40 research outputs found
Solvated interaction energy: from small-molecule to antibody drug design
Scoring functions are ubiquitous in structure-based drug design as an aid to predicting binding modes and estimating binding affinities. Ideally, a scoring function should be broadly applicable, obviating the need to recalibrate and refit its parameters for every new target and class of ligands. Traditionally, drugs have been small molecules, but in recent years biologics, particularly antibodies, have become an increasingly important if not dominant class of therapeutics. This makes the goal of having a transferable scoring function, i.e., one that spans the range of small-molecule to protein ligands, even more challenging. One such broadly applicable scoring function is the Solvated Interaction Energy (SIE), which has been developed and applied in our lab for the last 15Â years, leading to several important applications. This physics-based method arose from efforts to understand the physics governing binding events, with particular care given to the role played by solvation. SIE has been used by us and many independent labs worldwide for virtual screening and discovery of novel small-molecule binders or optimization of known drugs. Moreover, without any retraining, it is found to be transferrable to predictions of antibody-antigen relative binding affinities and as accurate as functions trained on protein-protein binding affinities. SIE has been incorporated in conjunction with other scoring functions into ADAPT (Assisted Design of Antibody and Protein Therapeutics), our platform for affinity modulation of antibodies. Application of ADAPT resulted in the optimization of several antibodies with 10-to-100-fold improvements in binding affinity. Further applications included broadening the specificity of a single-domain antibody to be cross-reactive with virus variants of both SARS-CoV-1 and SARS-CoV-2, and the design of safer antibodies by engineering of a pH switch to make them more selective towards acidic tumors while sparing normal tissues at physiological pH
Molecular dynamics and virtual screening approaches in drug discovery
Computer-aided drug discovery (CADD) methods are now routinely used in the
preclinical phase of drug development. Powerful high-performance computing
facilities and the extremely fast CADD methods constantly scale up the coverage of
drug-like chemical space achievable in rational drug development. In this thesis,
CADD approaches were applied to address several early-phase drug discovery
problems. Namely, small molecule binding site detection on a novel target protein,
virtual screening (VS) of molecular databases, and characterization of small
molecule interactions with metabolic enzymes were studied. Various CADD
methods, including molecular dynamics (MD) simulations in mixed solvents,
molecular docking, and binding free energy calculations, were employed. Co-solvent
MD simulations detected biologically relevant binding sites and provided guidance
for screening potential protein-protein interaction inhibitors for a crucial protein of
the SARS-CoV-2. VS with fragment- and negative image-based (F-NIB) models
identified three active and structurally novel inhibitors of the putative drug target
phosphodiesterase 10A. MD simulations and docking provided detailed insights on
the effects of active site structural flexibility and variation on the binding and
resultant metabolism of small molecules with the cytochrome P450 enzymes. The
results presented in this thesis contribute to the increasing evidence that supports
employment and further development of CADD approaches in drug discovery.
Ultimately, rational drug development coupled with CADD may enable higher
quality drug candidates to the human studies in the future, reducing the risk of
financially and temporally costly clinical failure.
KEYWORDS: Structure-based drug development, Computer-aided drug discovery
(CADD), Molecular dynamics (MD) simulation, Virtual screening (VS), Fragmentand
negative image-based (F-NIB) model, Structure-activity relationship (QSAR),
Cytochrome P450 ligand binding predictionMolekyylidynamiikka- ja virtuaaliseulontamenetelmÀt lÀÀkeaine-etsinnÀssÀ
Tietokoneavusteista lÀÀkeaine-etsintÀÀ kÀytetÀÀn nykyisin yleisesti prekliinisessÀ lÀÀketutkimuksessa. Suurteholaskenta ja ÀÀrimmÀisen nopeat tietokoneavusteiset lÀÀkeaine-etsintÀmenetelmÀt mahdollistavat jatkuvasti kattavamman lÀÀkkeenkaltaisten molekyylien kemiallisen avaruuden seulonnan. TÀssÀ vÀitöskirjassa tietokonepohjaisia menetelmiÀ hyödynnettiin lÀÀketutkimuksen prekliiniseen vaiheeseen liittyvissÀ tyypillisissÀ tutkimusongelmissa. Työhön kuului pienmolekyylien sitoutumisalueiden tunnistus uuden kohdeproteiinin rakenteesta, molekyylitietokantojen virtuaaliseulonta sekÀ pienmolekyylien ja metabolian entsyymien vÀlisten vuorovaikutusten tietokonemallinnus. TyössÀ kÀytettiin useita tietokoneavusteisen lÀÀkeaine-etsinnÀn menetelmiÀ, sisÀltÀen molekyylidynamiikkasimulaatiot (MD-simulaatiot) vaihtuvissa liuottimissa, molekulaarisen telakoinnin ja sitoutumisenergian laskennan. Orgaanisen liuottimen ja veden sekoituksessa tehdyt MD-simulaatiot tunnistivat biologisesti merkittÀviÀ sitoutumisalueita SARS-CoV-2:n tÀrkeÀstÀ proteiinista ja ohjasivat infektioon liittyvÀn proteiini-proteiinivuorovaikutuksen potentiaalisten estÀjien etsintÀÀ. Virtuaaliseulonnalla tunnistettiin kolme rakenteellisesti uudenlaista tunnetun lÀÀkekehityskohteen, fosfodiesteraasi 10A:n, estÀjÀÀ hyödyntÀen fragmentti- ja negatiivikuvamalleja. MD-simulaatiot ja telakointi tuottivat yksityiskohtaista tietoa sytokromi P450 entsyymien aktiivisen kohdan rakenteen jouston ja muutosten vaikutuksesta pienmolekyylien sitoutumiseen ja metaboliaan. TÀmÀn vÀitöskirjan tulokset tukevat kasvavaa todistusaineistoa tietokoneavusteisen lÀÀkeaine-etsinnÀn kÀytön ja kehityksen hyödyllisyydestÀ prekliinisessÀ lÀÀketutkimuksessa. Tietokoneavusteinen lÀÀkeaine-etsintÀ voi lopulta mahdollistaa korkeampilaatuisten lÀÀkekandidaattien pÀÀtymisen ihmiskokeisiin, pienentÀen taloudellisesti ja ajallisesti kalliin kliinisen tutkimuksen epÀonnistumisen riskiÀ.
AVAINSANAT: Rakennepohjainen lÀÀkeainekehitys, Tietokoneavusteinen lÀÀkeaine-etsintÀ, Molekyylidynamiikkasimulaatio (MD-simulaatio), Virtuaaliseulonta, Fragmentti- ja negatiivikuvamalli, Rakenne-aktiivisuussuhde, Sytokromi P450 ligandien sitoutumisen ennustu
Improving Docking Performance Using Negative Image-Based
Despite the large computational costs of molecular docking, the default scoring functions are often unable to recognize the active hits from the inactive molecules in large-scale virtual screening experiments. Thus, even though a correct binding pose might be sampled during the docking, the active compound or its biologically relevant pose is not necessarily given high enough score to arouse the attention. Various rescoring and post-processing approaches have emerged for improving the docking performance. Here, it is shown that the very early enrichment (number of actives scored higher than 1% of the highest ranked decoys) can be improved on average 2.5-fold or even 8.7-fold by comparing the docking-based ligand conformers directly against the target protein's cavity shape and electrostatics. The similarity comparison of the conformers is performed without geometry optimization against the negative image of the target protein's ligand-binding cavity using the negative image-based (NIB) screening protocol. The viability of the NIB rescoring or the R-NiB, pioneered in this study, was tested with 11 target proteins using benchmark libraries. By focusing on the shape/electrostatics complementarity of the ligand-receptor association, the R-NiB is able to improve the early enrichment of docking essentially without adding to the computing cost. By implementing consensus scoring, in which the R-NiB and the original docking scoring are weighted for optimal outcome, the early enrichment is improved to a level that facilitates effective drug discovery. Moreover, the use of equal weight from the original docking scoring and the R-NiB scoring improves the yield in most cases
Structure-based Inhibitor Design for the Antimalarial Target Plasmepsin
Malaria stellt eine der weltweit am weitesten verbreiteten Krankheiten dar und ist nach Tuberkulose und AIDS die bedrohlichste aller Infektionskrankheiten. Heute gibt es eine Reihe von Medikamenten, die auf unterschiedliche Weise gegen Malaria wirken. Durch aufkommende Resistenzentwicklungen in vielen Regionen gegenĂŒber zahlreichen Wirkstoffen, ist es jedoch notwendig geworden, neue Arzneimittel zu entwickeln. Durch die Sequenzierung des Plasmodium falciparum Genoms wurden die Plasmepsine, die Hauptgegenstand dieser Arbeit waren, als neue potentielle Zielstrukturen identifiziert.
Zu Beginn der Arbeit waren bereits eine Reihe potenter, peptidomimetischer Inhibitoren und eine Kokristallstruktur von Plasmepsin II in Komplex mit Pepstatin A in der Literatur beschrieben. Um einen Beitrag im Bereich des strukturbasierten Designs von Plasmepsin Inhibitoren leisten zu können, wurden zunĂ€chst die erforderlichen Rahmenbedingungen geschaffen. Dazu gehörte die Etablierung eines Expressionssystems fĂŒr Plasmepsin II und IV, was im Rahmen einer vorausgegangenen Diplomarbeit geschah. WĂ€hrend der Doktorarbeit ist es gelungen einen Assay zu entwickeln, der die Gesetze der Michaelis-Menten Kinetik befolgt. Weiterhin wurde versucht, ein funktionsfĂ€higes Kristallisationssystem fĂŒr Plasmespin II zu entwickeln, das die AufklĂ€rung von Protein-Ligand-Bindungsmoden mittels Röntgenbeugungsexperimenten ermöglichen sollte. Obwohl die Reproduktion der in der Literatur bekannten Kristallstruktur mit Pepstatin A gelang, erwies es sich im Verlauf der Arbeit als schwierig, Kristalle des Zielproteins in Komplex mit neuartigen Inhibitoren zu erhalten.
Nach Etablierung der fĂŒr das strukturbasierte Liganden-Design erforderlichen Methoden galt es in Zusammenarbeit mit synthetisch arbeitenden Gruppen, Inhibitoren mit neuartigen, nichtpeptidomimetischen Eigenschaften zu entwerfen und zu optimieren.
Erste Modelling-Studien ergaben, dass 2,3,4,7-Tetrahydro-1H-Azepin ein geeignetes GrundgerĂŒst fĂŒr die Entwicklung von Plasmepsin II und IV Inhibitoren darstellen könnte. Durch eine Kombination aus einer Datenbankanalyse mit dem Programm Cavbase und einem kombinatorischen Docking mit FlexXC wurden erste Liganden vorgeschlagen, die sich im Enzymassay als Hemmstoffe fĂŒr Plasmepsin II und IV mit Ki-Werten im niedrig mikromolaren Bereich erwiesen. Eine ausgiebige Analyse theoretisch möglicher Wechselwirkungen zum Protein fĂŒhrte zu VorschlĂ€gen fĂŒr weitere Inhibitoren, wodurch die AffinitĂ€tsdaten bis in den nanomolaren Bereich verbessert werden konnten. Insgesamt standen die experimentell bestimmten Ki Werte von 13 Inhibitoren gut im Einklang mit der Design Hypothese und die beobachteten Struktur-Wirkbeziehungen stĂŒtzen dabei den vorhergesagten Bindungsmodus.
In einem weiteren Ansatz wurden Pyrrolidin Derivate, die ursprĂŒnglich als HIV-1 Protease Inhibitoren synthetisiert wurden, auf Plasmepsin-Hemmung getestet. In der Literatur sind einige FĂ€lle beschrieben, bei denen HIV-1 Protease Inhibitoren gleichzeitig gute Hemmstoffe fĂŒr Plasmepsine darstellen. Von insgesamt zwölf MolekĂŒlen zeigten die affinsten Liganden Ki Werte im nanomolaren Bereich. FĂŒr zwei Verbindungsklassen (Pyrrolidin-diol-diester und Pyrrolidindiamide) gelang es, plausible Bindungsmoden zu generieren. Mit den vorhandenen kristallographischen Informationen war es jedoch nicht möglich, einen sinnvollen Bindungsmodus fĂŒr Pyrrolidindimethylenediamin-Derivate, die die dritte Verbindungsklasse darstellen, vorherzusagen. Da die Plasmepsine als sehr flexible Proteine bekannt sind, wurden MolekĂŒldynamik (MD) Simulationen durchgefĂŒhrt und mobile Bereiche innerhalb der Bindetasche genauer untersucht. Unter BerĂŒcksichtigung der Ergebnisse der MD Simulation konnte so ein Bindungsmodus fĂŒr den Typ der Pyrrolidindimethylenediamin-Derivate erzeugt werden. Auch bei der AffinitĂ€tsdaten-Analyse einer Norstatin-Bibliothek, bei der die Verbindungen sich nur im P1-Substituenten unterschieden, konnten zunĂ€chst ĂŒberraschende Ergebnisse mit Hilfe der MD Simulation erklĂ€rt werden. Eine konformative Ăffnung der Plasmepsin II S1 Tasche wurde beobachtet, wodurch eine Anpassung an sperrige P1 Substituenten ermöglicht wird. So wurden durch die durchgefĂŒhrte MD Simulation zahlreiche Erkenntnisse gewonnen, die von Relevanz fĂŒr das strukturbasierte Wirkstoffdesign sind.
Ausgehend von den Azepin Derivaten wurde in dieser Arbeit ein dritter Ansatz verfolgt, Plasmepsin Inhibitoren mit neuartigen GrundgerĂŒsten zu entwickeln. Zu diesem Zweck wurde Ftrees (Feature Trees) verwendet, ein Computerprogramm, das durch seine Funktionsweise einen Ăbergang von einem SuchmolekĂŒl zu strukturell unĂ€hnlichen MolekĂŒlen âScaffold Hoppingâ ermöglichen soll. Eine Verbindung mit einem ThiophengrundgerĂŒst fiel bei der Auswertung als besonders interessant auf und wurde auf die Plasmepsine getestet. AnfĂ€ngliche Ki Werte im zweistellig mikromolaren Bereich konnten durch gezieltes Design um den Faktor 110 verbessert werden, was zu ersten Inhibitoren im nanomolaren Bereich fĂŒhrte.
Insgesamt gelang es in dieser Arbeit durch unterschiedliche AnsĂ€tze Hemmstoffe fĂŒr Plasmepsin II und IV vorherzusagen, bei denen die Struktur-Wirkbeziehungen mit den erzeugten Bindungsmoden weitestgehend ĂŒbereinstimmten. Strukturlösungsversuche gelangen nur mit Pepstatin A, MolekĂŒldynamiksimulationen gewĂ€hrten dennoch einen Einblick in die hohe FlexibilitĂ€t und stabile Konformationen der Plasmepsine. Daraus wurden Erkenntnisse fĂŒr das strukturbasierte Wirkstoffdesign gewonnen, was zur Entwicklung neuer Malaria-Medikamente einen Beitrag leisten könnte
Computational Approaches: Drug Discovery and Design in Medicinal Chemistry and Bioinformatics
This book is a collection of original research articles in the field of computer-aided drug design. It reports the use of current and validated computational approaches applied to drug discovery as well as the development of new computational tools to identify new and more potent drugs
Theoretical screening of organic conjugated materials
As the urgency to address the effects of climate change increases, so does the need to discover new materials for the generation of renewable energy, to replace the environmentally dam- aging combustion of fossil fuels. Two main areas of research are focused on the design and discovery of photoactive materials for photovoltaics and for photocatalysts for water splitting. Although there are currently high-efficiency photovoltaics commercially available, there is mo- tivation to replace them with materials with non-toxic and earth-abundant compositions. As they generally meet these criteria, organic conjugated materials are very desirable candidates for these applications. Computational chemistry methods can accelerate materials discovery, eliminating the need to synthesize large libraries of molecules in the preliminary screening stages. Both high-accuracy, expensive methods and fast, cheap, lower-accuracy methods have their merits and in conjunction with one another can provide a detailed and informative description of chemical systems. The high-throughput virtual screening methodology used throughout this thesis provides the opportunity to efficiently explore property space and high- light potential candidates for given applications, such as polymeric photocatalysts, organic photovoltaics and dye sensitizers in solar cells. In this thesis this methodology is explored for a small aromatic molecules, diketopyrrolopyrrole-based dyes and both ordered and disordered polymers. Through the high-throughput virtual screening, large datasets of chemical com- pounds were investigated and analysed, highlighting the patterns in the optical and electronic properties influenced by building block sequence, conformerism and composition. The use of high-accuracy, expensive methods is also explored in this thesis, demonstrating the difficulties in pushing such methods to larger chemical structures
IN SILICO METHODS FOR DRUG DESIGN AND DISCOVERY
Computer-aided drug design (CADD) methodologies are playing an ever-increasing role in drug discovery that are critical in the cost-effective identification of promising drug candidates. These computational methods are relevant in limiting the use of animal models in pharmacological research, for aiding the rational design of novel and safe drug candidates, and for repositioning marketed drugs, supporting medicinal chemists and pharmacologists during the drug discovery trajectory.Within this field of research, we launched a Research Topic in Frontiers in Chemistry in March 2019 entitled âIn silico Methods for Drug Design and Discovery,â which involved two sections of the journal: Medicinal and Pharmaceutical Chemistry and Theoretical and Computational Chemistry. For the reasons mentioned, this Research Topic attracted the attention of scientists and received a large number of submitted manuscripts. Among them 27 Original Research articles, five Review articles, and two Perspective articles have been published within the Research Topic. The Original Research articles cover most of the topics in CADD, reporting advanced in silico methods in drug discovery, while the Review articles offer a point of view of some computer-driven techniques applied to drug research. Finally, the Perspective articles provide a vision of specific computational approaches with an outlook in the modern era of CADD
Computational methods applied to drug discovery: the rational design of dual inhibitors of FAAH and COX
The search for effective and safe drugs in pain-relief treatment represents a great challenge for medicinal chemists. Lipid derived mediators, such as endocannabinoids, may have different roles as agonists of cannabinoid receptors, relieving pain, or as substrates of cyclooxygenase (COX), generating the pro-inflammatory prostamides. Moreover, the tissue-protective endocannabinoid anandamide is metabolised by fatty acid amide hydrolase (FAAH). Therefore, a new challenging approach in pain-relief might be the development of dual action FAAH/COX inhibitors.
The purpose of this thesis is to apply computational methods in drug discovery to assist medicinal chemistry studies targeting the rational design of novel FAAH/COX inhibitors, and to exploit structural studies relative to two side projects on other biological targets.
The wider project of this thesis explores the mechanism of action and the rational design of novel FAAH/COX dual inhibitors. The reversible mixed type inhibitors Flu-AM1 and Ibu-AM5, derivatives of flurbiprofen and ibuprofen, respectively, retain similar COX inhibitory properties and are more potent FAAH inhibitors than the parent compounds. Applying a combination of molecular docking, MD simulations and free energy evaluation of the ligand-receptor complex, the binding mode of the enantiomer forms of Flu-AM1 and Ibu-AM5 has been found in the substrate access channel of FAAH
and has been supported by studies of site-directed mutagenesis. The substitution of the isobutyl group of Ibu-AM5 with 4-(2-(trifluoromethyl)pyridin-4-yl)amino group led to the design of TPA5 derivative, which showed an inhibitory activity (IC50 = 0.59 ÎŒM) similar to the lead compound (Ibu-AM5, IC50 = 0.52 ÎŒM). Kinetic studies of TPA5
revealed that it is a pure competitive inhibitor of rat FAAH and molecular modeling studies supported a binding mode that overlap the anandamide analog MAFP. Among TPA5 derivatives, compound TPA27 exhibited a 10-fold enhancement in the inhibitory profile against FAAH (IC50 = 0.058 ÎŒM).
Thermodynamic Integration calculations performed to complete the transformation of TPA5 in TPA27 yielded a free energy difference of 0.3 kcal/mol, which indicates a slight lower affinity of TPA27 with respect to TPA5, in the competitive binding site. Kinetics studies showed that TPA27
could be considered the first non-competitive reversible FAAH inhibitor reported so far, and that it more likely binds to an allosteric site.
Differences in the inhibitory potency against rat and mouse FAAH for all compounds studied suggested different aminoacid composition of both competitive and non-competitive binding sites. This information was used as
criteria of selection for a putative allosteric site found between the cytosolic port and the interface of the FAAH monomers. Computational studies in the allosteric site allowed the definition of the binding mode of Ibu-AM5 and TPA27. Nevertheless, a series of derivatives of Ibu-AM5 and Flu-AM1 were designed in order to get more information on the structure-activity relationships, leading to the identification of novel derivatives with improved activity against FAAH (Ibu-AM56, IC50 = 0.08 ÎŒM; Ibu-AM57, IC50 = 0.1
ÎŒM; Flu-AM3, IC50 = 0.02 ÎŒM.
Finally, the thesis also reports the results of two other projects: i) the design of potential anticancer peptides that interfere in the formation of the tetrameric complex hUbA1/UbcH10/Ub2, key intermediate of the ubiquitination
cascade.; ii) structural studies on the hybridization of PNA of different length with miR-509-3p, involved in regulating the expression of the CFTR gene, as a way to validate a potential new strategy for the treatment of Cystic Fibrosis
Do All Roads Really Lead to Rome? Learnings from Comparative Analysis using SPR, NMR, & X-Ray Crystallography to Optimize Fragment Screening in Drug Discovery.
There are several biophysical methods developed to rapidly identify weakly binding fragments to a
target protein. X-ray crystallography provides structural information that is crucial for fragment
optimization, however there are several criteria that must be met for a successful fragment screening
including the production of soakable and well-diffracting crystals. Therefore, having a reliable cascade
of screening methods to be used as pre-screens prior to labor-intensive X-ray crystallography would
be extremely beneficial. This would allow the filtering of compounds as the screening progresses so
that only the most promising hits remain. But which method should be the one to start the screening
cascade? In this work, various sets of fragment libraries were screened against three different proteins;
namely tRNA guanine transglycosylase (TGT) an important protein in Shigella, membrane associated
protein peroxin 14 (PEX14) of T. Brucei, and endothiapepsin (EP), to investigate whether different
screening methods will reveal similar collections of putative binders. The detailed comparative analysis
of the findings obtained by the different methods is discussed in this thesis.
Shigellosis, an acute bacterial infection of the intestine, is caused by the gram-negative Shigella
bacterium whose pathogenicity is reliant on virulence factors (VirF) required to invade epithelial cells.
The expression of these VirF is modulated by TGT. Strategies developed to inhibit TGT include potent
active-site inhibitors to block the binding of tRNA, thereby preventing the transcription of the virulence
factors. Our 96-fragment library was screened against TGT using SPR, NMR, and X-ray crystallography,
as described in Chapter 2. A total of 81 fragments were screened in SPR using a direct binding assay
approach, revealing a hit rate of 12%. A total of 77 fragments were screened in NMR revealing a hit
rate of 29%. High-resolution crystal structures were also collected for the entire fragment library by
soaking, revealing a hit rate of 8%. Upon comparison of all discovered fragment hits no overlaps from
all three methods were found. Several factors are responsible for this finding such as exclusion of
fragments from individual screens due to technical reasons. In detail, four X-ray hits were excluded
from the SPR and NMR screens, two SPR hits were discarded from the NMR screen, and five NMR hits
were never subjected to the SPR screen. SPR and NMR are currently the most commonly applied
primary fragment screening techniques, however, our results suggest that if they would have been
applied as incipient methods of a screening cascade, they would have missed three binders discovered
by a subsequently applied, more elaborate crystallographic screen. X-ray crystallography allows the
detection of specific binders that may be too weak binders to be detected by SPR and even by NMR
but can still provide valid structural information to support the search for appropriate starting points
in lead discovery. Additionally, MD simulations of the apo wild type TGT have predicted the opening
of a transient sub-pocket located above the guanine/preQ1 pocket, which suggested a strategy to
target this new binding site for the design of new inhibitors against TGT following a structure- based
drug design concept which is also discussed in section 2.3.
The human African trypanosomiasis (HAT), also known as the sleeping sickness, is a vector-borne
parasitic disease caused by T. brucei and transmitted to humans by bites of the tsetse fly. T. brucei
lacks feedback allosteric regulation of early steps in glycolysis but compartmentalizes the relevant
enzymes within organelles called glycosomes. PEX14, a peroxin protein essential for biogenesis of
glycosomes, forms an important protein-protein interaction with PEX5, an import receptor that
transports cytoplasmic glycosomal enzymes into the organelle. Disrupting the PEX14/PEX5 interaction
leads to the accumulation of glycosomal enzymes in the cytosol, depletion of ATP, glucose toxicity,
metabolic collapse and death of T. brucei. This disruption can be achieved through small molecules
that bind to and block PEX14, preventing PEX5 binding. A previous NMR screening of a fragment library
resulted in fragment hits that bind to the N-terminal domain (NTD) of T. brucei PEX14. In this project,
we attempted to validate these hits through X-ray crystallography by soaking, to allow visualization of
the fragment interactions. The promising fragment hits would then be optimized into more potent
lead compounds. Crystallization of the NTD PEX14 with a mutation in the first residue (E1W) revealed
blocked binding pockets, as described in Chapter 3. The purpose of the added tryptophan was to
render fluorescent properties to the short NTD construct which lacked fluorescent amino acids.
However, this tryptophan was found to block the binding pockets of its neighboring crystal mates in
the protein crystal, rendering a crystal form impossible to use for soaking. Attempts to find new crystal
forms with free pockets were unsuccessful, as the small size of the protein and the hydrophobic nature
of tryptophan rendered tightly packed protein crystals that block the binding pockets of neighboring
crystal mates. Virtual Screening to discover novel ligands for co-crystallization revealed a ligand that
aids the crystallization of the E1W PEX14 variant in the same space group but with a slightly different
packing. This produced a crystal form that proved successful for fragment soaking as it enabled the
binding of two additional fragment hits binding to further protein pockets. Additionally, the wild type
form of PEX14 which lacks the tryptophan residue and thus has free binding pockets was crystallized.
This enabled the soaking of a previously designed lead compound in different pockets of the PEX5
binding site. By obtaining a crystal structure of this complex at a resolution of 1.8 Ă
, the feasibility of
using wild type PEX14 crystals for further fragment screening has been demonstrated.
Endothiapepsin is a member of the pepsin-like aspartic proteases responsible for the hydrolytic
cleavage of peptide substrates. Owing to its high degree of similarity to other pharmacologically
relevant aspartic proteases, it has served as the model enzyme for studying their mechanism and to
discover first lead structures. In previous work done by other members from our group to identify and
characterize endothiapepsin binders, X-ray crystallography was consulted as a primary fragment
screening method and its hit identification potential was compared to several biochemical and
biophysical screening methods. The fragment library screened was designed for general purposes and
contained 361 entries. Comparison of the overlap in the hit rates of the different methods to that of
X-ray crystallography revealed a low overlap, with the RDA having the highest overlap at 7% and MS
having the lowest overlap at 1% followed by STD NMR at 3%. To understand the reason behind the low
overlap, two of these screening techniques were prioritized for closer analysis as described in Chapter
4. The 71 X-ray detected fragment hits were selected and rescreened again with STD NMR under
slightly different buffer conditions, in addition to WaterLOGSY NMR experiments. The second STD NMR
screen detected almost double the amount of hits as the initial one, and the Water LOGSY screen had
the highest correlation from the NMR methods to the X-ray hits at 69%. This comparative analysis also
revealed the phenomena of active site fragment displacement by use of so-called reporter ligands and
that non-deuterated water in STD NMR may lead to false negatives. The entire 361 fragment library
was also screened with SPR using an inhibition in solution assay, adding another biophysical method
for our comparative analysis to give us further insight of which conditions are crucial to maintain while
transferring across different techniques. The resulting hit rate from SPR was 34%, correlating to an
overlap of 11% with the X-ray hits - the highest correlation between screening methods reported by
us thus far. Finally, we also studied fragment detection and cocktailing in crystallography in comparison
to fragment cocktailing in NMR. From this we concluded that cocktailing in crystallography can also
lead to false negatives due to fragment competitive behavior and can reveal a different binding mode
for a given fragment compared to the adopted geometry found when soaked individually. As for NMR,
despite the ability to detect competitive binding of fragments due to the temporary binding and
unbinding events, the parallel binding and thus detection of fragments is not always guaranteed as
seen in 20% of the fragments we screened, in addition to our observation that the detection of
fragments in cocktail NMR may also depend on the comparison of the cocktail set they are a part of