotic Hybrids: A Path to Clinical Candidates.

The tale of abate in antibiotics continued defense mechanisms that chaperone the rise of drug-defying superbugs-on the other hand, the astray in antibacterial drug discovery and development. Our salvation lies in circumventing the genesis of resistance. Considering the competitive advantages of antibacterial chemotherapeutic agents equipped with multiple warheads against resistance, the development of hybrids has rejuvenated. The adoption of antibiotic hybrid paradigm to macrocycles has advanced novel chemical entities to clinical trials. The multi-targeted TD-1792, for instance, retained potent antibacterial activities against multiple strains that are resistant to its constituent, vancomycin. Moreover, the antibiotic conjugation of rifamycins has provided hybrid clinical candidates with desirable efficacy and safety profiles. In 2020, the U.S. FDA has granted an orphan drug designation to TNP-2092, a conjugate of rifamycin and fluoroquinolone, for the treatment of prosthetic joint infections. DSTA4637S is a pioneer antibacterial agent under clinical development and represents a novel class of bacterial therapy, that is, antibody-antibiotic conjugates. DSTA4637S is effective against the notorious persistent S. aureus bacteremia, a revelation of the abracadabra potential of antibiotic hybrid approaches.