Designing antitrypanosomal and antileishmanial BODIPY derivatives: A computational and in vitro assessment
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This article belongs to the Special Issue Boron Dipyrromethene (BODIPY) Dyes and Their Derivatives.
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Citation: Gonçalves, R.C.R.; Teixeira, F.; Peñalver, P.; Costa, S.P.G.; Morales, J.C.; Raposo, M.M.M. Designing Antitrypanosomal and Antileishmanial BODIPY Derivatives: A Computational and In Vitro Assessment. Molecules 2024,29, 2072. https://doi.org/10.3390/ molecules29092072 Academic Editor: Jarosław Piskorz Received: 13 March 2024 Revised: 23 April 2024 Accepted: 25 April 2024 Published: 30 April 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). molecules Article Designing Antitrypanosomal and Antileishmanial BODIPY Derivatives: A Computational and In Vitro Assessment Raquel C. R. Gonçalves 1,2,†, Filipe Teixeira 1,† , Pablo Peñalver 3, Susana P. G. Costa 1, Juan C. Morales 3 and M. Manuela M. Raposo 1,* 1Centre of Chemistry, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal; [email protected] (F.T.); [email protected] (S.P.G.C.) 2Advanced (Magnetic) Theranostic Nanostructures Lab, International Iberian Nanotechnology Laboratory, Av. Mestre JoséVeiga s/n, 4715-330 Braga, Portugal 3Instituto de Parasitología y Biomedicina López Neyra, CSIC, PTS Granada, Avenida del Conocimiento, 17, 18016 Armilla, Granada, Spain; [email protected] (P.P.) *Correspondence: [email protected] †These authors contributed equally to this work. Abstract: Leishmaniasis and Human African trypanosomiasis pose significant public health threats in resource-limited regions, accentuated by the drawbacks of the current antiprotozoal treatments and the lack of approved vaccines. Considering the demand for novel therapeutic drugs, a series of BODIPY derivatives with several functionalizations at the meso, 2 and/or 6 positions of the core were synthesized and characterized. The in vitro activity against Trypanosoma brucei and Leishmania major parasites was carried out alongside a human healthy cell line (MRC-5) to establish selectivity indices (SIs). Notably, the meso-substituted BODIPY, with 1-dimethylaminonaphthalene (1b) and anthracene moiety (1c), were the most active against L. major, displaying IC 50 = 4.84 and 5.41 µ M, with a 16 and 18-fold selectivity over MRC-5 cells, respectively. In contrast, the mono-formylated analogues 2b and 2c exhibited the highest toxicity (IC 50 = 2.84 and 6.17 µ M, respectively) and selectivity (SI = 24 and 11, respectively) against T. brucei. Further insights on the activity of these compounds were gathered from molecular docking studies. The results suggest that these BODIPYs act as competitive inhibitors targeting the NADPH/NADP + linkage site of the pteridine reductase (PR) enzyme. Additionally, these findings unveil a range of quasi-degenerate binding complexes formed between the PRs and the investigated BODIPY derivatives. These results suggest a potential correlation between the anti-parasitic activity and the presence of multiple configurations that block the same site of the enzyme. Keywords: antiprotozoal agent; BODIPY derivatives; Leishmania major; molecular modeling; Trypanosoma brucei 1. Introduction Leishmania major and Trypanosoma brucei are the pathogenic parasites responsible for two neglected tropical diseases (NTDs): leishmaniasis and Human African trypanosomiasis (HAT, also known as sleeping sickness). Due to the absence of an approved vaccination for these pathogenic diseases and the high toxicity and drug resistance associated with current antileishmanial and antitrypanosomal drugs, ongoing efforts are being dedicated to develop improved molecules to address the limitations of the existing treatments [ 1 ]. Notably, in 2021, the US Food and Drug Administration (FDA) approved fexinidazole as the first all-oral treatment for both stages of the Trypanosoma brucei gambiense form of sleeping sickness. In 2023, the approval by the European Medicines Agency followed. In any case, new potential treatments are advisable for HAT and also for leishmaniasis. Recently, our group reported the investigation of bis(indolyl)methane (BIM) derivatives substituted with different (hetero)aromatic moieties as antiparasitic agents. We found Molecules 2024,29, 2072. https://doi.org/10.3390/molecules29092072 https://www.mdpi.com/journal/molecules
Molecules 2024,29, 2072 2 of 18 that the triphenylamine-functionalized compound exhibited an IC 50 of 3.21 and 3.30 µ M against Trypanosoma brucei (T. brucei) and Leishmania major (L. major), respectively, and an eight-fold selectivity over a healthy cell line (MRC-5) [2]. Identifying and understanding molecular targets associated with the survival of parasites plays an important role in advancing drug discovery towards antiparasitic agents. Numerous studies have elucidated the potential of target-based drug discovery for parasitic diseases, including HAT and leishmaniasis. These findings have been comprehensively reviewed previously [ 3 , 4 ]. For example, dihydrofolate reductase (DHFR) and pteridine reductase 1 (PTR1) are NADPH-dependent enzymes involved in the reduction of pterins and folates of the trypanosomatid parasites. A greater understanding of the folates pathway in trypanosomatid biology highlighted their crucial role in cellular mechanisms, such as DNA and protein synthesis [ 5 ]. Therefore, by disrupting these cellular processes, drugs targeting the folate pathway (also known as antifolates) have the potential to effectively combat trypanosomatid infections. Inhibitors of DHFR, such as trimethoprim and chloroguanide, have been used for the treatment of bacterial infections and malaria, respectively [ 6 – 8 ]. However, blockers of the DHRF have shown less efficacy against Trypanosoma and Leishmania parasites, mainly due to the presence of the PTR1 enzyme [ 9 ]. Studies demonstrated that PTR1 is upregulated under DHFR inhibition, thus contributing to the parasites’ resistance to antifolates [ 10 ]. Hence, additional PTR1 inhibition has been proposed as an alternative to suppress the trypanosomatidae folate pathway in order to avoid this mechanism of resistance [ 11 , 12 ]. In the past years, several compounds targeting PTR1 have been developed, for example, quinoxaline-based, 2,4-diaminopteridine-based and thiadiazoles-based derivatives [ 11 , 13 – 15 ]. However, additional advancements are required to enhance their selectivity and effectiveness in combating these pathogenic parasites. The derivatives based on the boron-dipyrromethene (BODIPY) scaffold represent a versatile class of fluorophores widely employed in different scientific fields. The first BODIPY was synthesized by Alfred Treibs and Franz-Heinreich Kreuzer in 1968 [ 16 ] and since then, there has been an exponential emergence in the design and synthesis of new compounds. The relevance of these derivatives arises from their facile synthesis and structural versatility. There are numerous functionalization strategies to develop a desirable framework pattern with specific features, including pre-functionalization of the pyrrole/aldehyde and post-functionalization of each position of the core [ 17 , 18 ]. The synthetic flexibility of this scaffold has given rise to a plethora of derivatives with finetuned properties, making them versatile tools for diverse applications, such as organic light-emitting diodes (OLEDs), chemosensors, fluorescence probes for bioimaging and photosensitizers in Photodynamic Therapy (PDT) [19–23]. As a matter of fact, BODIPY derivatives are demonstrating a growing success in the field of Photodynamic Therapy (PDT) and Photodynamic Inactivation (PDI) [ 24 , 25 ]. These two therapeutic approaches have become an alternative, especially for cancer treatment and against resistant pathogenic agents, such as bacteria, viruses and fungi. Several approaches have been attempted to fine-tune the BODIPY framework and improve its efficiency in generating reactive oxygen species [26–28]. BODIPY-based molecules are also increasingly prominent in the bioimaging field, ascribed to their excellent photophysical properties and great photostability in biological conditions [ 20 ]. Interestingly, BODIPY-based fluorescence probes have been developed to investigate the cellular internalization and biodistribution of antiparasitic drugs [ 29 – 31 ]. A BODIPY-fluorophore based probe for an anti-Chagas agent was developed and investigated for its potential as an in vivo theranostic probe [ 30 ]. Moreover, fluorescent analogues of the leishmanicidal drug miltefosine were successfully developed by integrating a BODIPY moiety on the alkyl chain. These derivatives demonstrated in vitro antiparasitic activity, comparable to that of the original alkylphosphocholine compound [ 31 ]. Nevertheless, to the best of our knowledge, the investigation of BODIPY derivatives as the active moiety of antiparasitic agents has not been reported previously.
Molecules 2024,29, 2072 3 of 18 In this work, we report on the therapeutic activity of BODIPY derivatives against protozoan parasites. We prepared a series of BODIPY derivatives featuring different functionalizations at the meso, 2 and/or 6 positions of the scaffold structure (Cf. Figure 1). The in vitro effectiveness of these compounds was assessed against T. brucei and L. major, along with the human lung fibroblast cell line (MRC-5), to establish their selectivity indices (SIs). Additional understanding regarding the antitrypanosomal and antileishmanial activity of the BODIPY derivatives was obtained by employing molecular modeling methods to analyze the interactions of the compounds with the pteridine reductase (PR) enzyme. Molecules2024,29,xFORPEERREVIEW3of19 antiparasiticactivity,comparabletothatoftheoriginalalkylphosphocholinecompound [31].Nevertheless,tothebestofourknowledge,theinvestigationofBODIPYderivatives astheactivemoietyofantiparasiticagentshasnotbeenreportedpreviously. Inthiswork,wereportonthetherapeuticactivityofBODIPYderivativesagainst protozoanparasites.WepreparedaseriesofBODIPYderivativesfeaturingdifferent functionalizationsatthemeso,2and/or6positionsofthescaffoldstructure(Cf.Figure1). TheinvitroeffectivenessofthesecompoundswasassessedagainstT.bruceiandL.major, alongwiththehumanlungfibroblastcellline(MRC‐5),toestablishtheirselectivityin‐ dices(SIs).Additionalunderstandingregardingtheantitrypanosomalandantileishma‐ nialactivityoftheBODIPYderivativeswasobtainedbyemployingmolecularmodeling methodstoanalyzetheinteractionsofthecompoundswiththepteridinereductase(PR) enzyme. Figure1.FunctionalizationoftheBODIPYcoreatmesoposition,2positionand6positionwith differentsubstituentsgroups. 2.ResultsandDiscussion 2.1.SynthesisandCharacterizationofBODIPYDerivatives AseriesofBODIPYderivativeswerepreparedandcharacterizedinordertoinves‐ tigatetheeffectsofthedifferentchemicalmodificationsatpositions2,6andmesoofthe coreontheirbiologicalactivityagainstT.bruceiandL.majorparasites.Therouteforthe synthesisoftheBODIPYderivativesisrepresentedinScheme1,andthefunctionalization groupsateachpositionoftheBODIPYcore(R 1 ,R 2 andR 3 ),thereactionyieldsandthe photophysicalcharacterizationinacetonitrilesolutionarecompiledinTable1.Inthis work,thenomenclatureemployedisasfollows:theprecursorcompounds(R 2 =R 3 =H, Cf.Figure1)ofthecorearedenominatedwithnumber1;thesuccessorcompounds functionalizedatR 2 and/orR 3 aredenominatedwithnumbersfrom2to4;andthesuffix lettersatoidenotethemesosubstituent(R 1 ).Thesynthesisandcharacterizationofthe BODIPYderivatives1a,2a,3a,1b,2b,3b,1c,2c,4c,1d,2d,1eand2ehavebeenprevi‐ ouslyreportedbyourgroup[27,28,32–40].ThesynthesisoftheBODIPYderivatives1f–h hasbeenreportedpreviouslyelsewhere[41–43]. Figure 1. Functionalization of the BODIPY core at meso position, 2 position and 6 position with different substituents groups. 2. Results and Discussion 2.1. Synthesis and Characterization of BODIPY Derivatives A series of BODIPY derivatives were prepared and characterized in order to investigate the effects of the different chemical modifications at positions 2, 6 and meso of the core on their biological activity against T. brucei and L. major parasites. The route for the synthesis of the BODIPY derivatives is represented in Scheme 1, and the functionalization groups at each position of the BODIPY core (R 1 , R 2 and R 3 ), the reaction yields and the photophysical characterization in acetonitrile solution are compiled in Table 1. In this work, the nomenclature employed is as follows: the precursor compounds ( R2= R3= H , Cf. Figure 1) of the core are denominated with number 1; the successor compounds functionalized at R 2 and/or R 3 are denominated with numbers from 2to 4; and the suffix letters ato idenote the meso substituent (R 1 ). The synthesis and characterization of the BODIPY derivatives 1a,2a,3a,1b,2b,3b,1c,2c,4c,1d,2d,1e and 2e have been previously reported by our group [ 27 , 28 , 32 – 40 ]. The synthesis of the BODIPY derivatives 1f–hhas been reported previously elsewhere [41–43]. Firstly, to obtain the BODIPY scaffold substituted at meso position with several aromatic and heteroaromatic groups (1a–i), we followed the established Lindsey’s method, as reported by our group previously [ 27 , 32 – 36 , 44 ], with yields ranging from 3% to 74%, probably explained by the lower reactivity of the precursor aldehydes, and the difficulty in the purification, by silica gel column chromatography, especially for the thiophene, thiazole and thieno[3,2-b]thiophene derivatives 1f–i. The BODIPYs formylated in position 2 and/or 6 (2a–f) were obtained from the precursors 1a–fvia the Vilsmeier-Haack reaction [ 27 , 28 , 32 , 36 – 39 ] in moderate to excellent yields (47–91%). In the case of the formylation of precursor 1d, the reaction resulted in two products: the monoformylated compound 2d (yield 25%), with an aldehyde group on the BODIPY core and the diformylated compound 3d (yield 26%), with a second aldehyde group on the triphenylamine moiety. The diformylated BODIPY 3c substituted with two aldehydes groups on the BODIPY core was obtained by a second formylation of its precursor 2c, with a lower yield (10%). The derivatives functionalized with a benzimidazole at position 2 of the core (3a and 3b) were obtained, in fair to good yields (77% and 31%, respectively), by the condensation between the aldehyde group and o-phenylenediamine, followed by an intramolecular cyclisation reaction [ 32 , 36 ]. Finally, derivative 4c was obtained by the direct halogenation of the precursor 1c with N-iodosuccinimide (NIS), with a yield of 57% [ 28 ]. The NMR spectroscopic characterization of compounds 1f,1g and 1h were in agreement with the previously published data [ 41 – 43 ]. The new compounds 3c,3d,2f and 1i were characterized through NMR spectroscopy
Molecules 2024,29, 2072 4 of 18 and high-resolution mass spectrometry. The synthetic methods and characterizations are described in the supporting information. A comprehensive study focused on the influence of the electron donor/withdrawing substituents at the meso and 2 positions of the BODIPY core and solvent polarity on the photophysical properties of the BODIPY derivatives 3a,1b,2b,1c and 2c has been recently reported by our research group [ 27 , 36 ]. Overall, the derivatives are characterized by an absorption band, with maxima at 492–512 nm, and emission maxima at 507–524 nm, the typical features for the BODIPY chromophore [ 19 ]. Nevertheless, the anthracene-BODIPY substituted with iodine atoms at positions 2 and 6 (compound 4c) displayed the highest absorption maxima of the series at 540 nm and emission maxima at 559 nm. Comparison of derivatives 1d and 3d shows that the introduction of the electron-withdrawing formyl group, at position 2 of the BODIPY core and at the triphenylamine moiety, leads to a red-shift of the maximum emission band ( λfluo = 519 nm vs. λfluo = 549 nm, respectively), whereas the absorption maximum did not suffer significant changes. Regarding the relative fluorescence quantum yield ( ΦF ), in general, the BODIPY derivatives demonstrated low fluorescence intensity, with the exception of compounds 1a,2a and 2f, which exhibited values of 0.68, 0.84 and 0.30, respectively. Comparison between compounds 1a and 2a with their counterpart 3a shows that the modification with the benzimidazole induces a fluorescence quenching ( ΦF = 0.68 and ΦF = 0.84 vs. ΦF = 0.10, respectively). In the case of derivative 2f, bearing a thiophene moiety at the meso position of the BODIPY core and an electron acceptor formyl group at position 2, the compound demonstrated a higher fluorescence emission’s efficiency when compared to its analogue 1f, non-substituted with the formyl group (ΦF= 0.30 vs. ΦF= 0.045, respectively). Molecules2024,29,xFORPEERREVIEW5of19 Scheme1.SyntheticroutefortheBODIPYderivativesfunctionalizedatmesopositionand2and/or 6positions. Firstly,toobtaintheBODIPYscaffoldsubstitutedatmesopositionwithseveralar‐ omaticandheteroaromaticgroups(1a–i),wefollowedtheestablishedLindsey’smethod, asreportedbyourgrouppreviously[27,32–36,44],withyieldsrangingfrom3%to74%, probablyexplainedbythelowerreactivityoftheprecursoraldehydes,andthedifficulty inthepurification,bysilicagelcolumnchromatography,especiallyforthethiophene, thiazoleandthieno[3,2‐b]thiophenederivatives1f–i.TheBODIPYsformylatedinposition 2and/or6(2a–f)wereobtainedfromtheprecursors1a–fviatheVilsmeier‐Haackreac‐ tion[27,28,32,36–39]inmoderatetoexcellentyields(47–91%).Inthecaseoftheformyla‐ tionofprecursor1d,thereactionresultedintwoproducts:themonoformylatedcom‐ pound2d(yield25%),withanaldehydegroupontheBODIPYcoreandthediformylated compound3d(yield26%),withasecondaldehydegrouponthetriphenylaminemoiety. ThediformylatedBODIPY3csubstitutedwithtwoaldehydesgroupsontheBODIPY corewasobtainedbyasecondformylationofitsprecursor2c,withaloweryield(10%). Thederivativesfunctionalizedwithabenzimidazoleatposition2ofthecore(3aand3b) wereobtained,infairtogoodyields(77%and31%,respectively),bythecondensation betweenthealdehydegroupando‐phenylenediamine,followedbyanintramolecular cyclisationreaction[32,36].Finally,derivative4cwasobtainedbythedirecthalogenation oftheprecursor1cwithN‐iodosuccinimide(NIS),withayieldof57%[28].TheNMR spectroscopiccharacterizationofcompounds1f,1gand1hwereinagreementwiththe previouslypublisheddata[41–43].Thenewcompounds3c,3d,2fand1iwerecharac‐ terizedthroughNMRspectroscopyandhigh‐resolutionmassspectrometry.Thesyn‐ theticmethodsandcharacterizationsaredescribedinthesupportinginformation. Acomprehensivestudyfocusedontheinfluenceoftheelectrondonor/withdrawing substituentsatthemesoand2positionsoftheBODIPYcoreandsolventpolarityonthe photophysicalpropertiesoftheBODIPYderivatives3a,1b,2b,1cand2chasbeenre‐ centlyreportedbyourresearchgroup[27,36].Overall,thederivativesarecharacterized byanabsorptionband,withmaximaat492–512nm,andemissionmaximaat507–524 Scheme 1. Synthetic route for the BODIPY derivatives functionalized at meso position and 2 and/or 6 positions.
Molecules 2024,29, 2072 5 of 18 Table 1. Synthesis and photophysical data for the BODIPY derivatives. The photophysical studies were conducted in acetonitrile solutions with concentrations of the compounds ranging from 10 −5 to 10 −6 M (absorption, λabs , and fluorescence emission maxima, λfluo , and fluorescence quantum yields, ΦF). Compound R1R2R3Yield (%) λabs (nm) λfluo (nm) ΦF 1a Molecules2024,29,xFORPEERREVIEW4of19 Table1.SynthesisandphotophysicaldatafortheBODIPYderivatives.Thephotophysicalstudies wereconductedinacetonitrilesolutionswithconcentrationsofthecompoundsrangingfrom10−5 to10−6M(absorption,λabs,andfluorescenceemissionmaxima,λfluo,andfluorescencequantum yields,ΦF). CompoundR1R2R3Yield(%)λabs(nm)λfluo(nm)ΦF 1a HH214975130.68 2aHCHO914925080.84 3aHBenzimidazole775075170.10 1b HH225025150.03 2bHCHO594975090.01 3bHBenzimidazole315125140.03 1c HH745025150.03 2cHCHO474975090.01 3cCHOCHO105005240.009 4cII575405590.003 1d HH204975190.005 2dHCHO254915150.01 3d HCHO264935460.011 1e HH174965070.019 2eHCHO574925060.024 1f HH85085190.045 2fHCHO495025170.30 1g HH75125240.009 1h HH375005120.026 1i HH35115220.036 H H 21 497 513 0.68 2a H CHO 91 492 508 0.84 3a H Benzimidazole 77 507 517 0.10 1b Molecules2024,29,xFORPEERREVIEW4of19 Table1.SynthesisandphotophysicaldatafortheBODIPYderivatives.Thephotophysicalstudies wereconductedinacetonitrilesolutionswithconcentrationsofthecompoundsrangingfrom10−5 to10−6M(absorption,λabs,andfluorescenceemissionmaxima,λfluo,andfluorescencequantum yields,ΦF). CompoundR1R2R3Yield(%)λabs(nm)λfluo(nm)ΦF 1a HH214975130.68 2aHCHO914925080.84 3aHBenzimidazole775075170.10 1b HH225025150.03 2bHCHO594975090.01 3bHBenzimidazole315125140.03 1c HH745025150.03 2cHCHO474975090.01 3cCHOCHO105005240.009 4cII575405590.003 1d HH204975190.005 2dHCHO254915150.01 3d HCHO264935460.011 1e HH174965070.019 2eHCHO574925060.024 1f HH85085190.045 2fHCHO495025170.30 1g HH75125240.009 1h HH375005120.026 1i HH35115220.036 H H 22 502 515 0.03 2b H CHO 59 497 509 0.01 3b H Benzimidazole 31 512 514 0.03 1c Molecules2024,29,xFORPEERREVIEW4of19 Table1.SynthesisandphotophysicaldatafortheBODIPYderivatives.Thephotophysicalstudies wereconductedinacetonitrilesolutionswithconcentrationsofthecompoundsrangingfrom10−5 to10−6M(absorption,λabs,andfluorescenceemissionmaxima,λfluo,andfluorescencequantum yields,ΦF). CompoundR1R2R3Yield(%)λabs(nm)λfluo(nm)ΦF 1a HH214975130.68 2aHCHO914925080.84 3aHBenzimidazole775075170.10 1b HH225025150.03 2bHCHO594975090.01 3bHBenzimidazole315125140.03 1c HH745025150.03 2cHCHO474975090.01 3cCHOCHO105005240.009 4cII575405590.003 1d HH204975190.005 2dHCHO254915150.01 3d HCHO264935460.011 1e HH174965070.019 2eHCHO574925060.024 1f HH85085190.045 2fHCHO495025170.30 1g HH75125240.009 1h HH375005120.026 1i HH35115220.036 H H 74 502 515 0.03 2c H CHO 47 497 509 0.01 3c CHO CHO 10 500 524 0.009 4c I I 57 540 559 0.003 1d Molecules2024,29,xFORPEERREVIEW4of19 Table1.SynthesisandphotophysicaldatafortheBODIPYderivatives.Thephotophysicalstudies wereconductedinacetonitrilesolutionswithconcentrationsofthecompoundsrangingfrom10−5 to10−6M(absorption,λabs,andfluorescenceemissionmaxima,λfluo,andfluorescencequantum yields,ΦF). CompoundR1R2R3Yield(%)λabs(nm)λfluo(nm)ΦF 1a HH214975130.68 2aHCHO914925080.84 3aHBenzimidazole775075170.10 1b HH225025150.03 2bHCHO594975090.01 3bHBenzimidazole315125140.03 1c HH745025150.03 2cHCHO474975090.01 3cCHOCHO105005240.009 4cII575405590.003 1d HH204975190.005 2dHCHO254915150.01 3d HCHO264935460.011 1e HH174965070.019 2eHCHO574925060.024 1f HH85085190.045 2fHCHO495025170.30 1g HH75125240.009 1h HH375005120.026 1i HH35115220.036 H H 20 497 519 0.005 2d H CHO 25 491 515 0.01 3d Molecules2024,29,xFORPEERREVIEW4of19 Table1.SynthesisandphotophysicaldatafortheBODIPYderivatives.Thephotophysicalstudies wereconductedinacetonitrilesolutionswithconcentrationsofthecompoundsrangingfrom10−5 to10−6M(absorption,λabs,andfluorescenceemissionmaxima,λfluo,andfluorescencequantum yields,ΦF). CompoundR1R2R3Yield(%)λabs(nm)λfluo(nm)ΦF 1a HH214975130.68 2aHCHO914925080.84 3aHBenzimidazole775075170.10 1b HH225025150.03 2bHCHO594975090.01 3bHBenzimidazole315125140.03 1c HH745025150.03 2cHCHO474975090.01 3cCHOCHO105005240.009 4cII575405590.003 1d HH204975190.005 2dHCHO254915150.01 3d HCHO264935460.011 1e HH174965070.019 2eHCHO574925060.024 1f HH85085190.045 2fHCHO495025170.30 1g HH75125240.009 1h HH375005120.026 1i HH35115220.036 H CHO 26 493 546 0.011 1e Molecules2024,29,xFORPEERREVIEW4of19 Table1.SynthesisandphotophysicaldatafortheBODIPYderivatives.Thephotophysicalstudies wereconductedinacetonitrilesolutionswithconcentrationsofthecompoundsrangingfrom10−5 to10−6M(absorption,λabs,andfluorescenceemissionmaxima,λfluo,andfluorescencequantum yields,ΦF). CompoundR1R2R3Yield(%)λabs(nm)λfluo(nm)ΦF 1a HH214975130.68 2aHCHO914925080.84 3aHBenzimidazole775075170.10 1b HH225025150.03 2bHCHO594975090.01 3bHBenzimidazole315125140.03 1c HH745025150.03 2cHCHO474975090.01 3cCHOCHO105005240.009 4cII575405590.003 1d HH204975190.005 2dHCHO254915150.01 3d HCHO264935460.011 1e HH174965070.019 2eHCHO574925060.024 1f HH85085190.045 2fHCHO495025170.30 1g HH75125240.009 1h HH375005120.026 1i HH35115220.036 H H 17 496 507 0.019 2e H CHO 57 492 506 0.024 1f Molecules2024,29,xFORPEERREVIEW4of19 Table1.SynthesisandphotophysicaldatafortheBODIPYderivatives.Thephotophysicalstudies wereconductedinacetonitrilesolutionswithconcentrationsofthecompoundsrangingfrom10−5 to10−6M(absorption,λabs,andfluorescenceemissionmaxima,λfluo,andfluorescencequantum yields,ΦF). CompoundR1R2R3Yield(%)λabs(nm)λfluo(nm)ΦF 1a HH214975130.68 2aHCHO914925080.84 3aHBenzimidazole775075170.10 1b HH225025150.03 2bHCHO594975090.01 3bHBenzimidazole315125140.03 1c HH745025150.03 2cHCHO474975090.01 3cCHOCHO105005240.009 4cII575405590.003 1d HH204975190.005 2dHCHO254915150.01 3d HCHO264935460.011 1e HH174965070.019 2eHCHO574925060.024 1f HH85085190.045 2fHCHO495025170.30 1g HH75125240.009 1h HH375005120.026 1i HH35115220.036 H H 8 508 519 0.045 2f H CHO 49 502 517 0.30 1g Molecules2024,29,xFORPEERREVIEW4of19 Table1.SynthesisandphotophysicaldatafortheBODIPYderivatives.Thephotophysicalstudies wereconductedinacetonitrilesolutionswithconcentrationsofthecompoundsrangingfrom10−5 to10−6M(absorption,λabs,andfluorescenceemissionmaxima,λfluo,andfluorescencequantum yields,ΦF). CompoundR1R2R3Yield(%)λabs(nm)λfluo(nm)ΦF 1a HH214975130.68 2aHCHO914925080.84 3aHBenzimidazole775075170.10 1b HH225025150.03 2bHCHO594975090.01 3bHBenzimidazole315125140.03 1c HH745025150.03 2cHCHO474975090.01 3cCHOCHO105005240.009 4cII575405590.003 1d HH204975190.005 2dHCHO254915150.01 3d HCHO264935460.011 1e HH174965070.019 2eHCHO574925060.024 1f HH85085190.045 2fHCHO495025170.30 1g HH75125240.009 1h HH375005120.026 1i HH35115220.036 H H 7 512 524 0.009 1h Molecules2024,29,xFORPEERREVIEW4of19 Table1.SynthesisandphotophysicaldatafortheBODIPYderivatives.Thephotophysicalstudies wereconductedinacetonitrilesolutionswithconcentrationsofthecompoundsrangingfrom10−5 to10−6M(absorption,λabs,andfluorescenceemissionmaxima,λfluo,andfluorescencequantum yields,ΦF). CompoundR1R2R3Yield(%)λabs(nm)λfluo(nm)ΦF 1a HH214975130.68 2aHCHO914925080.84 3aHBenzimidazole775075170.10 1b HH225025150.03 2bHCHO594975090.01 3bHBenzimidazole315125140.03 1c HH745025150.03 2cHCHO474975090.01 3cCHOCHO105005240.009 4cII575405590.003 1d HH204975190.005 2dHCHO254915150.01 3d HCHO264935460.011 1e HH174965070.019 2eHCHO574925060.024 1f HH85085190.045 2fHCHO495025170.30 1g HH75125240.009 1h HH375005120.026 1i HH35115220.036 H H 37 500 512 0.026 1i Molecules2024,29,xFORPEERREVIEW4of19 Table1.SynthesisandphotophysicaldatafortheBODIPYderivatives.Thephotophysicalstudies wereconductedinacetonitrilesolutionswithconcentrationsofthecompoundsrangingfrom10−5 to10−6M(absorption,λabs,andfluorescenceemissionmaxima,λfluo,andfluorescencequantum yields,ΦF). CompoundR1R2R3Yield(%)λabs(nm)λfluo(nm)ΦF 1a HH214975130.68 2aHCHO914925080.84 3aHBenzimidazole775075170.10 1b HH225025150.03 2bHCHO594975090.01 3bHBenzimidazole315125140.03 1c HH745025150.03 2cHCHO474975090.01 3cCHOCHO105005240.009 4cII575405590.003 1d HH204975190.005 2dHCHO254915150.01 3d HCHO264935460.011 1e HH174965070.019 2eHCHO574925060.024 1f HH85085190.045 2fHCHO495025170.30 1g HH75125240.009 1h HH375005120.026 1i HH35115220.036 H H 3 511 522 0.036 2.2. Antiparasitic and Cytotoxicity Activity The in vitro antitrypanosomal and antileishmanial activity of the BODIPY derivatives were evaluated on the bloodstream form (BSF) of T. brucei and the promastigote form of L. major parasites. The toxicity of the compounds was determined after 72 h of incubation using the alamarBlue assay (for T. brucei) and the MTT assay (for L. major). The cytotoxicity of the compounds on a healthy human cell line (human lung fibroblast, MRC-5) was also determined after 72 h of incubation using the alamarBlue assay. The data were expressed as the half maximal inhibitory concentration (IC 50 ). The selectivity index (SI) of each derivative was calculated by dividing the IC 50 of MRC-5 cells into the IC 50 of T. brucei or L. major. The results are shown in Table 2.
Molecules 2024,29, 2072 6 of 18 Table 2. Antiparasitic activity against T. brucei BSF and L. major promastigotes and cytotoxicity in MRC-5 represented as IC 50 values ( µ M) with the error reported as σ . The selectivity index was calculated related to a healthy cell line (MRC-5). The best selectivity values (SI > 10) are highlighted in bold. Compounds IC50 µM Selectivity Index (SI) T. brucei L. major MRC 5 T. brucei L. major 1a 38.20 ±7.29 19.65 ±3.94 67.16 ±4.39 1.76 3.42 2a 74.68 ±6.93 >100 91.33 ±0.66 1.22 <0.91 3a 5.5 ±0.21 >100 62.61 ±3.27 11.38 <0.63 1b 18.24 ±3.63 4.84 ±1.56 76.84 ±5.33 4.21 15.93 2b 2.84 ±0.04 15.75 ±1.31 69.12 ±4.90 24.34 4.39 3b 27.87 ±5.42 84.39 ±0.32 >100 >3.59 >1.18 1c 77.11 ±2.98 5.41 ±0.43 82.14 ±0.03 1.07 17.90 2c 6.17 ±2.35 10.33 ±1.59 66.89 ±7.65 10.84 6.47 3c 14.15 ±0.31 3.13 ±0.13 21.65 ±3.46 1.53 6.91 4c 28.05 ±1.77 >50 48.22 ±1.13 1.72 <0.96 1d 48.84 ±3.29 >100 91.29 ±6.31 1.87 <0.91 2d 22.74 ±1.88 >100 81.06 ±3.90 3.56 <0.81 3d 38.26 ±0.93 >100 87.13 ±3.28 2.28 <0.87 1e 18.65 ±2.67 >100 88.89 ±3.82 4.77 <0.89 2e 15.23 ±0.86 20.71 ±1.79 89.65 ±9.08 5.89 4.33 1f 36.93 ±7.43 64.55 ±8.43 57.96 ±4.76 1.57 0.90 2f 12.37 ±4.02 19.72 ±3.21 80.89 ±1.06 6.54 4.10 1g 55.67 ±1.65 >100 82.82 ±1.20 1.49 <0.83 1h 77.36 ±3.46 >100 95.58 ±3.45 1.24 <0.96 1i 61.47 ±10.94 92.21 ±10.02 96.82 ±4.50 1.58 1.05 SI = (IC50 MRC-5/IC50 parasite). We observed that the BODIPY derivatives 1b,meso-substituted with 1-dimethylaminonaphthalene, and 1c,meso-substituted with anthracene moiety, were the most active of the series against L. major, exhibiting IC 50 values of 4.84 and 5.41 µ M, together with a 16 and 18-fold selectivity over the healthy cell line (MRC-5), respectively. In contrast, the cytotoxicity of the derivative 3a, functionalized at the meso-position with a phenyl group and with a benzimidazole moiety at position 2, was higher for the healthy cell line than towards the L. major (IC 50 = 63 µ M vs. IC 50 > 100 µ M, respectively), which resulted in a SI lower than one. Interestingly, the compound exhibited an inhibitory effect on T. brucei with an IC 50 value of 5.5 µ M and an SI value of 11.38. The formylated derivatives 2b and 2c also exhibited good inhibitory activity against T. brucei, particularly compound 2b (substituted with 1-dimethylaminonaphthalene at meso position). This compound displayed the best IC 50 2.84 µ M and selectivity index of the series and a 24-fold selectivity over MRC-5, which suggests an interesting therapeutic potential of 2b against T. brucei parasites. Moreover, inhibitory concentration values of 3.6 µ M against L. major and 2.7 µ M against T. brucei have been reported for methotrexate, a reference antifolate drug [ 12 , 45 ]. These values fall within the range of the toxicity exhibited by the most active BODIPY derivatives 1b,2c and 3c towards L. major and compound 2b towards T. brucei. The functionalization of the BODIPY core with a formyl group seems to influence, in certain cases, their antiparasitic activity. The unsubstituted compounds 1b and 1c were more active against L. major, whilst the mono-formyl analogues 2b and 2c were more cytotoxic against T. brucei parasites. Additionally, in T. brucei, the BODIPYs functionalized at R 3 with formyl were overall more toxic than the unsubstituted analogues, hence with higher selectivity indices (C.f. Figure S1). For instance, compound 2b has an IC 50 of 2.84 µ M (SI = 24.34), whilst its precursor 1b has an IC 50 = 18.24 µ M (SI = 4.21); compound 2c shows an IC 50 = 6.17 µ M (SI = 10.84) and its unsubstituted analogue 1c an IC 50 = 77.11 µ M (SI = 1.07); compound 2f displays an IC 50 of 12.37 µ M (SI = 6.54), whilst its counterpart 1f shows an IC 50 = 36.93 µ M (SI = 1.57). However, this tendency seems not to be so relevant for antileishmanial activity (C.f. Figure S2).
Molecules 2024,29, 2072 7 of 18 2.3. Molecular Docking Studies In order to rationalize the antileishmanial and antitrypanosomal activity data presented in Table 2for the 20 BODIPY derivatives, their interaction with pteridine reductase native to L. major (PRLm) and T. brucei (PRTb) was studied using a molecular docking protocol. The selection of Pteridine reductase as the probable target for these BODIPY derivatives was predicated on its structural resemblance to known antileishmanial and antitrypanosomal drugs. A quantitative assessment of the structural similarities was conducted through the analysis of Morgan fingerprint bit differences (Cf. Table S1). Figure 2summarizes the main results from the molecular docking studies. In general, all BODIPY derivatives present good binding energies for the most stable binding mode, and ∆bindG is generally lower for PRTb than for PRLm, with the noticeable exception of 1d. As it will be detailed later, the attachment of the BODYPY derivatives is mostly established via hydrophobic interactions between the sp 2 carbon atoms of the BODIPY derivative and the carbon atoms on the side chains of a handful of amino acid residues (Cf. Figures S3–S11). The only significant exceptions noted involved the possible hydrogen bonding between PRLm and compounds 1b,2b and 3b, involving the H atoms of the amine group and the backbone N of Ala15, the heterocyclic N of Hys241 and the O atom of Ser111, respectively. Compound 3b also forms a hydrogen bond with the amino moiety at the side chain of Arg14 of PRTb. Molecules2024,29,xFORPEERREVIEW8of19 andtheOatomofSer111,respectively.Compound3balsoformsahydrogenbondwith theaminomoietyatthesidechainofArg14ofPRTb. Figure2.BindingGibbsenergies(∆ 𝐺,inkcal/mol)forthebestbindingmodeofeachen‐ zyme‐BODIPYderivative.Compounds1b,1c,2b,2c,and3cpresentantileishmanialactivity, whereascompounds2b,2c,2f,3a,and3cpresentantitrypanosomalactivity. AlthoughtheresultsdepictedinFigure2donotallowforacleardistinctionbe‐ tweenactiveandinactivecompounds,thedockingstudiesfoundadditionallow‐energy bindingmodes,someofwhichtranslateintoasignificantlydifferentgeometryofthe enzyme‐ligandcomplex. Inmostcases,thesetof30bindingmodeswithbindingenergieslowerthan5 kcal.mol −1 ,relativetothemoststablemode,couldbeclassifiedinto2or3familiesof nearlydegeneratemodes.Theimportanceoftheseobservationswasfirstaccessedbyes‐ timatingtheprobability𝑝offindingeachenzyme‐BODIPYderivativecomplexata givenbindingmodei: 𝑝𝑔𝑒∆ ∑𝑔𝑒∆ (1) where𝑇=298.15K,𝑅isthegasconstant(inkcal/mol)and𝑔isthedegeneracyofeach bindingmode.Becausethemodesarenearly,butnotcompletely,degenerate,allcalcu‐ lationswerecarriedoutusing𝑔=1.Assuminganexhaustivesearchoftheconforma‐ tionalspaceandthatthecollectedmodesarerepresentativeofthelowestenergystatesof thesystem,theseprobabilitiesreflectthepopulationofeachbindingmode[46].The completeresultsfromthesecalculationsintheformofthepopulationofeachbinding modeforeachenzyme‐BODIPYderivativearegiveninTablesS2andS3intheSupport‐ ingInformation. Ingeneral,theresultsshowthatthereisahighprobabilitythatthemoststable bindingmodeisnotrepresentativeofthethermallyavailablecomplexes.Indeed,the higher𝑝valueswerefoundforthelowestenergybindingmodeofPRLm‐3d(48%)and PRTb‐3b(44%).These,however,areexceptions,withthesecondhigher𝑝beingob‐ servedforthecomplexesof2dwithPRLmandPRTb,whichcorrespondedtopopula‐ tionsofonly26%and39%,respectively.Itshouldbenotedthattheligandineachofthese highlystablecomplexesisnotactiveagainstL.majornorT.brucei.Ontheotherhand,the resultsshowthatmostcomplexeswithantileishmanialandantitrypanosomalcom‐ poundspresentapopulationofthelowestenergybindingmodesmallerthan20%,with Figure 2. Binding Gibbs energies ( ∆bindG , in kcal/mol) for the best binding mode of each enzymeBODIPY derivative. Compounds 1b,1c,2b,2c, and 3c present antileishmanial activity, whereas compounds 2b,2c,2f,3a, and 3c present antitrypanosomal activity. Although the results depicted in Figure 2do not allow for a clear distinction between active and inactive compounds, the docking studies found additional low-energy binding modes, some of which translate into a significantly different geometry of the enzyme-ligand complex. In most cases, the set of 30 binding modes with binding energies lower than 5 kcal · mol −1 , relative to the most stable mode, could be classified into 2 or 3 families of nearly degenerate modes. The importance of these observations was first accessed by estimating the probability pi of finding each enzyme-BODIPY derivative complex at a given binding mode i: pi=gie −∆bindGi RT ∑Nmodes jgje −∆bindGj RT (1) where T = 298.15 K, R is the gas constant (in kcal/mol) and gi is the degeneracy of each binding mode. Because the modes are nearly, but not completely, degenerate, all calculations were carried out using gi = 1. Assuming an exhaustive search of the conformational space and that the collected modes are representative of the lowest energy states of the system, these probabilities reflect the population of each binding mode [ 46 ]. The complete
Molecules 2024,29, 2072 8 of 18 results from these calculations in the form of the population of each binding mode for each enzyme-BODIPY derivative are given in Tables S2 and S3 in the Supporting Information. In general, the results show that there is a high probability that the most stable binding mode is not representative of the thermally available complexes. Indeed, the higher pi values were found for the lowest energy binding mode of PRLm-3d (48%) and PRTb-3b (44%). These, however, are exceptions, with the second higher pi being observed for the complexes of 2d with PRLm and PRTb, which corresponded to populations of only 26% and 39%, respectively. It should be noted that the ligand in each of these highly stable complexes is not active against L. major nor T. brucei. On the other hand, the results show that most complexes with antileishmanial and antitrypanosomal compounds present a population of the lowest energy binding mode smaller than 20%, with the exception of PRTb-2b, for which the lowest energy binding mode represents 36% of the population. Thus far, the results from the molecular docking studies strongly suggest that most BODIPY derivatives have some freedom to move within the binding cavity of the tested enzymes, making the lowest energy binding mode less representative than what ought to be expected. As a consequence, the affinity of each compound for the enzyme cannot be rationalized solely based on the geometry of this binding mode, prompting us to explore more sophisticated ways of understanding the formation of these complexes; why are only a handful of BODIPY derivatives biologically active, and why some are specific to a given parasite? For this purpose, the geometry of each binding mode was analyzed in order to find the 10 closest contacts Nc between the enzyme and the ligand; for each contact, we recorded the amino acid involved, the atoms of the ligand and amino acid involved, as well as the distance between the atoms at close contact. For each ligand, the contact to each amino acid in the enzyme was weighted by the pi of the binding mode. Thus, for each amino acid A and ligand L , the overall affinity between a given amino acid, and a ligand is the number of closest contacts between Aand L, weighted by pi: A f f (A,L)= Nmodes ∑ j 100.0 ×picard(A∈Ci)(2) where Ci is the set of amino acids containing atoms in close contact with the ligand in the binding mode i (limited to the Nc closest contacts) and card(A∈Ci) —cardinality of A∈Ci—denotes the count of amino acid Ain the set of closest contacts. The results from using this unitless affinity metric were quite interesting, as they highlighted the affinity of the BODIPY derivatives to certain amino acids in and around the binding pocket. Considering all the 20 BODIPY derivatives, only 57 of the 301 amino acids in PRLm’s chain A have some affinity for these ligands, representing about 19% of the enzyme’s primary structure. In the case of PRTb, affinity toward the BODIPY derivatives was found for 61 of the 267 amino acids in chain A, which covers about 23% of the primary structure. Nevertheless, the values of A f f (A,L) vary by more than three orders of magnitude, from less than 0.1 to about 325 (measured for A f f (Val206, 2b) in PRTb). Although some patterns in A f f (A,L) were apparent by a simple visual depiction of the data, a Principal Component Analysis (PCA) of the affinity data provided a muchneeded simplification of the data. In the case of PRLm, the first Principal Component (PC), representing 43% of the variance in the data, clearly classifies the active from the inactive compounds, as shown in Figure 3a, with the exception of 4c. This erroneous classification of 4c may be due to its low solubility in water, which may have prevented it from reaching PRTb during the biological assays.
Molecules 2024,29, 2072 9 of 18 Molecules2024,29,xFORPEERREVIEW9of19 theexceptionofPRTb‐2b,forwhichthelowestenergybindingmoderepresents36%of thepopulation. Thusfar,theresultsfromthemoleculardockingstudiesstronglysuggestthatmost BODIPYderivativeshavesomefreedomtomovewithinthebindingcavityofthetested enzymes,makingthelowestenergybindingmodelessrepresentativethanwhatoughtto beexpected.Asaconsequence,theaffinityofeachcompoundfortheenzymecannotbe rationalizedsolelybasedonthegeometryofthisbindingmode,promptingustoexplore moresophisticatedwaysofunderstandingtheformationofthesecomplexes;whyare onlyahandfulofBODIPYderivativesbiologicallyactive,andwhysomearespecifictoa givenparasite?Forthispurpose,thegeometryofeachbindingmodewasanalyzedin ordertofindthe10closestcontacts𝑁𝑐betweentheenzymeandtheligand;foreach contact,werecordedtheaminoacidinvolved,theatomsoftheligandandaminoacid involved,aswellasthedistancebetweentheatomsatclosecontact.Foreachligand,the contacttoeachaminoacidintheenzymewasweightedbythe𝑝ofthebindingmode. Thus,foreachaminoacid𝐴andligand𝐿,theoverallaffinitybetweenagivenamino acid,andaligandisthenumberofclosestcontactsbetween𝐴and𝐿,weightedby𝑝: 𝐴 𝑓𝑓 𝐴 ,𝐿 100.0 𝑝𝑐𝑎𝑟𝑑 𝐴 ∈𝐶 (2) where𝐶isthesetofaminoacidscontainingatomsinclosecontactwiththeligandinthe bindingmode𝑖(limitedtothe𝑁𝑐closestcontacts)and𝑐𝑎𝑟𝑑𝐴∈𝐶 —cardinalityof 𝐴∈𝐶 —denotesthecountofaminoacidAinthesetofclosestcontacts. Theresultsfromusingthisunitlessaffinitymetricwerequiteinteresting,asthey highlightedtheaffinityoftheBODIPYderivativestocertainaminoacidsinandaround thebindingpocket.Consideringallthe20BODIPYderivatives,only57ofthe301amino acidsinPRLm’schainAhavesomeaffinityfortheseligands,representingabout19%of theenzyme’sprimarystructure.InthecaseofPRTb,affinitytowardtheBODIPYderiva‐ tiveswasfoundfor61ofthe267aminoacidsinchainA,whichcoversabout23%ofthe primarystructure.Nevertheless,thevaluesof𝐴𝑓𝑓𝐴,𝐿varybymorethanthreeorders ofmagnitude,fromlessthan0.1toabout325(measuredfor𝐴𝑓𝑓(Val206,2b)inPRTb). Althoughsomepatternsin𝐴𝑓𝑓𝐴,𝐿wereapparentbyasimplevisualdepictionof thedata,aPrincipalComponentAnalysis(PCA)oftheaffinitydataprovideda much‐neededsimplificationofthedata.InthecaseofPRLm,thefirstPrincipalCompo‐ nent(PC),representing43%ofthevarianceinthedata,clearlyclassifiestheactivefrom theinactivecompounds,asshowninFigure3a,withtheexceptionof4c.Thiserroneous classificationof4cmaybeduetoitslowsolubilityinwater,whichmayhavepreventedit fromreachingPRTbduringthebiologicalassays. Figure3.PrincipalComponentAnalysis(PCA)scoresoftheaffinitydataforPRLm(a)andPRTb (b)fromthemoleculardockingstudiesofthe20BODIPYderivativesbindingtotheactivesiteat Figure 3. Principal Component Analysis (PCA) scores of the affinity data for PRLm (a) and PRTb (b) from the molecular docking studies of the 20 BODIPY derivatives binding to the active site at chain A of each enzyme. The variance explained by each PC is given in parenthesis, as a percentage of the total variance in each data set. Compounds with antileishmanial (a) or antitrypanosomal (b) activity are depicted in green and are further labeled in (b), and inactive ones are depicted in red. On the other hand, PCA was unable to resolve the compounds for which antitrypanosomal activity was observed from those lacking it, as shown in Figure 3b, which depicts the distribution of the data along PC1 and PC2, covering 62% of the variance in the data. This intriguing result prompted more details of the loadings associated with PC1 for the data concerning PRLm. Table 3shows the seven most prominent loadings along the positive (pointing toward antileishmanial activity) and negative (pointing toward lack of antileishmanial activity) directions of PC1. These results highlight that affinity toward Ser40 and Ala15 of PRLm are desirable traits when searching for antileishmanial activity. On the other hand, significant affinity toward Phe113, as well as affinity toward a number of hydrophobic amino acids in the 224 to 230 positions, appear to hinder the antileishmanial activity of the BODIPY derivatives. Table 3. Most prominent PCA loadings associated with PC1 of the affinity data for PRLm and the set of 20 BODIPY derivatives. Positive Loadings Negative Loadings Amino Acid Value Amino Acid Value Ser40 +0.43 Phe113 −0.49 Ala15 +0.40 Arg17 −0.23 Ser111 +0.23 Tyr194 −0.23 His38 +0.23 Gly225 −0.15 Tyr37 +0.17 Pro224 −0.14 Ser112 +0.16 Val230 −0.11 Ser146 +0.14 Leu229 −0.10 With the results from PRLm in mind, we sought to find in the primary structure of PRTb, the homologous amino acids to those listed in Table 3using the aligned primary structures of both compounds, as shown in Figure 4. As shown in Figure 4, almost all amino acids listed in Table 3are present in both PRLm and PRTb. Despite that, a few of these amino acids are replaced by similar ones: Ser112 is replaced in PRTb by alanine (Ala96) and Ser146 is replaced by threonine (Thr126), only one amino acid mentioned in Table 3(Val230) is replaced by a substantially different one (Pro210). Moreover, the two enzymes align quite well, with the amino acids highlighted by PCA in PRLm overlaying their homologues in PRTb, as shown in Figure 5.
Molecules 2024,29, 2072 16 of 18 Author Contributions: Conceptualization, R.C.R.G., P.P., J.C.M. and M.M.M.R.; methodology, R.C.R.G., P.P., J.C.M. and M.M.M.R.; validation, P.P. and M.M.M.R.; formal analysis, F.T., R.C.R.G., P.P., J.C.M. and M.M.M.R.; investigation, R.C.R.G., F.T. and P.P.; resources, S.P.G.C., J.C.M. and M.M.M.R.; writing—original draft preparation, R.C.R.G. and F.T.; writing—review and editing, R.C.R.G., P.P., J.C.M. and M.M.M.R.; supervision, S.P.G.C., J.C.M. and M.M.M.R.; project administration, S.P.G.C. and M.M.M.R.; funding acquisition, S.P.G.C. and M.M.M.R. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Fundação para a Ciência e Tecnologia (FCT) and FEDER (European Fund for Regional Development)-COMPETE-QRENEU through the Chemistry Research Centre of the University of Minho (ref. CQ/UM (UID/QUI/00686/2020), a contract CEECINST/00156/2018/ CP1642/CT0011, and a PhD grant of R.C.R. Gonçalves (SFRH/BD/05278/2020, https://doi.org/ 10.54499/2020.05278.BD). The NMR spectrometer Bruker Avance III 400 is part of the National NMR Network and was purchased within the framework of the National Program for Scientific Re-equipment, contract REDE/1517/RMN/2005 with funds from POCI 2010 (FEDER) and FCT. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data are contained within the article and Supplementary Materials. Conflicts of Interest: The authors declare no conflicts of interest. References 1. De Rycker, M.; Wyllie, S.; Horn, D.; Read, K.D.; Gilbert, I.H. Anti-Trypanosomatid Drug Discovery: Progress and Challenges. Nat. Rev. Microbiol. 2023,21, 35–50. [CrossRef] 2. Gonçalves, R.C.R.; Peñalver, P.; Costa, S.P.G.; Morales, J.C.; Raposo, M.M.M. Polyaromatic Bis(Indolyl)Methane Derivatives with Antiproliferative and Antiparasitic Activity. Molecules 2023,28, 7728. [CrossRef] [PubMed] 3. 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