50-Godišnjica Cambridge Structural Database i 30-godišnjica uporabe u Hrvatskoj

This article is dedicated to the memory of Dr. F. H. Allen and the 50th anniversary of the Cambridge Crystallographic Data Centre (CCDC); the world-renowned centre for deposition and control of crystallographic data including atomic coordinates that define the three-dimensional structures of organic molecules and metal complexes containing organic ligands. The mission exposed at the web site (http://www.ccdc.cam.ac.uk) is clearly stated: “The Cambridge Crystallographic Data Centre (CCDC) is dedicated to the advancement of chemistry and crystallography for the public benefit through providing high quality information, software and services.” The Cambridge Structural Database (CSD), one among the first established electronic databases, nowadays is one of the most significant crystallographic databases in the world. In the International Year of Crystallography 2014, the CSD announced in December over 750,000 deposited structures. The use of the extensive and rapidly growing database needs support of sophisticated and efficient software for checking, searching, analysing, and visualising structural data. The seminal role of the CSD in researches related to crystallography, chemistry, materials science, solid state physics and chemistry, (bio)technology, life sciences, and pharmacology is widely known. The important issues of the CCDC are the accuracy of deposited data and development of software for checking the data. Therefore, the Crystallographic Information File (CIF) is introduced as the standard text file format for representing crystallographic information. Among the most important software for users is ConQuest, which enables searching all the CSD information fields, and the web implementation WebCSD software. Mercury is available for visualisation of crystal structures and crystal morphology including intra- and intermolecular interactions with graph-set notations of hydrogen bonds, and analysis of geometrical parameters. The CCDC gives even more options to the users developing sophisticated software such as GOLD, IsoStar and SuperStar, DASH, and extensive knowledge electronic libraries such as Mogul and Relibase. The CCDC released the new facility – Mercury’s Solid Form module. Such demanding projects require a highly competent team of experts with a scientific approach based on the long tradition in crystallography, modelling and informatics. The Cambridge Structural Database and diversified software and searching engines are useful tools in research and teaching. The use of electronic media and computer graphics makes “data mining” very efficient and useful, but also aesthetically appealing due to the molecular architecture. One can expect even more advanced approaches using cloud computing and ‘Big Data’ management; merging data from related databases will enable to recognize hidden molecular and crystal properties and information that could bring new important knowledge. Since 1985, the CSD has been available to users in Croatia. The use of the CSD in Croatia is illustrated by a few examples performed and published by the presenting authors and colleagues.Prikaz je posvećen uspomeni na dr. F. H. Allena i obilježavanju 50-godišnjice The Cambridge Crystallographic Data Centre (CCDC), svjetski poznatog centra, koji pohranjuje i provjerava kristalografske podatke, uključujući koordinate atoma koje određuju trodimenzijsku strukturu organskih molekula i metalnih kompleksa s organskim ligandima. Misija institucije istaknuta na njezinim web-stranicama je jasna: “Centar za kristalografske podatke u Cambridgeu (CCDC) potpomaže napredak kemije i kristalografije za sveopću dobrobit pružajući kvalitetne informacije, softver i podršku.” Baza strukturnih podataka u Cambridgeu (CSD), jedna je od prvih elektroničkih i najznačajnijih baza u svijetu. U Međunarodnoj godini kristalografije 2014., u prosincu, Centar je obznanio 750.000. pohranjenu strukturu. Upotreba opsežne baze podataka, koja se brzo povećava, zahtijeva podršku u dobro osmišljenom i učinkovitom softveru za provjeru, pretraživanje, analizu i vizualizaciju strukturnih podataka. Prestižna je uloga te baze u istraživanjima povezanim s kristalografijom, kemijom, znanošću o materijalima, fizikom i kemijom krutog stanja, (bio)tehnologijom, znanošću o životu, te farmakologijom, posebno u oblikovanju lijekova. CCDC posvećuje posebnu pažnju točnosti pohranjenih podataka i razvoju softvera za tu namjenu (CIF). Jedan od najvažnijih softvera za korisnike je ConQuest, koji omogućava pretraživanje svih informacija u bazi CSD-a, te njegovo postavljanje na mreži u interaktivnom obliku pod imenom WebCSD. Za vizualizaciju kristalnih struktura i morfologije kristala, uključujući analizu intramolekularnih i intermolekularnih interakcija te topološke oznake vodikovih veza, kao i svih geometrijskih podataka, na raspolaganju je Mercury. Centar (CCDC) pruža još veće mogućnosti korisnicima razvijajući kompleksne softvere poput GOLD, IsoStar i SuperStar te DASH, kao i opsežne elektroničke biblioteke poput Mogula i Relibase. CCDC je pružio novu mogućnost korištenjem Mercuryjeva modula Solid Form. Takvi složeni i zahtjevni projekti mogu se ostvarivati samo s veoma kvalitetnim timom stručnjaka sa znanstvenim pristupom, temeljeći se na dugogodišnjoj tradiciji u kristalografiji, modeliranju i informatici. Sama baza, raznolik softver i alati za pretraživanje afirmirali su se u istraživanju, nastavi i stručnoj izobrazbi. Primjena elektroničkih medija i računalne grafike čini “data mining” učinkovitima i korisnima, ali i estetski privlačnim zbog molekularne arhitekture. Možemo očekivati još više unapređenja zbog uporabe “računarstva u oblacima” (cloud computing) i rukovanja opsežnim podacima (Big Data); umrežavanje podataka iz srodnih baza pomoći će da prepoznamo sakrivena svojstva molekula i kristala i informacija koje mogu otkriti nove važne spoznaje. Od 1985. Strukturna baza podataka u Cambridgeu (CSD) dostupna je korisnicima u Hrvatskoj. Upotreba te baze u Hrvatskoj ilustrirana je s nekoliko primjera autora ovog prikaza i nekih korisnika

Open science discovery of potent noncovalent SARS-CoV-2 main protease inhibitors

INTRODUCTION COVID-19 became a global pandemic partially as a result of the lack of easily deployable, broad-spectrum oral antivirals, which complicated its containment. Even endemically, and with effective vaccinations, it will continue to cause acute disease, death, and long-term sequelae globally unless there are accessible treatments. COVID-19 is not an isolated event but instead is the latest example of a viral pandemic threat to human health. Therefore, antiviral discovery and development should be a key pillar of pandemic preparedness efforts. RATIONALE One route to accelerate antiviral drug discovery is the establishment of open knowledge bases, the development of effective technology infrastructures, and the discovery of multiple potent antivirals suitable as starting points for the development of therapeutics. In this work, we report the results of the COVID Moonshot—a fully open science, crowdsourced, and structure-enabled drug discovery campaign—against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro). This collaboration may serve as a roadmap for the potential development of future antivirals. RESULTS On the basis of the results of a crystallographic fragment screen, we crowdsourced design ideas to progress from fragment to lead compounds. The crowdsourcing strategy yielded several key compounds along the optimization trajectory, including the starting compound of what became the primary lead series. Three additional chemically distinct lead series were also explored, spanning a diversity of chemotypes. The collaborative and highly automated nature of the COVID Moonshot Consortium resulted in >18,000 compound designs, >2400 synthesized compounds, >490 ligand-bound x-ray structures, >22,000 alchemical free-energy calculations, and >10,000 biochemical measurements—all of which were made publicly available in real time. The recently approved antiviral ensitrelvir was identified in part based on crystallographic data from the COVID Moonshot Consortium. This campaign led to the discovery of a potent [median inhibitory concentration (IC50) = 37 ± 2 nM] and differentiated (noncovalent and nonpeptidic) lead compound that also exhibited potent cellular activity, with a median effective concentration (EC50) of 64 nM in A549-ACE2-TMPRSS2 cells and 126 nM in HeLa-ACE2 cells without measurable cytotoxicity. Although the pharmacokinetics of the reported compound is not yet optimal for therapeutic development, it is a promising starting point for further antiviral discovery and development. CONCLUSION The success of the COVID Moonshot project in producing potent antivirals, building open knowledge bases, accelerating external discovery efforts, and functioning as a useful information-exchange hub is an example of the potential effectiveness of open science antiviral discovery programs. The open science, patent-free nature of the project enabled a large number of collaborators to provide in-kind support, including synthesis, assays, and in vitro and in vivo experiments. By making all data immediately available and ensuring that all compounds are purchasable from Enamine without the need for materials transfer agreements, we aim to accelerate research globally along parallel tracks. In the process, we generated a detailed map of the structural plasticity of Mpro, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data to spur further research into antivirals and discovery methodologies. We hope that this can serve as an alternative model for antiviral discovery and future pandemic preparedness. Further, the project also showcases the role of machine learning, computational chemistry, and high-throughput structural biology as force multipliers in drug design. Artificial intelligence and machine learning algorithms help accelerate chemical synthesis while balancing multiple competing molecular properties. The design-make-test-analyze cycle was accelerated by these algorithms combined with planetary-scale biomolecular simulations of protein-ligand interactions and rapid structure determination

Modelling the binding mode of macrocycles: Docking and conformational sampling

Drug discovery is increasingly tackling challenging protein binding sites regarding molecular recognition and druggability, including shallow and solvent-exposed protein-protein interaction interfaces. Macrocycles are emerging as promising chemotypes to modulate such sites. Despite their chemical complexity, macrocycles comprise important drugs and offer advantages compared to non-cyclic analogs, hence the recent impetus in the medicinal chemistry of macrocycles. Elaboration of macrocycles, or constituent fragments, can strongly benefit from knowledge of their binding mode to a target. When such information from X-ray crystallography is elusive, computational docking can provide working models. However, few studies have explored docking protocols for macrocycles, since conventional docking methods struggle with the conformational complexity of macrocycles, and also potentially with the shallower topology of their binding sites. Indeed, macrocycle binding mode prediction with the mainstream docking software GOLD has hardly been explored. Here, we present an in-depth study of macrocycle docking with GOLD and the ChemPLP scores. First, we summarize the thorough curation of a test set of 41 protein-macrocycle X-ray structures, raising the issue of lattice contacts with such systems. Rigid docking of the known bioactive conformers was successful (three top ranked poses) for 92.7% of the systems, in absence of crystallographic waters. Thus, without conformational search issues, scoring performed well. However, docking success dropped to 29.3% with the GOLD built-in conformational search. Yet, the success rate doubled to 58.5% when GOLD was supplied with extensive conformer ensembles docked rigidly. The reasons for failure, sampling or scoring, were analyzed, exemplified with particular cases. Overall, binding mode prediction of macrocycles remains challenging, but can be much improved with tailored protocols. The analysis of the interplay between conformational sampling and docking will be relevant to the prospective modelling of macrocycles in general

Structural characterization of the Mycobacterium tuberculosis ABC transporter Rv1747 : studies on interaction with the PknF protein and conformational

Orientador: Andrea Balan FernandesTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A tuberculose, é uma doença infecciosa que geralmente afeta os pulmões e é causada pelo bacilo Mycobacterium tuberculosis (Mtb). Sua complexidade, duração do tratamento e o surgimento de cepas resistentes e multirresistentes, destacam a importância dos estudos sobre os mecanismos de resistência da bactéria, alvos para o desenvolvimento de novos medicamentos e inibidores de crescimento e infecção mais eficazes. Transportadores do tipo ABC (do inglês ATP-Binding Cassette) apresentam papel fundamental durante a infecção pois, estão relacionados aos mecanismos de aquisição de nutrientes (importadores) como também à extrusão de drogas (exportadores ou bombas de efluxo). O transportador ABC Rv1747 deMtb não tem função conhecida, mas sua presença é requerida para o crescimento da bactéria in vivo. Estudos prévios sugerem seu papel na reciclagem de compostos hidrofóbicos, como drogas e lipídios. Adicionalmente, sua estrutura apresenta dois domínios do tipo Forkhead Associated (FHA-1 e FHA-2), fosforilados pela proteína PknF, uma serina/treonina quinase, possível reguladora da atividade do transportador. Neste contexto, esta pesquisa teve como objetivo caracterizar estruturalmente o transportador ABC Rv1747, especificamente, a sua interação com a proteína PknF (cPknF) e avaliar os possíveis efeitos estruturais e funcionais da fosforilação nos domínios FHA. Nesse sentido, produzimos o domínio FHA e três mutantes fosfomiméticos (FHA_T152E, e FHA_T210E e FHA_T152E/T210E) e o domínio catalítico de PknF (cPknF) na forma solúvel e estável. As alterações estruturais induzidas no domínio FHA como consequência da emulação da fosforilação, bem como da mistura FHA-cPknF, foram avaliadas por modelagem molecular, docking molecular, dicroísmo circular (CD), fluorimetria de varredura diferencial (DFS), fluorêscencia intrínseca de triptofanos, espalhamento de raios X a baixo ângulo (SAXS), cristalografia de raios-X e calorimetria de titulação isotérmica (ITC). Os resultados mostraram que os mutantes fosfomiméticos não sofrem alterações no conteúdo de estrutura secundária, mas diminuição (quenching) da fluorescência intrínseca dos triptofanos, similarmente ao que ocorreu com cPknF fosforilada (cPknF*). Tais alterações foram corroboradas pelas medidas de SAXS, que evidenciaram compactação do domínio FHA nos estados fosfomiméticos, tanto nos mutantes quanto na presença de cPknF*. As mutações nos domínios FHA também afetaram a afinidade pela cPknF, a qual exibiu maior afinidade pelo domínio selvagem (Kd = 0,4 µM) e menor pelo duplo mutante FHA_T152E/T210E (Kd = 9,4 µM).Finalmente, foi possível determinar a estrutura tridimensional de cPknF em complexo com o inibidor de quinases humanas IKK16 a 2.6 Å de resolução, a qual é caracterizada por arquitetura e topologia das quinases do tipo Hanks com os lobos amino (N) e carboxi (C) terminais, entre os quais forma-se a fenda que contém o sítio ativo. As análises de docking molecular com o ATP e a estrutura com o inibidor mostraram conservação em relação àsoutras quinases. O conjunto dos resultados permitiu-nos estabelecer um modelo para aativação do transportador pela quinase. Neste modelo, cPknF* teria como primeiro alvo a T152 próxima ao domínio FHA-1, desencadeando alterações estruturais que levariam à aproximação dos domínios FHA-1 e FHA-2 e, consequente fosforilação da T210. As mudanças conformacionais nos domínios FHA teriam como consequência, alterações nas ATPases, permeases e ativação do transportador. A estrutura da quinase e sua relevância na ativação do transportador abre perspectivas muito interessantes para o estudo destas proteínas comoalvos para o desenvolvimento de inibidores e papel funcional do transportador.Abstract: Tuberculosis is an infectious disease that usually affects the lungs and is caused by the bacillus Mycobacterium tuberculosis (Mtb). Its complexity, duration of treatment and the emergence of resistant and multiresistant strains, highlight the importance of studies on the bacteria'sresistance mechanisms, targets for the development of new drugs and more effective growth and infection inhibitors. ATP-Binding Cassette (ABC) transporters play a fundamental role during infection because they are related to the mechanisms of nutrient acquisition (importers) as well as to the extrusion of drugs (exporters or efflux pumps). The ABC Rv1747transporter from Mtb has no known function, but its presence is required for the growth of the bacteria in vivo. Previous studies suggest its role in recycling hydrophobic compounds, such as drugs and lipids. Additionally, its structure presents two Forkhead-Associated domains (FHA-1 and FHA-2), phosphorylated by the protein PknF, a serine/threonine kinase, a possible regulator of the activity of the transporter. In this context, this research aimed to structurally characterize the ABC transporter Rv1747, specifically, its interaction with the PknF protein and to evaluate the possible structural and functional effects of phosphorylation in the FHA domains. In this sense, we produced the FHA domain and three phosphomimetic mutants (FHA_T152E, and FHA_T210E and FHA_T152E / T210E) and the catalytic PknF domain (cPknF) in soluble and stable form. The structural changes induced in the FHA domain as a consequence of the emulation of phosphorylation, as well as the FHA-cPknF mixture, were evaluated by molecular modeling, molecular docking, circular dichroism (CD), differential scanning fluorimetry (DFS), intrinsic tryptophan fluoroscopy, scattering of X-rays at low angle (SAXS), X-ray crystallography and isothermal titration calorimetry (ITC). The results showed that the phosphomimetic mutants do not undergo changes in the content of secondary structure but decrease (quenching) of the intrinsic fluorescence of tryptophan, similarly to what occurred with phosphorylated cPknF (cPknF *). Such changes were corroborated by the SAXS measurements, which showed compaction of the FHA domain in phosphomimetic states and an increase in stiffness, both in the mutants and in the presence of cPknF*. Mutations in the FHA domains also affected the affinity for cPknF, which exhibited greater affinity for the wild domain (Kd = 0.4 µM) and lower for the double mutant FHA_T152E / T210E (Kd = 9.4 µM). Finally, it was possible to determine the three-dimensional structure of cPknF in complex with the human kinase inhibitor IKK16 at 2.6 Å resolution, which is characterized by the architecture and topology of Hanks-type kinases with the amino (N) and carboxy (C) lobe termini, including the cleft that contains the active site. The molecular docking analyzes with ATP and the structure with the inhibitor showed conservation in relation to other kinases. The set of results allowed us to establish a model for the activation of the transporter by the kinase. In this model, cPknF* would first target T152 close to the FHA-1 domain, triggering structural changes that would lead to the approximation of the FHA-1 domains of FHA-2 and, consequently, phosphorylation of T210. Conformational changes in the FHA domains would result in changes in ATPases, permeases and transporter activation. The structure of the kinase and its relevance in the activation of the transporter opens very interesting perspectives for the study of these proteins as targets for the development of inhibitors and the functional role of the transporterDoutoradoGenética de MicroorganismosDoutora em Genética e Biologia Molecular678COLCIENCIA

In Silico Design and Selection of CD44 Antagonists:implementation of computational methodologies in drug discovery and design

Drug discovery (DD) is a process that aims to identify drug candidates through a thorough evaluation of the biological activity of small molecules or biomolecules. Computational strategies (CS) are now necessary tools for speeding up DD. Chapter 1 describes the use of CS throughout the DD process, from the early stages of drug design to the use of artificial intelligence for the de novo design of therapeutic molecules. Chapter 2 describes an in-silico workflow for identifying potential high-affinity CD44 antagonists, ranging from structural analysis of the target to the analysis of ligand-protein interactions and molecular dynamics (MD). In Chapter 3, we tested the shape-guided algorithm on a dataset of macrocycles, identifying the characteristics that need to be improved for the development of new tools for macrocycle sampling and design. In Chapter 4, we describe a detailed reverse docking protocol for identifying potential 4-hydroxycoumarin (4-HC) targets. The strategy described in this chapter is easily transferable to other compounds and protein datasets for overcoming bottlenecks in molecular docking protocols, particularly reverse docking approaches. Finally, Chapter 5 shows how computational methods and experimental results can be used to repurpose compounds as potential COVID-19 treatments. According to our findings, the HCV drug boceprevir could be clinically tested or used as a lead molecule to develop compounds that target COVID-19 or other coronaviral infections. These chapters, in summary, demonstrate the importance, application, limitations, and future of computational methods in the state-of-the-art drug design process

Synthesis, crystal structures and molecular modelling of rare earth complexes with bis(2-pyridylmethyl)amine and its derivatives : a quantum chemical investigation of ligand conformational space, complex intramolecular rearrangement and stability

Limited research has been performed on the coordination behaviour of hybrid aliphatic and heterocyclic polyamines with trivalent rare earth elements. The rare earth coordination properties of several Nalkylated derivatives of the tridentate ligand bis(2-pyridylmethyl)amine (DPA, HL1) backbone - involving the rare earth elements Y, La-Nd, Sm, Eu and Tb-Lu - have been investigated in this study However, the structural and energetic characteristics of DPA coordination with rare earth elements (REE) have not been studied thus far. Potential applications of DPA-based rare earth complexes are primarily dependent on their electronic and magnetic characteristics, which are affected weakly by the coordination environment, where potential applications may include use as Lanthanide Shift Reagents (LSR), Luminescence probes and small-molecule magnets (SMM). A systematic conformational search of DPA was carried out in this study in order to identify the global minimum conformer and for comparison of the free and coordinated geometries, using the M06(D3) functional belonging to the Density Functional Theory (DFT) family of model chemistries. An understanding of the aforementioned would play an important role in analysis of bulk characterization and the prediction of the reactivity of DPA. Final geometries and electronic energies were obtained from the ‘domain based local pair natural orbital’ (DLPNO)-Møller-Plesset and -coupled cluster theoretical methods, as follows: DLPNO-CCSD(T0)/aug-cc-pVQZ//DLPNO-MP2/aug-cc-pVTZ. Fifteen Single-crystal X-ray diffractometer (SC-XRD) crystal structures of mononuclear rare earth chloride coordination complexes with DPA (RE = La-Nd, Sm, Eu, Tb-Lu and Y) were obtained and geometrically analysed in this study. Three isostructural series of constitutional isomers were identified, consisting of one series of nine-coordinate molecule (M1) and two series of eight-coordinate ion pairs (M2 and M3). This conformational diversity is most likely due the flexible nature of the DPA backbone, as well as the additional stability gained from reduced coordination spheres as a function of decreasing rare earth ionic radii across the lanthanide series (Lanthanide contraction). A Quantum Theory of Atoms-in-Molecules (QTAIM) topological analysis was performed in order to identify and characterise potential hydrogen bonding interactions in H-optimised crystal structures. The crystal structures of five dinuclear (RE = Tb-Tm) and two tetranuclear (RE = Yb and Lu) rare earth chloride complexes with DPA have also been structurally analysed. Furthermore, one mononuclear (RE = Dy), two dinuclear complexes (RE = Dy and Lu) with EtDPA, and one mononuclear complex with the DPA-derivative HL4 (RE = Dy) were structurally characterised. A DFT study of the theoretical interconversion of one real- and two hypothetical- mononuclear lanthanum containing isostructural series (cf. aforementioned crystal structures) was undertaken in order to gain a deeper understanding of the processes involved, in terms of the participating minimum energy paths (MEPs), intermediates and transition states – as determined via the Nudged-Elastic Band (NEB) procedure. This hypothesis is supported by the well-known conformational lability of rare earth complexes, due to the weak/minor covalency of their coordination bonds. An attempt was made to determine the respective energies of one real- and two hypothetical diamagnetic or ‘closed-shell’ constitutional isomers containing the rare earth ions La3+(M1), Y3+(M2) and Lu3+ (M3). It was assumed that the most stable isomers have a greater probability of being observed as the asymmetric unit of the complex crystal structure – assuming weak contributions of lattice or intermolecular interactions towards the geometry of the asymmetric unit