Combining automated peak tracking in SAR by NMR with structure-based backbone assignment from 15N-NOESY

BACKGROUND: Chemical shift mapping is an important technique in NMR-based drug screening for identifying the atoms of a target protein that potentially bind to a drug molecule upon the molecule's introduction in increasing concentrations. The goal is to obtain a mapping of peaks with known residue assignment from the reference spectrum of the unbound protein to peaks with unknown assignment in the target spectrum of the bound protein. Although a series of perturbed spectra help to trace a path from reference peaks to target peaks, a one-to-one mapping generally is not possible, especially for large proteins, due to errors, such as noise peaks, missing peaks, missing but then reappearing, overlapped, and new peaks not associated with any peaks in the reference. Due to these difficulties, the mapping is typically done manually or semi-automatically, which is not efficient for high-throughput drug screening. RESULTS: We present PeakWalker, a novel peak walking algorithm for fast-exchange systems that models the errors explicitly and performs many-to-one mapping. On the proteins: hBcl(XL), UbcH5B, and histone H1, it achieves an average accuracy of over 95% with less than 1.5 residues predicted per target peak. Given these mappings as input, we present PeakAssigner, a novel combined structure-based backbone resonance and NOE assignment algorithm that uses just (15)N-NOESY, while avoiding TOCSY experiments and (13)C-labeling, to resolve the ambiguities for a one-to-one mapping. On the three proteins, it achieves an average accuracy of 94% or better. CONCLUSIONS: Our mathematical programming approach for modeling chemical shift mapping as a graph problem, while modeling the errors directly, is potentially a time- and cost-effective first step for high-throughput drug screening based on limited NMR data and homologous 3D structures

Fast and Robust Mathematical Modeling of NMR Assignment Problems

NMR spectroscopy is not only for protein structure determination, but also for drug screening and studies of dynamics and interactions. In both cases, one of the main bottleneck steps is backbone assignment. When a homologous structure is available, it can accelerate assignment. Such structure-based methods are the focus of this thesis. This thesis aims for fast and robust methods for NMR assignment problems; in particular, structure-based backbone assignment and chemical shift mapping. For speed, we identified situations where the number of 15N-labeled experiments for structure-based assignment can be reduced; in particular, when a homologous assignment or chemical shift mapping information is available. For robustness, we modeled and directly addressed the errors. Binary integer linear programming, a well-studied method in operations research, was used to model the problems and provide practically efficient solutions with optimality guarantees. Our approach improved on the most robust method for structure-based backbone assignment on 15N-labeled data by improving the accuracy by 10% on average on 9 proteins, and then by handling typing errors, which had previously been ignored. We show that such errors can have a large impact on the accuracy; decreasing the accuracy from 95% or greater to between 40% and 75%. On automatically picked peaks, which is much noisier than manually picked peaks, we achieved an accuracy of 97% on ubiquitin. In chemical shift mapping, the peak tracking is often done manually because the problem is inherently visual. We developed a computer vision approach for tracking the peak movements with average accuracy of over 95% on three proteins with less than 1.5 residues predicted per peak. One of the proteins tested is larger than any tested by existing automated methods, and it has more titration peak lists. We then combined peak tracking with backbone assignment to take into account contact information, which resulted in an average accuracy of 94% on one-to-one assignments for these three proteins. Finally, we applied peak tracking and backbone assignment to protein-ligand docking to illustrate the potential for fast 3D complex determination

Automation of peak-tracking analysis of stepwise perturbed NMR spectra

6noWe describe a new algorithmic approach able to automatically pick and track the NMR resonances of a large number of 2D NMR spectra acquired during a stepwise variation of a physical parameter. The method has been named Trace in Track (TinT), referring to the idea that a gaussian decomposition traces peaks within the tracks recognised through 3D mathematical morphology. It is capable of determining the evolution of the chemical shifts, intensity and linewidths of each tracked peak.The performances obtained in term of track reconstruction and correct assignment on realistic synthetic spectra were high above 90% when a noise level similar to that of experimental data were considered. TinT was applied successfully to several protein systems during a temperature ramp in isotope exchange experiments. A comparison with a state-of-the-art algorithm showed promising results for great numbers of spectra and low signal to noise ratios, when the graduality of the perturbation is appropriate. TinT can be applied to different kinds of high throughput chemical shift mapping experiments, with quasi-continuous variations, in which a quantitative automated recognition is crucial.openopenBanelli, Tommaso; Vuano, Marco; Fogolari, Federico; Fusiello, Andrea; Esposito, Gennaro; Corazza, AlessandraBanelli, Tommaso; Vuano, Marco; Fogolari, Federico; Fusiello, Andrea; Esposito, Gennaro; Corazza, Alessandr

Liuostila NMR-spektroskopian pulssisarjojen suunnittelu : ominaisuuksia ja sovellutuksia

NMR spectroscopy enables the study of biomolecules from peptides and carbohydrates to proteins at atomic resolution. The technique uniquely allows for structure determination of molecules in solution-state. It also gives insights into dynamics and intermolecular interactions important for determining biological function. Detailed molecular information is entangled in the nuclear spin states. The information can be extracted by pulse sequences designed to measure the desired molecular parameters. Advancement of pulse sequence methodology therefore plays a key role in the development of biomolecular NMR spectroscopy. A range of novel pulse sequences for solution-state NMR spectroscopy are presented in this thesis. The pulse sequences are described in relation to the molecular information they provide. The pulse sequence experiments represent several advances in NMR spectroscopy with particular emphasis on applications for proteins. Some of the novel methods are focusing on methyl-containing amino acids which are pivotal for structure determination. Methyl-specific assignment schemes are introduced for increasing the size range of 13C,15N labeled proteins amenable to structure determination without resolving to more elaborate labeling schemes. Furthermore, cost-effective means are presented for monitoring amide and methyl correlations simultaneously. Residual dipolar couplings can be applied for structure refinement as well as for studying dynamics. Accurate methods for measuring residual dipolar couplings in small proteins are devised along with special techniques applicable when proteins require high pH or high temperature solvent conditions. Finally, a new technique is demonstrated to diminish strong-coupling induced artifacts in HMBC, a routine experiment for establishing long-range correlations in unlabeled molecules. The presented experiments facilitate structural studies of biomolecules by NMR spectroscopy.Liuostila NMR-spektroskopian pulssisarjojen suunnittelu: Ominaisuuksia ja sovellutuksia Proteiinit ovat solujen työjuhtia. Proteiinit osallistuvat esimerkiksi molekyylien kuljetukseen ja kemiallisten reaktioiden katalyysiin. Proteiinin rakenne kertoo, mikä on sen funktio. Tätä tietoa voidaan käyttää solujen sisäisen toiminnan ymmärtämiseen ja soveltaa uusien lääkeaineiden kehittämiseen. Proteiinit ovat liian pieniä havaittavaksi edes parhaalla mahdollisella mikroskoopilla. Proteiinien kolmiulotteinen rakenne voidaan kuitenkin määrittää atomitasolla ydinmagneettisen resonanssin (NMR) avulla. NMR tekniikka perustuu samaan periaatteeseen, jota käytetään aivojen magneettikuvantamisessa sairaaloissa, mutta proteiinien pienen koon takia laitteelta vaaditaan huomattavasti voimakkaampaa magneettikenttää. Kun proteiini laitetaan koeputkessa magneettikentään, proteiinien atomit alkavat käyttäytyä pienten antennien tavoin. Samalla tavalla kuin radioaaltoja vastaanottavat laitteet tunnistavat tiettyjä taajuuksia, proteiinit absorboivat vain rakenteeseensa sopivaa säteilyä. Tällä tavalla voidaan kustakin atomista syntyvät signaalit tunnistaa ja lopulta proteiinin koko rakenne määrittää. NMR:n avulla voidaan tutkia myös proteiinin muita ominaisuuksia kuten sen dynamiikkaa ja sen vuorovaikutuksia muiden molekyylien kanssa. Proteiineissa on kuitenkin paljon atomeja ja tästä johtuen myös niiden spektreissä on paljon signaaleja, jotka ovat usein päällekkäin. Näinollen signaalien alkuperän määrittäminen on vaikeaa. Useita radiopulsseja voi yhdistää pulssisarjaksi, joka on verrattavissa nuotteihin. Pulssisarjojen avulla on mahdollista tuottaa sellaisia spektrejä, joissa yksittäiset signaalit ovat erillään ja tunnistettavissa. Jo pari kymmentä vuotta pienten proteiinien rakenteen määrittäminen on ollut mahdollista NMR:n avulla. Suurempien molekyylien kolmiulotteisen rakenteen määrittäminen on kuitenkin edelleen haastavaa. Tässä suhteessa uusien pulssisarjojen kehittäminen on erityisen tärkeää sillä niiden avulla spektrien sisältämä tieto on helpommin tulkittavissa, sisältää enemmän yksityiskohtia ja lisäksi itse spektrien tuottaminen on tehokkaampaa. Tämä väitöstyö käsittelee ydinmagneettisen resonanssin menetelmien kehittämistä ja niiden soveltamista biomolekyylien rakenteen selvittämiseen. Työ mahdollistaa yhä kookkaampien proteiinien rakenteiden määrittämisen ja parantaa käytössäolevien menetelmien ominaisuuksia. Väitöskirja tarkastetaan perjantaina 7. marraskuuta 2008 klo 12 Viikin Biokeskus 2:ssa

NMR as a “gold standard” method in drug design and discovery

Studying disease models at the molecular level is vital for drug development in order to improve treatment and prevent a wide range of human pathologies. Microbial infections are still a major challenge because pathogens rapidly and continually evolve developing drug resistance. Cancer cells also change genetically, and current therapeutic techniques may be (or may become) ineffective in many cases. The pathology of many neurological diseases remains an enigma, and the exact etiology and underlying mechanisms are still largely unknown. Viral infections spread and develop much more quickly than does the corresponding research needed to prevent and combat these infections; the present and most relevant outbreak of SARS-CoV-2, which originated in Wuhan, China, illustrates the critical and immediate need to improve drug design and development techniques. Modern day drug discovery is a time-consuming, expensive process. Each new drug takes in excess of 10 years to develop and costs on average more than a billion US dollars. This demonstrates the need of a complete redesign or novel strategies. Nuclear Magnetic Resonance (NMR) has played a critical role in drug discovery ever since its introduction several decades ago. In just three decades, NMR has become a “gold standard” platform technology in medical and pharmacology studies. In this review, we present the major applications of NMR spectroscopy in medical drug discovery and development. The basic concepts, theories, and applications of the most commonly used NMR techniques are presented. We also summarize the advantages and limitations of the primary NMR methods in drug development

Identification and optimisation of ligands to target protein-protein interactions: EB1-SxIP proteins

End binding protein 1 (EB1) is a key element in the complex network of protein-protein interactions at microtubule growing ends which has a fundamental role in microtubule polymerisation. EB1 regulates the microtubule dynamic behaviour, through protein recruitment, and has been associated with several disease states, such as cancer and neuronal diseases. Diverse EB1 binding partners are recognised through a conserved SxIP motif within an intrinsically disordered region enriched with basic, serine and proline residues. Crystal structure of EB1 in complex with a peptide containing the SxIP motif demonstrated that the isoleucine-proline dipeptide is bound into a well‐defined cavity of EB1 that may be suitable for small molecule targeting. The research described herein reports the use of a multidisciplinary approach for the discovery of the first small molecule scaffold to target the EB1 recruiting domain. This approach included virtual screening (structure and ligand based design) and multiparameter compound selection. Solution NMR structures of the C-terminal domain of EB1 in the free form and in complex with the small molecule are also reported. A key finding from these structures is that the hydrophobic binding pocket reported to be essential for recruiting SxIP proteins is not pre-formed but highly dynamic in solution. This brings new insights to the protein recruitment mechanism regulated by EB1 and for the identification of new small molecule inhibitors for the EB1-SxIP protein interactions. The interaction of short length peptides containing the SxIP motif with EB1 was characterised through the use of solution NMR and ITC methods. The contributions for the binding of the SxIP motif and neighbouring residues to EB1 were quantified in terms of binding energy. A structural model shows that the binding pocket of EB1 is largely extended when in complex. This research describes not only the first chemical scaffold that targets EB1, it details important structural features of the interaction of this protein with SxIP containing peptides. This structural information provides fundamental understanding of this interaction that can be exploited in the future to discover higher affinity ligands

Entwicklung und Anwendung von Kernspinresonanz-Spektroskopie und Röntgenkristallographie in früher Wirkstoffentwicklung

Challenges in drug discovery including difficulties obtaining structural information on the ligand-protein complex are approached using multiple methods. Crystal structures of IMP-13, an antibiotic resistance protein, along with its natural antibiotic substrates are presented. Paramagnetic methods in drug discovery using NMR are investigated, and a deep learning approach for computational modelling positions of water molecules in protein structures for use in ligand optimisation is introduced.Herausforderungen in der Arzneimittelforschung einschließlich der Schwierigkeiten, strukturelle Informationen über den Ligand-Protein-Komplex zu erhalten, werden mit verschiedenen Methoden adressiert Kristallstrukturen von IMP-13, einem Antibiotikaresistenzprotein, zusammen mit seinen natürlichen Substraten werden vorgestellt. Paramagnetische Methoden in der Wirkstoffentwicklung mittels NMR werden untersucht, und die Anwendung von Deep Learning für die Modellierung der Positionen von Wassermolekülen in Proteinstrukturen für die Optimierung von Liganden wird vorgestell

Stability and Strain in Hisactophilin and Mechanism of the Myristoyl Switch

Hisactophilin is a myristoylated, histidine-rich, pH-dependent actin- and membrane-binding protein. In response to cellular changes in pH, this β-trefoil protein reversibly switches between cytosolic and membrane- bound forms. A key feature of the reversible membrane-binding is the covalent acylation of the N-terminal glycine with a C14 myristoyl group. At pH > 6.9, the myristoyl group favours sequestration in the barrel of the β-trefoil, whereas at pH < 6.9, the myristoyl group favours increased solvent accessibility and eventually anchors hisactophilin to the inner leaflet of the cell membrane. In Dictyostelium discoideum, membrane-bound hisactophilin also binds and bundles actin, contributing to cell locomotion. Despite widespread myristoylation of eukaryotic proteins, its effects on protein folding, stability, and function are still poorly understood and limits our understanding of a broader set of switches and the ability to design them. Combining equilibrium denaturation, folding kinetics, variable temperature and variable pH nuclear magnetic resonance (NMR), chemical shift perturbations, and reverse micelle encapsulation, we use hisactophilin as a model for characterizing the determinants of finely tuned myristoyl switches. Equilibrium stability measurements identified hisactophilin mutants with broken switches—in which pH ceases influencing conformational switching—and switches with tuned sensitivities. In a few cases, stunningly small changes to amino acid side chains broke the pH-dependent myristoyl switch. Interestingly, a thermodynamic switch broken by one mutation may be repaired by making additional mutations, illustrating novel synergistic contributions to global stability and switching. Studying the mutants also revealed that a predominant effector of switching appears to be strain from an overpacked core when the myristoyl group is sequestered in the binding pocket. Altering strain through changing the geometry of the myristoyl binding pocket offers a new approach for tuning the sensitivity of hisactophilin and other switch proteins, as well as to inform future design efforts. The temperature dependence of amide proton chemical shifts localized myristoyl-induced strain to a set of residues in wild-type hisactophilin’s myristoyl-binding pocket. The mutants with broken switches (I85L, which favours the accessible state, and F6L/I85L/I93L, which favours the sequestered state), however, no longer show evidence of strain. Nonlinear temperature dependence of chemical shifts indicate that dynamics in residues that report on switching adjacent to the myristoyl group are also attenuated in broken-switch mutants. Thus, the strained residues in the protein core appear to form part of the communication network between the proton binding site(s) and the myristoyl group. Apparent pKas of backbone amide protons for I85L and LLL obtained by NMR-monitored pH titrations further support the decoupling of residues adjacent to the myristoyl group from switching. While we have achieved a high resolution and unrivalled look at the mechanism of hisactophilin’s pH-dependent myristoyl switch, we have also shown the validity and utility of chemical shift temperature dependences for characterizing small, functionally relevant local stability changes and gaining insight to the near-native energy landscape and its relation to protein function. Myristoyl switches participate in important cell signalling cascades by forming reversible protein- membrane or protein-protein interactions in response to environmental stimuli. Notwithstanding their prevalence, the high sensitivity and cooperativity of myristoyl switches complicates their study, resulting in poorly understood determinants and mechanisms. By employing thermodynamic measurements and developing a new, general application of an NMR technique, we have developed a detailed picture of the mechanism of a pH-dependent myristoyl switch