University of Groningen
Antimalarial Drug Discovery: Structural Insights
Lunev, Sergey
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Chapter 8
Summary
Despite reported elimination from many regions, malaria remains a dev-astating burden to the human population, annually infecting millions and killing hundreds of thousands people, predominantly in Africa, and delay-ing economic development in endemic regions[1, 2].
Rising phenotypic and clinical resistance against all chemical entities used against malarial parasites urgently requires a continuous supply of novel antimalarial drugs [3-9]. Recently Verlinden et al. stated that clin-ical lifespan of novel antimalarials must at least exceed the time required for drug development[3]. This task requires significant efforts in speeding discovery and validation of new antimalarial drug targets, parallel to the development of novel drugs, delivery strategies and resistance preven-tion. This thesis was primarily focused on new drug target identification and providing means and structural information for target validation in Human malaria. The introductory Chapter 1 presents an overview of the academic publications addressing these topics in detail.
In Chapters 2 & 3, we stress the urgent requirement of novel probe tech-niques to be developed in order to tackle yet far more rapid rate of the drug resistance development of the parasite.
Chapter 2 reviews the pool of the malarial mitochondrial and carbon
metabolism targets that have received attention in recent years in order to draw more attention to unexplored areas [10]. We discuss the “Har-low-Knapp” effect in antimalarial research, as scientists tend to further research well-known targets and pathways, improving existing drugs rather than exploring less studied directions. Plasmodial bc1 complex (a target of Atovaquone) and a validated antimalarial target dihydroorotate dehydrogenase (PfDHODH) are striking examples of the Harlow-Knapp effect and the number of articles featuring both enzymes is continuous-ly rising [11-14]. The main reason of such a tendency is lack of specific probe-tools in the antimalarial “toolbox” allowing targeted interference with previously unexplored enzymes and pathways of interest.
We propose a novel method for specific modulation of target protein ac-tivity using a common biological feature – oligomerization. In this
meth-od the target proteins are probed using their own mutagenic copies, thus bypassing common challenges such as expensive inhibitor design, (often) unreliable genetic manipulation techniques or ambiguous in vivo results of the drug tests due to the poor transport, drug degradation or localiza-tion. An example of such ambiguity is given in the Chapter 2, where oro-tate phosphoribosyltransferase from Plasmodium falciparum (PfOPRT) has not yet been validated as drug target despite the predictions and iden-tification of tight-binding in vitro inhibitors. Although these inhibitors were able to clear parasitemia from P. berghei –infected mice, no in vivo efficacy against P. falciparum was observed [15]. It is unknown wheth-er PfOPRT was dispensable for parasites survival or whethwheth-er the assayed drugs were simply non-effective, degraded or couldn’t reach their target
in vivo.
The Protein Interference Assay (PIA), introduced in Chapter 2, requires structural information on the target system, which is used to generate functionally affected mutant species. These mutant proteins are able to recombine with their wild type counterparts, modulating their activity and thus providing an opportunity for highly specific interference with the target proteins in vitro. Furthermore, overexpression of such mutants within Plasmodium parasites using transfection technique would allow
in(ex) vivo interference and subsequent phenotypic analysis and drug
tar-get validation. The facts that the majority of the protein structures depos-ited in the Protein Data Bank [16], belong to oligomeric species, as well as the overall lower evolutional conservation of oligomeric interfaces, com-pared to often highly conserved active sites [17-20], make PIA a promising addition to the antimalarial “toolbox”. We believe that PIA would aid in validation of previously unexplored targets as well as re-evaluation of al-ready studied systems, where current validation approaches have failed. In Chapter 3, we continue the review of the current drug target validation tools and the potential drug targets from various malarial pathways [21]. Analysis of the essential genes remains highly challenging despite great improvements in the molecular genetics toolset in recent years. These methods are often ineffective in challenging organisms with multiple
stag-es in their life-cycle, such as Plasmodium parasitstag-es. New methods that can provide reliable analysis of the essential genes are urgently required. In this chapter, we specifically focus on the oligomeric surfaces and inter-actions and report the initial progress in utilizing structural information on such surfaces in drug target validation. We use our previous data as an example of specific target inhibition through Protein Interference Assay:
in vitro inhibition of Aspartate Aminotransferase (PfAspAT) [22] and in vivo inhibition of PfPdx1/PfPdx2 complex (Plasmodial PLP-synthase)
re-sulting in significantly increased sensitivity of the transfected parasites to reactive oxygen species [23].
Using PIA in challenging and complex systems, such as malarial parasite, would provide a number of advantages compared to the conventional val-idation methods. The complex lifecycle of the malarial parasite renders stage-specific target analysis highly challenging. As parasite cultivation is extremely difficult in such cases as mosquito- and dormant liver-stages, the isolation of the parasites with integrated DNA modifications would be nearly impossible, making the essential gene analysis using “standard” techniques highly difficult.
High specificity of PIA is achieved due to the ability of the oligomeric sur-faces to bind only to the “correct” partners, making potential cross-re-activity highly unlikely. Furthermore, such specificity allows target val-idation prior to initiating often expensive and laborious small-molecule inhibitor design.
Controlled expression of the mutant probes within the parasite is facilitat-ed through transfection [24-27], a well-understood technique that allows quantitative analysis based on actual protein levels.
PIA also allows bypassing common and often host-specific target vali-dation limitations, such as degravali-dation of the inhibitor, poor membrane transport or localization. The described PIA approach generates complete mutant proteins directly within the parasite. These mutants are practi-cally indistinguishable from their wild type targets, contain the necessary targeting sequences and are constantly expressed.
In Chapter 4, further example of PIA modulation of the target enzyme
dehy-drogenase from Plasmodium falciparum (PfMDH) and identification of the oligomeric interfaces, based on our previously collected X-ray data [29]. We designed a number of point mutations interfering with the na-tive tetrameric state of PfMDH. Introduction of a steric clash at one of the interfaces, distal from the active sites, resulted in dimeric species with significantly reduced specific activity. Furthermore, we show that mutant dimers can recombine with the wild type PfMDH in vitro, rendering the wild type-mutant chimera inactive. As both substrate and cofactor bind-ing sites of PfMDH are highly conserved, design of a specific small mole-cule inhibitor would be extremely challenging and risky, as no evidence is yet available to validate PfMDH as antimalarial drug target. However, the use of PIA-generated PfMDH mutants provides an opportunity for rap-id and an inexpensive acquisition of such evrap-idence. Further experiments, confirming PIA-inhibition of PfAspAT and PfMDH activity in vivo and survivability of transgenic parasites are underway (Batista, Bosch, Lunev
et al., manuscript in preparation).
In Chapter 5, the crystal structure and preliminary characterization of another promising antimalarial target, aspartate transcarbamoylase (PfATC), is reported [30]. Based on the analysis of the crystal structure of
PfATC as well as previous reports on homologous ATC’s [31], we have
de-signed mutant version of PfATC, lacking two key active site residues with significantly reduced specific activity.
These results provide a basis for further PIA validation of PfATC as a drug target. Firstly, recombinant co-expression of both wild type and mutant
PfATC would confirm the proposed ability of the mutant PfATC to
incor-porate into the native assembly, as neither introduced mutation was de-signed to interfere with the oligomeric state. Subsequent activity assays are required to confirm the proposed inhibitory effect of introduction of the mutant copy into the native assembly. As introduction of one mutant copy would impair two out of three active sites, controlled overexpres-sion of the mutant veroverexpres-sion would ensure that in each PfATC (mutant-wild type) chimeric assembly at least two out of three subunits are mutated. Furthermore, survivability of the transfected malarial parasites express-ing mutated PfATC in addition to the endogenous wild type enzyme would
show phenotypic effect of PIA-mediated specific inhibition of PfATC. Such experiments are underway (Bosch, Lunev, et al., in preparation).
In addition to the efforts aimed at the validation of PfATC as a drug target using PIA, conventional drug discovery approach was also used. In
Chap-ter 6, identification of the lead compound inhibiting PfATC activity is
re-ported [32]. Based on (semi)high-throughput screening, 2,3-napthalene-diol was shown to significantly stabilize PfATC, bind and inhibit PfATC at low micromolar concentrations. As observed in the crystal structure of
PfATC-inhibitor complex, 2,3-napthalenediol does not bind in the active
site cavity and does not seem to affect the binding of the first substrate, carbamoyl-phosphate, suggesting an allosteric mode of inhibition. These data provide structural basis for further rational drug or tool design. In Chapter 7, expression, purification and initial X-ray structure solu-tion of pyridoxal kinase from Plasmodium falciparum (PfPdxK) is report-ed [33]. This enzyme, catalysing the phosphorylation of the PLP (vitamin B6) precursors, is suggested to be a promising drug target, as PLP is es-sential for the oxidative stress protection in malaria [23, 34] as well as other metabolic processes. For example, the previously mentioned plas-modial aspartate aminotransferase (PfAspAT), bridging carbon metabo-lism, nucleotide biosynthesis and mitochondrial TCA cycle requires PLP as a cofactor [22, 35, 36]. The data provided in this chapter is another step in understanding the plasmodial metabolism.
Future perspectives
Our proposed PIA approach should be by no means limited to the ma-laria parasite. Other pathogenic systems where currently available ge-netic manipulations allow transfection and overexpression of modified genes can be analysed using PIA. Cases where no such tools available yet, should therefore receive additional attention in order to bypass the “Har-low-Knapp” effect and expand the drug discovery toolset.
We believe that the synergistic combination of multiple orthogonal ap-proaches will drive the research towards elimination of malaria and other
devastating diseases. Recent advances in such fields as systems biology, high-throughput screening, metabolomics and genomic profiling in anti-malarial research support this statement.
Indeed, several genome-scale models have already been generated for P.
falciparum in order to integrate the available knowledge and guide the
fu-ture malaria research [37-40]. Although, systems biology has not yet been widely acknowledged nor used as a research tool in anti-malarial drug target validation, the field of in silico metabolic characterization is rapidly evolving, as supported by reports of in silico elucidation of the chloro-quine action in malaria [40] or prediction of the essential genes within P.
falciparum with little or no homology to human proteins [37-39]. The
in-evitable disagreements between in silico predictions and the experimental data can and will lead to the improvement of current models.
Target re-evaluation strategy based on the published data can help prior-itize the research in well-explored areas. For example, Chaparro and col-leagues structured the current antimalarial portfolio in terms of complex-ity, safety implications as well as genetic, pharmacological and chemical validation [41]. Moreover, investigation of parasitic resistome in addition to the novel compound development can provide valuable insights. A re-cent systematic study of the genomic evolutionary response of P.
falci-parum towards small molecule treatments in order to map the genes
re-sponsible for drug resistance as well as to identify promising antimalarial targets is such an example [42].
High-throughput screening of existing drug libraries [43, 44], path-way-targeted screening [45-48], screening of natural products and me-tabolites for antimalarial activity [49] as well as in-silico profiling [50] have proved to be a valuable source of novel lead scaffolds, often provid-ing highly promisprovid-ing antimalarial hits with low-nanomolar efficacy [45, 46].
(High-throughput) Protein X-Ray Crystallography, the most widely used technique for high-resolution protein structure determination, remains a working horse of drug discovery. The challenges it faced a few years ago [51-53], such as technical and computational limitations in data collection, interpretation and subsequent analysis, have been extensively addressed. Significant advances in crystallographic methods, such as
high-through-put crystal soaking using acoustic liquid dispensers [54], automated X-ray data collection techniques and hardware [55-60] as well as rapidly evolv-ing X-ray data processevolv-ing methods [61, 62] now allow routine large-scale experiments designed to screen thousands of compounds in a matter of days. Additionally, insights into processes underlying the protein crys-tallization would allow reliable prediction and tailored design of optimal crystallization conditions. Recently, Adawy & Groves reported a SLS/ SEC-monitoring approach that could predict the phase diagram param-eters for the target protein at chosen conditions [63]. Furthermore, im-proved protein refolding techniques allow systematic analysis and design of optimal refolding conditions for a wide range of insolubly expressed proteins [64, 65].
Despite numerous reports of recent developments in CryoEM [66-70], NMR spectroscopy [71-74] and XFEL [75-77], challenging the dominance of classic X-ray protein crystallography [53, 78], it is unlikely to become obsolete. Advantages of other methods, such as low sample requirements and ability to monitor a near-natural state of the target can and should be used to compliment crystallography, especially in cases where mac-romolecular systems cannot be crystallized. The fundamental differenc-es between protein crystallography and other emerging methods provide significant advantages such as tunable wavelength for anomalous data collection. For the near future, crystallography will continue to play a ma-jor role [78], as a straightforward, well known, quick and easily accessible high-throughput technique.
The future of drug discovery likely lies in synergistic use of orthogonal approaches, confirming and complementing each other.
Samenvattingen
Ondanks berichten over eliminatie van malaria in vele regio’s, blijft de ziekte een verwoestende belasting geven op de wereldbevolking [1, 2]. Het uitroeien van malaria vereist een onafgebroken aanvoer van vernieuwende anti-malaria medicijnen [3-9]. Aanzienlijke inspanning in het versnellen van ontdekking en validatie van nieuwe anti-malaria geneesmiddel targets zijn nodig, parallel aan de ontwikkeling van vernieuwende geneesmiddel-en, afgifte-strategieën en het voorkomen van resistentie. Dit proefschrift was voornamelijk gericht op het identificeren van nieuwe drug targets en het leveren van structuren en structuur informatie voor target validatie van malaria bij mensen. Het introducerende Hoofdstuk 1 presenteert een overzicht van academische publicaties die deze onderwerpen tot in detail behandelen.
In Hoofdstuk 2 & 3, wordt de dringende behoefte benadrukt aan het ontwikkelen van nieuwe probe technieken om de snellere medicijn resis-tentie ontwikkeling van de parasiet onderuit te halen.
Hoofdstuk 2 behandeld de mitogondrische en koolstof metaboliserende
malaria targets die recentelijk veel aandacht hebben gekregen om zo uit te lichten welke onontdekte gebieden meer aandacht behoeven [10]. We dis-cussiëren het “Harlow-Knapp” effect in antimalaria onderzoek, aangezien wetenschappers vaak neigen naar onderzoek van bekende targets en routes en het verbeteren van bestaande medicijnen, in plaats van het verkennen van minder bestudeerde richtingen. De hoofdreden van deze trend is het gebrek aan specifieke probe-tools in de anti-malaria “toolbox” die gerichte tussenkomst van interessante enzymen en routes toestaan. In Hoofdstuk 3, wordt het overzicht vervolgd van de huidige medicijn target validatietools vanuit de verschillende malaria routes [21]. Analyse van de essentiële genen blijft een zeer grote uitdaging, ondanks de enorme verbeteringen in de moleculaire genetische toolset van de afgelopen jaren. Deze methoden zijn vaak niet effectief in het aanpakken van organismes met meerdere stadia in hun levenscyclus, zoals Plasmodium parasieten. Er is een dringende vraag naar nieuwe methoden die een betrouwbare
analyse kunnen geven van de essentiële genen. In dit hoofdstuk, ligt de focus voornamelijk of de oligomere oppervlakken en interacties en wordt de initiële voortgang van het benutten van de structuur informatie op dergelijke oppervlakken gerapporteerd.
In Hoofdstukken 2 & 3 wordt een nieuwe methode aangedragen voor specifieke modulatie van target eiwit activiteit door gebruik te maken van een gezamenlijke biologische eigenschap – oligomerisatie. Gebaseerd op beschikbare structurele informatie worden target eiwitten ge-probed met hun eigen mutagene kopie om zodoende de gebruikelijke uitdagin-gen te omzeilen, zoals kostbare remmer design, (vaak) onbetrouwbare genetische manipulatie technieken, of dubbelzinnige in vivo resultaten van de geneesmiddel proeven door slechte transport, medicijn degradatie of lokalisatie. Verder laat over-expressie van dergelijke mutanten binnen
Plasmodium parasieten, gebruik makend van een transfectie techniek, in(ex) vivo interferentie toe en de daarop volgende fenotype analyse en
geneesmiddel target validatie. We geloven dat PIA helpt in de validatie van eerder nog niet bestudeerde targets en ook re-evaluatie van eerder bestudeerde targets, waar huidige validatie aanpakken tot dusver hebben gefaald.
In Hoofdstuk 4, wordt nog een voorbeeld van PIA modulatie van het target enzym in vitro beschreven [28]. We rapporteren het kristal struc-tuur van malaat dehydrogenase van Plasmodium falciparum (PfMDH) en identificatie van de oligomere interfaces, gebaseerd op onze eerder ver-zamelde röntgendiffractie gegevens [29]. We laten zien hoe het gebruik van PIA-gegenereerde PfMDH mutanten toegepast kunnen worden voor snelle en goedkope in vivo target enzym inhibitie. Vervolg experimenten
in vivo PIA-inhibitie van PfAspAT en PfMDH activiteit die en de mate
van overleven van transgene parasieten bevestigen is onderweg (Batista, Bosch, Lunev et al., manuscript in preparation).
In Hoofdstuk 5, wordt d kristalstructuur en de voorlopige karakterisa-tie van nog een veelbelovend antimalaria target, spartate transcarbamoy-lase (PfATC) beschreven [30]. Deze data levert een basis voor verdere PIA
validatie van PfATC als medicijn target. Dergelijke experimenten zijn in voorbereiding (Bosch, Lunev, et al., in preparation). Bovenop de inspan-ningen gericht of de validatie van PfATC als medicijn target, gebruik mak-end van PIA, wordt ook de conventionele medicijn ontwikkeling aanpak toegepast.
In Hoofdstuk 6, wordt de identificatie van de lead verbinding die
PfATC activiteit inhibeert beschreven [32]. Gebaseerd op een (semi)
high-throughput screening, laat 2,3-napthftaleendiol een significante sta-bilisatie, binding en inhibitie van PfATC zien bij laag micromolaire con-centraties. Zoals geobserveerd in de kristalstructuur van het PfATC-in-hibitor complex, bindt 2,3-naftaleendiol niet in de holte van de actieve site en heeft geen invloed op de binding van het eerste substraat, carba-moyl-fosfaat, wat een allostere inhibitie suggereert. Deze data levert een structurele basis voor verdere rationele geneesmiddelen en tool design. In Hoofdstuk 7 wordt de expressie, opzuivering en eerste röntgendif-fractie structuur opheldering van Plasmodium falciparum (PfPdxK) bes-chreven [33]. Dit enzym, welke de fosforylatie van de PLP (vitamine B6) precursoren katalyseert, wordt gesuggereerd als een veelbelovend medici-jn target, aangezien PLP essentieel is voor de oxidatieve stress bescherm-ing bij zowel malaria [23, 34] als andere metabolische processen. De in dit hoofdstuk geleverde data is wederom een stap voorwaarts in het begri-jpen van het plasmodiale metabolisme.
Toekomst perspectieven
Onze beoogde PIA aanpak zou in geen geval beperkt moeten zijn tot de malariaparasiet. Andere pathogene systemen waarbij momenteel beschik-bare genetische manipulaties, transfectie en over expressie van gemodi-ficeerde genen mogelijk maken en kunnen met behulp van PIA worden geanalyseerd. De gevallen waar dergelijke tools nog niet beschikbaar zijn, moeten daarom extra aandacht krijgen om het “Harlow-Knapp” effect te omzeilen en zo de toolset voor het ontdekken van geneesmiddelen uit te breiden.
Wij geloven dat de synergetische combinatie van meerdere orthogonale benaderingen, het onderzoek richting eliminatie van malaria en andere verwoestende ziektes bevorderd. Recente ontwikkelingen op het gebied van systems biology, high-throughput screening, metabolomics en ge-noom profilering bij antimalaria onderzoek ondersteunen deze stelling. (High-throughput) Eiwit röntgendiffractie kristallografie, de meest geb-ruikte techniek voor het bepalen van eiwitstructuren met hoge resolutie, blijft een werkpaard voor het ontdekken van geneesmiddelen. De uit-dagingen van een paar jaar geleden [20-22] zoals technische en computa-tionele beperkingen bij het verzamelen van gegevens, interpretatie en de daarop volgende analyse, zijn uitgebreid behandeld.
Aanzienlijke vooruitgang in kristallografische methoden, waaronder high-throughput kristal soaking met behulp van akoestische vloeistof dis-pensers [54], geautomatiseerde röntgen datacollectie technieken en hard-ware [55-60] alsmede snel evoluerende röntgen diffractie dataverwerk-ing methodes [61, 62] maken het nu mogelijk om routinematig op grote schaal duizenden verbindingen te screenen in slechts dagen. Ondanks talrijke rapporten over recente ontwikkelingen in CryoEM [66-70], NMR spectroscopie [71-74] en XFEL [75-77], welke de dominantie van klassieke röntgendiffractie uitdagen [53, 78], is het onwaarschijnlijk dat deze tech-niek verouderd raakt. Voordelen van andere techtech-nieken, zoals lage beno-digde monsterhoeveelheid en de mogelijkheid om een zo goed als natuur-getrouwe staat van de target te monitoren, zouden zeker gebruikt moeten worden als aanvulling op kristallografische technieken, vooral in gevallen waarin macromoleculaire systemen niet gekristalliseerd kunnen worden. De fundamentele verschillen tussen eiwit kristallografie en andere op-komende technieken, leveren een aanzienlijk voordeel zoals aanpasbare golflengte voor afwijkende data verzameling. Voor de nabije toekomst zal kristallografie een grote rol blijven spelen [78], als een eenvoudige, bek-ende, snelle en gemakkelijk toegankelijke high-throughput techniek. De toekomst van het ontdekken van geneesmiddelen ligt waarschijnlijk in synergetisch gebruik van orthogonale benaderingen, die elkaar beves-tigen en aanvullen.
Resumo e Perspectivas futuras
Embora existam atualmente relatos da eliminação da malária em muitas regiões, a doença continua trazendo consequências devastadoras para a população humana [1, 2]. Erradicar a malária requer um fornecimento contínuo de novos medicamentos antimaláricos [3-9]. São necessários es-forços significativos para acelerar a descoberta e a validação de novos al-vos de drogas antimaláricas, paralelamente ao desenvolvimento de noal-vos medicamentos, estratégias de entrega e prevenção de resistência.
O foco principal desta tese foi a identificação de novos alvos de drogas e a disponibilização de meios e informações estruturais para a validação de alvos terapêuticos para o tratamento da Malária Humana. O Capítulo 1 apresenta uma visão geral das publicações acadêmicas que abordam esses temas detalhadamente.
Nos Capítulos 2 e 3, enfatizamos a necessidade urgente de desenvolvi-mento de novas técnicas de sondagem, a fim de enfrentar uma taxa ainda mais rápida do desenvolvimento de resistência a fármacos do parasita. O Capítulo 2 analisa o grupo dos alvos pertencentes ao metabolismo mi-tocondrial e do carbono do parasita Plasmodium que receberam atenção nos últimos anos, a fim de chamar mais atenção para áreas inexploradas [10]. Discutimos o efeito “Harlow-Knapp” na pesquisa de antimaláricos, já que os cientistas tendem a pesquisar ainda mais alvos e caminhos bem conhecidos, melhorando as drogas existentes ao invés de explorar as dire-ções menos estudadas. O principal motivo dessa tendência é a falta de fer-ramentas de sondagem específicas na “caixa de ferfer-ramentas” antimalárica que permitam uma interferência direcionada a enzimas e vias de interesse previamente inexploradas.
No Capítulo 3, continuamos a revisão das ferramentas atuais de valida-ção de alvos de drogas e os possíveis alvos pertencentes as diversas vias da malária [21]. A análise dos genes essenciais permanece altamente desafia-dora apesar das grandes melhorias no conjunto de ferramentas de genéti-ca molecular nos últimos anos. Esses métodos são muitas vezes inefigenéti-cazes em organismos com múltiplos estágios em seu ciclo de vida, como o
para-sita Plasmodium. Torna-se urgentemente necessário o desenvolvimento de novas técnicas que possam fornecer uma análise confiável de tais ge-nes essenciais. Neste capítulo, focamos especificamente nas superfícies e interações oligoméricas e relatamos o progresso inicial na utilização de informações estruturais sobre essas superfícies na validação de alvos de drogas.
Nos Capítulos 2 e 3, propomos um novo método para a modulação es-pecífica da atividade de proteínas alvo usando uma característica bioló-gica comum – a oligomerização. Com base nas informações estruturais disponíveis, as proteínas alvo são analisadas usando suas próprias cópias mutagênicas, burlando dificuldades comumente encontradas neste tipo de análise, como o design custoso de inibidores, técnicas de manipula-ção genética (frequentemente) não confiáveis ou resultados ambíguos dos exames de drogas in vivo devido a transporte deficiente, degradação ou localização da droga. Além disso, a superexpressão de tais mutantes nos parasitas Plasmodium através da técnica de transfecção, possibilitaria a interferência in(ex) vivo, posterior análise fenotípica e validação de alvo terapêuticos.
Acreditamos que o ensaio de interferência proteica (protein interference
assay, PIA) possa auxiliar na validação de alvos anteriormente
inexplora-dos, bem como na reavaliação de sistemas já estudainexplora-dos, onde as aborda-gens de validação atuais falharam.
No Capítulo 4, relatamos outro exemplo de modulação PIA in vitro [28]. Relatamos a estrutura cristalina da enzima malato desidrogenase de Plasmodium falciparum (PfMDH) e a identificação das interfaces oli-goméricas, com base em nossos dados de raios-X previamente coletados [29]. Mostramos, como o uso de mutantes PfMDH gerados por PIA pos-sibilitam a inibição rápida e econômica de enzimas alvo in vitro. Outros experimentos confirmando a inibição da atividade das enzimas aspartato aminotransferase de Plasmodium falciparum (PfAspAT) e PfMDH por PIA in vivo e avaliando a sobrevivência de parasitas transgênicos estão em andamento (Bosch, Batista, Lunev et al., manuscrito em preparação). No Capítulo 5, são relatadas a estrutura cristalina e a caracterização
pre-liminar de outro alvo antimalárico promissor, a enzima aspartato trans-carbamoilase (PfATC) [30]. Esses dados fornecem uma base para a va-lidação da PfATC como alvo de drogas por PIA. Tais experimentos já se encontram em andamento (Bosch, Lunev, et al., em preparação).
Além dos esforços voltados para a validação da PfATC como alvo de dro-gas usando PIA, a abordagem convencional de descoberta de drodro-gas tam-bém foi utilizada. No Capítulo 6 relatamos a identificação do composto principal que inibe a atividade da PfATC [32]. Com base em um (semi)
high-throughput screening, o composto 2,3-Napthalenediol demonstrou
significativa estabilização, ligação e inibição da PfATC em baixas concen-trações micromolares. Conforme observado na estrutura cristalina do complexo inibidor da PfATC, o 2,3-naftalenodiol não se liga à cavidade do sítio ativo e não parece afetar a ligação do primeiro substrato, o car-bamoil-fosfato, sugerindo um modo de inibição alostérico. Esses dados fornecem uma base estrutural para o planejamento racional de drogas. No Capítulo 7 relatamos a expressão, purificação e solução inicial da es-trutura de raio-X da enzima piridoxal quinase de Plasmodium falciparum (PfPdxK) [33]. Esta enzima, que catalisa a fosforilação dos precursores de piridoxal fosfato (PLP, vitamina B6), é sugerida como um alvo de drogas promissor, pois o PLP é essencial para a proteção contra o estresse oxi-dativo no parasita [23, 34] entre outros processos metabólicos. Os dados fornecidos neste capítulo são um passo em direção à melhor compreensão do metabolismo plasmodial.
Perspectivas futuras
A abordagem de PIA proposta neste trabalho não deve ser limitada de for-ma algufor-ma ao parasita da for-malária. Outros sistefor-mas patogênicos onde as manipulações genéticas atualmente disponíveis permitem a transfecção e a superexpressão de genes modificados podem ser analisados usando o PIA. Casos em que ainda não existam ferramentas disponíveis, devem, portanto, receber atenção adicional para burlar o efeito “Harlow-Knapp” e expandir o conjunto de ferramentas para descoberta de medicamentos.
Acreditamos que a combinação sinergética de múltiplas abordagens or-togonais impulsionará a pesquisa para a eliminação da malária e outras doenças devastadoras. Os avanços recentes em campos como biologia de sistemas, triagem de alto rendimento, metabolómica e perfil genômico na pesquisa de antimaláricos apoiam esta afirmação.
A Cristalografia de Raio-X de Proteína (de alto rendimento), a técnica mais utilizada para a determinação da estrutura proteica de alta resolu-ção, continua sendo a ferramenta básica para o desenvolvimento de fár-macos. Os desafios já enfrentados há alguns anos [51-53], como limita-ções técnicas e computacionais na coleta de dados, interpretação e análise subsequente, tem sido amplamente abordados. Avanços significativos em métodos cristalográficos, como soaking de cristais de alto rendimento usando distribuidores de líquidos acústicos [54], técnicas automatizadas de coleta de dados de raios-X e hardware [55-60] bem como métodos de processamento de dados de raios-X que evoluem rapidamente [61, 62] agora permitem experiências de rotina em larga escala projetadas para exibir milhares de compostos em questão de dias. Apesar de numerosos relatos de desenvolvimentos recentes em CryoEM [66-70], espectrosco-pia de RMN [71-74] e XFEL [75-77], desafiando o domínio da cristalo-grafia de proteína de raio X clássica [53, 78], é improvável que este se torne obsoleto. As vantagens de outros métodos, como possibilidade de se trabalhar com pequenas quantidades de amostra e capacidade de mo-nitorar um estado quase natural do alvo, podem e devem ser utilizadas para complementar a cristalografia, especialmente nos casos em que os sistemas macromoleculares não podem ser cristalizados. As diferenças fundamentais entre cristalografia de proteínas e outros métodos emer-gentes proporcionam vantagens significativas, como o comprimento de onda ajustável para a coleta de dados anômalos. Para o futuro próximo, a cristalografia continuará a desempenhar um papel importante [78], como uma técnica de alto rendimento de uso direto, bem conhecida, rápida e facilmente acessível.
O futuro da descoberta de fármacos provavelmente reside no uso sinérgi-co de abordagens ortogonais, sinérgi-confirmando e sinérgi-complementando-se.
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