This document discusses various NMR techniques for ligand screening in drug discovery. It begins by providing background on the increasing role of NMR in drug research due to its ability to sensitively detect molecular interactions and provide structural information. The document then reviews both ligand-observed and target-observed NMR screening techniques, describing methods based on changes in molecular diffusion, relaxation, and intramolecular or intermolecular magnetization transfer upon ligand binding. Specific techniques discussed include saturation transfer difference (STD) NMR, transferred nuclear Overhauser effect (trNOE), and NOE pumping. The review concludes by noting the dual importance of NMR for drug screening and structure-based drug design.
Limitations of in silico drug discovery methodsAlichy Sowmya
In drug discovery there are various in silico approaches such as Virtual high throughput screening, Molecular docking, Homology modelling, QSAR, CoMFA, Molecular Dynamics, and Pharmacophore mapping. In this presentation various limitations of these approaches are given
Limitations of in silico drug discovery methodsAlichy Sowmya
In drug discovery there are various in silico approaches such as Virtual high throughput screening, Molecular docking, Homology modelling, QSAR, CoMFA, Molecular Dynamics, and Pharmacophore mapping. In this presentation various limitations of these approaches are given
PRESENTED BY: HARSHPAL SINGH WAHI, SHIKHA D. POPALI
USEFUL FOR PHARMACY STUDENTS AND ACADEMICS, INDUSTRIALS FOR MOLECULE DEVELOPMENT, MODELING, DRUG DISCOVERY, COMPUTATIONAL TOOLS, MOLECULAR DOCKING ITS TYPES, FACTORS AFFECTING, DIFFERENT STAGES, QSAR ADVANTAGES, NEED
Gel matrix dependence on the dose response properties and diffusion phenomena...inventionjournals
Fricke-gel dosimeters proved to be suitable tools to perform 3D radiotherapy pre-treatment dosimetry. The tissue equivalent gel matrix helpsto preserve the spatial information of the dose. Several gel matrices proved to be suitable for dosimetric purposes. The influence of the gel matrices on both system dose response and diffusion processwasinvestigated. Three gel matrices were considered: Gelatinfrom porcine skin, Agaroseand polyvinyl alcohol (PVA) cross-linked with glutaraldehyde(GTA). In these systems, Xylenol-Orange (XO), an iron(III)chelator, forms red-colored complexeswith Fe3+ that eases the optical determination of the dose. However, the dose evaluationresults to be affectedbydifferent XO-Fe3+ complexes that absorb at different wavelengths. In particular,this phenomenon influences the dose response, the calibration curveand the dose threshold.Therefore, a deeper study of the XO-Fe3+ speciation mechanism could lead to a more accurate evaluation of the dose. A novel procedure, based on a laser-beam irradiation, was implemented for the diffusion process evaluation. The diffusion coefficients were calculated for the three gel matrices tested. PVAmatrix proved to highly limit the diffusion with respect to the other matrices. Further investigations are needed to verify the influence of XO-Fe3+complexeson the diffusion phenomenon.
Computer Added Drug Design is one of the latest technology of medicine world. This short slide will help you to know a little about CADD.If you want to know a vast plz go throw the reference book.
Superimposition method- ligand based drug designIshpreet Sachdev
superimposition or alignment methods used in the ligand based drug designing approach.classification of superimposition Rigid alignment method semiflexible alignment method and flexible method. application of superimposition method in pharmacoinformatics or bioinformatics
Simultaneous loading of 5-florouracil and SPIONs in HSA nanoparticles: Optimi...Nanomedicine Journal (NMJ)
Objective(s): Over the past two decades, considerable interest has been focused on utilizing biocompatible magnetic nanoparticles (MNPs) for biomedical applications. In this study, production of human serum albumin (HSA) nanoparticles using desolvation technique that were simultaneous loaded with high amounts of superparamagnetic iron oxide nanoparticles (SPIONs) and 5-flourouracil (5-FU) was investigated.
Materials and Methods: 5-FU loading (%) and SPIONs entrapment efficiency (%) were optimized using response surface methodology (RSM). The design expert software used to analyse the interactive effects of pH, 5-FU and SPIONs concentrations.
Results:The optimum conditions found to be pH of 8.2, drug concentration of 1.5 mg/ml and SPIONs concentration of 2.79 mg/ml. Under the mentioned optimum conditions, particles with the size of 111.8 nm, zeta potential of -37.1 mV, 5-FU loading of 15.8% and SPIONs entrapment efficiency of 41.1% were obtained. In vitro cumulative release of 5-FU from the nanoparticles was evaluated in phosphate buffer saline (pH 7.4, 37 °C). Results indicated that 85% of the 5-FU released during 95 h, which revealed a sustained release profile. In addition, Vibrating Sample Magnetometer (VSM) analyses confirmed the superparamagnetic properties of magnetic albumin nanoparticles manufactured under the optimum conditions.
Conclusion: According to the findings,SPIONs and 5-FU loaded HAS nanoparticles arepromising for use as novel targeted delivery system due to proper magnetic and drug release behaviours.
Image analysis and Laser Particle Diffraction study of ProRoot-MTA, Portland ...CrimsonPublishersRDMS
Image analysis and Laser Particle Diffraction study of ProRoot-MTA, Portland cement and Bismuth Oxide-A comparative study by MMA Rafique* in Crimson Publishers: Peer Reviewed Material Science Journals
Cadd and molecular modeling for M.PharmShikha Popali
THE CADD IS FOR THE DRUG DEVELOPMENT THE DIFFERENT STRATEGIES ARE MENTIONED LIKE QSAR MOLECULAR DOCKING, THE DIFFERENT DIMNSIONAL FORMS OF QSAR , THE ADVANCE SAR of it.
PRESENTED BY: HARSHPAL SINGH WAHI, SHIKHA D. POPALI
USEFUL FOR PHARMACY STUDENTS AND ACADEMICS, INDUSTRIALS FOR MOLECULE DEVELOPMENT, MODELING, DRUG DISCOVERY, COMPUTATIONAL TOOLS, MOLECULAR DOCKING ITS TYPES, FACTORS AFFECTING, DIFFERENT STAGES, QSAR ADVANTAGES, NEED
Gel matrix dependence on the dose response properties and diffusion phenomena...inventionjournals
Fricke-gel dosimeters proved to be suitable tools to perform 3D radiotherapy pre-treatment dosimetry. The tissue equivalent gel matrix helpsto preserve the spatial information of the dose. Several gel matrices proved to be suitable for dosimetric purposes. The influence of the gel matrices on both system dose response and diffusion processwasinvestigated. Three gel matrices were considered: Gelatinfrom porcine skin, Agaroseand polyvinyl alcohol (PVA) cross-linked with glutaraldehyde(GTA). In these systems, Xylenol-Orange (XO), an iron(III)chelator, forms red-colored complexeswith Fe3+ that eases the optical determination of the dose. However, the dose evaluationresults to be affectedbydifferent XO-Fe3+ complexes that absorb at different wavelengths. In particular,this phenomenon influences the dose response, the calibration curveand the dose threshold.Therefore, a deeper study of the XO-Fe3+ speciation mechanism could lead to a more accurate evaluation of the dose. A novel procedure, based on a laser-beam irradiation, was implemented for the diffusion process evaluation. The diffusion coefficients were calculated for the three gel matrices tested. PVAmatrix proved to highly limit the diffusion with respect to the other matrices. Further investigations are needed to verify the influence of XO-Fe3+complexeson the diffusion phenomenon.
Computer Added Drug Design is one of the latest technology of medicine world. This short slide will help you to know a little about CADD.If you want to know a vast plz go throw the reference book.
Superimposition method- ligand based drug designIshpreet Sachdev
superimposition or alignment methods used in the ligand based drug designing approach.classification of superimposition Rigid alignment method semiflexible alignment method and flexible method. application of superimposition method in pharmacoinformatics or bioinformatics
Simultaneous loading of 5-florouracil and SPIONs in HSA nanoparticles: Optimi...Nanomedicine Journal (NMJ)
Objective(s): Over the past two decades, considerable interest has been focused on utilizing biocompatible magnetic nanoparticles (MNPs) for biomedical applications. In this study, production of human serum albumin (HSA) nanoparticles using desolvation technique that were simultaneous loaded with high amounts of superparamagnetic iron oxide nanoparticles (SPIONs) and 5-flourouracil (5-FU) was investigated.
Materials and Methods: 5-FU loading (%) and SPIONs entrapment efficiency (%) were optimized using response surface methodology (RSM). The design expert software used to analyse the interactive effects of pH, 5-FU and SPIONs concentrations.
Results:The optimum conditions found to be pH of 8.2, drug concentration of 1.5 mg/ml and SPIONs concentration of 2.79 mg/ml. Under the mentioned optimum conditions, particles with the size of 111.8 nm, zeta potential of -37.1 mV, 5-FU loading of 15.8% and SPIONs entrapment efficiency of 41.1% were obtained. In vitro cumulative release of 5-FU from the nanoparticles was evaluated in phosphate buffer saline (pH 7.4, 37 °C). Results indicated that 85% of the 5-FU released during 95 h, which revealed a sustained release profile. In addition, Vibrating Sample Magnetometer (VSM) analyses confirmed the superparamagnetic properties of magnetic albumin nanoparticles manufactured under the optimum conditions.
Conclusion: According to the findings,SPIONs and 5-FU loaded HAS nanoparticles arepromising for use as novel targeted delivery system due to proper magnetic and drug release behaviours.
Image analysis and Laser Particle Diffraction study of ProRoot-MTA, Portland ...CrimsonPublishersRDMS
Image analysis and Laser Particle Diffraction study of ProRoot-MTA, Portland cement and Bismuth Oxide-A comparative study by MMA Rafique* in Crimson Publishers: Peer Reviewed Material Science Journals
Cadd and molecular modeling for M.PharmShikha Popali
THE CADD IS FOR THE DRUG DEVELOPMENT THE DIFFERENT STRATEGIES ARE MENTIONED LIKE QSAR MOLECULAR DOCKING, THE DIFFERENT DIMNSIONAL FORMS OF QSAR , THE ADVANCE SAR of it.
SAR BY NMR (Structure Activity Relationship by Using NMR)SAKEEL AHMED
SAR by NMR is the Nuclear Magnetic Resonance (NMR) based method in which small organic molecules that bind to the proximal site are identified, optimized and finally linked together to produce high-affinity ligands.
It is called “SAR by NMR” because the structure-activity relationship (SAR) is obtained by the Nuclear Magnetic Resonance (NMR).
It is based on the fragments approaches to drug design.
With this technique, compounds with nanomolar affinity for a target protein can be rapidly discovered by tethering two ligands with micromolar affinities.
The method reduces the chemical synthesis and the time required for the discovery of high-affinity ligands and is particularly useful in target-directed drugs research.
Fragment screening library workshop (IQPC 2008)Peter Kenny
I also ran a workshop on selection of compounds for fragment screening just before the 2008 IQPC compound library conference and these are the slides I used.
Characterizing aptamer small molecule interactions with back-scattering inter...Melodie Benford
New paper resulting from joint collaboration with Base Pair Bio and the Bornhop group at Vanderbilt University. Using aptamers and back scattering interferometry (BSI) to detect small molecules, such as TFV, BPA, and norepinephrine.
Crimson Publishers-Potential Application of Raman Micro-Spectroscopy as an In...CrimsonPublishersMAPP
Potential Application of Raman Micro-Spectroscopy as an In vitro Drug Screening and Companion Diagnostic Tool for Clinical Application: Chemotherapeutic Drug Mechanism of Action, Cellular Effects and Resistance by Z Farhane in Modern Applications in Pharmacy & Pharmacology
About NMR, Fundamental Principle and Theory, Instrumentation, Solvent, Chemical Shift, Factor Affecting Chemical Shift, Spin-spin Coupling, Application of NMR, Reference, Acknowledgment
SSNM is more precise, effective, and sensitive. It is one of the most powerful techniques for structural elucidating solids and other compounds. Whereas in the solution NMR, there are limitations for some solids. So SSNMR is in high demand in various areas.
Proteomics Practical (NMR and Protein 3D softwareiqraakbar8
Nuclear magnetic resonance (NMR) is a physical phenomenon in which nuclei in a strong constant magnetic field are perturbed by a weak oscillating magnetic field (in the near field) and respond by producing an electromagnetic signal with a frequency characteristic of the magnetic field at the nucleus.
Two dimensional Nuclear Magnetic Resonance (2D NMR) refers to a set of multi pulse techniques which were introduced to overcome the complex spectra obtained with NMR.
It is a set of NMR methods which give data plotted in a space defined by two frequency axes rather than one.
Design of fragment screening libraries (IQPC 2008)Peter Kenny
These were the slides that I used for the 2008 IQPC compound libraries conference which was the first external lecture on fragment screening libraries.
GDG Cloud Southlake #33: Boule & Rebala: Effective AppSec in SDLC using Deplo...James Anderson
Effective Application Security in Software Delivery lifecycle using Deployment Firewall and DBOM
The modern software delivery process (or the CI/CD process) includes many tools, distributed teams, open-source code, and cloud platforms. Constant focus on speed to release software to market, along with the traditional slow and manual security checks has caused gaps in continuous security as an important piece in the software supply chain. Today organizations feel more susceptible to external and internal cyber threats due to the vast attack surface in their applications supply chain and the lack of end-to-end governance and risk management.
The software team must secure its software delivery process to avoid vulnerability and security breaches. This needs to be achieved with existing tool chains and without extensive rework of the delivery processes. This talk will present strategies and techniques for providing visibility into the true risk of the existing vulnerabilities, preventing the introduction of security issues in the software, resolving vulnerabilities in production environments quickly, and capturing the deployment bill of materials (DBOM).
Speakers:
Bob Boule
Robert Boule is a technology enthusiast with PASSION for technology and making things work along with a knack for helping others understand how things work. He comes with around 20 years of solution engineering experience in application security, software continuous delivery, and SaaS platforms. He is known for his dynamic presentations in CI/CD and application security integrated in software delivery lifecycle.
Gopinath Rebala
Gopinath Rebala is the CTO of OpsMx, where he has overall responsibility for the machine learning and data processing architectures for Secure Software Delivery. Gopi also has a strong connection with our customers, leading design and architecture for strategic implementations. Gopi is a frequent speaker and well-known leader in continuous delivery and integrating security into software delivery.
SAP Sapphire 2024 - ASUG301 building better apps with SAP Fiori.pdfPeter Spielvogel
Building better applications for business users with SAP Fiori.
• What is SAP Fiori and why it matters to you
• How a better user experience drives measurable business benefits
• How to get started with SAP Fiori today
• How SAP Fiori elements accelerates application development
• How SAP Build Code includes SAP Fiori tools and other generative artificial intelligence capabilities
• How SAP Fiori paves the way for using AI in SAP apps
Welocme to ViralQR, your best QR code generator.ViralQR
Welcome to ViralQR, your best QR code generator available on the market!
At ViralQR, we design static and dynamic QR codes. Our mission is to make business operations easier and customer engagement more powerful through the use of QR technology. Be it a small-scale business or a huge enterprise, our easy-to-use platform provides multiple choices that can be tailored according to your company's branding and marketing strategies.
Our Vision
We are here to make the process of creating QR codes easy and smooth, thus enhancing customer interaction and making business more fluid. We very strongly believe in the ability of QR codes to change the world for businesses in their interaction with customers and are set on making that technology accessible and usable far and wide.
Our Achievements
Ever since its inception, we have successfully served many clients by offering QR codes in their marketing, service delivery, and collection of feedback across various industries. Our platform has been recognized for its ease of use and amazing features, which helped a business to make QR codes.
Our Services
At ViralQR, here is a comprehensive suite of services that caters to your very needs:
Static QR Codes: Create free static QR codes. These QR codes are able to store significant information such as URLs, vCards, plain text, emails and SMS, Wi-Fi credentials, and Bitcoin addresses.
Dynamic QR codes: These also have all the advanced features but are subscription-based. They can directly link to PDF files, images, micro-landing pages, social accounts, review forms, business pages, and applications. In addition, they can be branded with CTAs, frames, patterns, colors, and logos to enhance your branding.
Pricing and Packages
Additionally, there is a 14-day free offer to ViralQR, which is an exceptional opportunity for new users to take a feel of this platform. One can easily subscribe from there and experience the full dynamic of using QR codes. The subscription plans are not only meant for business; they are priced very flexibly so that literally every business could afford to benefit from our service.
Why choose us?
ViralQR will provide services for marketing, advertising, catering, retail, and the like. The QR codes can be posted on fliers, packaging, merchandise, and banners, as well as to substitute for cash and cards in a restaurant or coffee shop. With QR codes integrated into your business, improve customer engagement and streamline operations.
Comprehensive Analytics
Subscribers of ViralQR receive detailed analytics and tracking tools in light of having a view of the core values of QR code performance. Our analytics dashboard shows aggregate views and unique views, as well as detailed information about each impression, including time, device, browser, and estimated location by city and country.
So, thank you for choosing ViralQR; we have an offer of nothing but the best in terms of QR code services to meet business diversity!
Builder.ai Founder Sachin Dev Duggal's Strategic Approach to Create an Innova...Ramesh Iyer
In today's fast-changing business world, Companies that adapt and embrace new ideas often need help to keep up with the competition. However, fostering a culture of innovation takes much work. It takes vision, leadership and willingness to take risks in the right proportion. Sachin Dev Duggal, co-founder of Builder.ai, has perfected the art of this balance, creating a company culture where creativity and growth are nurtured at each stage.
The Art of the Pitch: WordPress Relationships and SalesLaura Byrne
Clients don’t know what they don’t know. What web solutions are right for them? How does WordPress come into the picture? How do you make sure you understand scope and timeline? What do you do if sometime changes?
All these questions and more will be explored as we talk about matching clients’ needs with what your agency offers without pulling teeth or pulling your hair out. Practical tips, and strategies for successful relationship building that leads to closing the deal.
Securing your Kubernetes cluster_ a step-by-step guide to success !KatiaHIMEUR1
Today, after several years of existence, an extremely active community and an ultra-dynamic ecosystem, Kubernetes has established itself as the de facto standard in container orchestration. Thanks to a wide range of managed services, it has never been so easy to set up a ready-to-use Kubernetes cluster.
However, this ease of use means that the subject of security in Kubernetes is often left for later, or even neglected. This exposes companies to significant risks.
In this talk, I'll show you step-by-step how to secure your Kubernetes cluster for greater peace of mind and reliability.
Observability Concepts EVERY Developer Should Know -- DeveloperWeek Europe.pdfPaige Cruz
Monitoring and observability aren’t traditionally found in software curriculums and many of us cobble this knowledge together from whatever vendor or ecosystem we were first introduced to and whatever is a part of your current company’s observability stack.
While the dev and ops silo continues to crumble….many organizations still relegate monitoring & observability as the purview of ops, infra and SRE teams. This is a mistake - achieving a highly observable system requires collaboration up and down the stack.
I, a former op, would like to extend an invitation to all application developers to join the observability party will share these foundational concepts to build on:
UiPath Test Automation using UiPath Test Suite series, part 4DianaGray10
Welcome to UiPath Test Automation using UiPath Test Suite series part 4. In this session, we will cover Test Manager overview along with SAP heatmap.
The UiPath Test Manager overview with SAP heatmap webinar offers a concise yet comprehensive exploration of the role of a Test Manager within SAP environments, coupled with the utilization of heatmaps for effective testing strategies.
Participants will gain insights into the responsibilities, challenges, and best practices associated with test management in SAP projects. Additionally, the webinar delves into the significance of heatmaps as a visual aid for identifying testing priorities, areas of risk, and resource allocation within SAP landscapes. Through this session, attendees can expect to enhance their understanding of test management principles while learning practical approaches to optimize testing processes in SAP environments using heatmap visualization techniques
What will you get from this session?
1. Insights into SAP testing best practices
2. Heatmap utilization for testing
3. Optimization of testing processes
4. Demo
Topics covered:
Execution from the test manager
Orchestrator execution result
Defect reporting
SAP heatmap example with demo
Speaker:
Deepak Rai, Automation Practice Lead, Boundaryless Group and UiPath MVP
Smart TV Buyer Insights Survey 2024 by 91mobiles.pdf91mobiles
91mobiles recently conducted a Smart TV Buyer Insights Survey in which we asked over 3,000 respondents about the TV they own, aspects they look at on a new TV, and their TV buying preferences.
State of ICS and IoT Cyber Threat Landscape Report 2024 previewPrayukth K V
The IoT and OT threat landscape report has been prepared by the Threat Research Team at Sectrio using data from Sectrio, cyber threat intelligence farming facilities spread across over 85 cities around the world. In addition, Sectrio also runs AI-based advanced threat and payload engagement facilities that serve as sinks to attract and engage sophisticated threat actors, and newer malware including new variants and latent threats that are at an earlier stage of development.
The latest edition of the OT/ICS and IoT security Threat Landscape Report 2024 also covers:
State of global ICS asset and network exposure
Sectoral targets and attacks as well as the cost of ransom
Global APT activity, AI usage, actor and tactic profiles, and implications
Rise in volumes of AI-powered cyberattacks
Major cyber events in 2024
Malware and malicious payload trends
Cyberattack types and targets
Vulnerability exploit attempts on CVEs
Attacks on counties – USA
Expansion of bot farms – how, where, and why
In-depth analysis of the cyber threat landscape across North America, South America, Europe, APAC, and the Middle East
Why are attacks on smart factories rising?
Cyber risk predictions
Axis of attacks – Europe
Systemic attacks in the Middle East
Download the full report from here:
https://sectrio.com/resources/ot-threat-landscape-reports/sectrio-releases-ot-ics-and-iot-security-threat-landscape-report-2024/
UiPath Test Automation using UiPath Test Suite series, part 3DianaGray10
Welcome to UiPath Test Automation using UiPath Test Suite series part 3. In this session, we will cover desktop automation along with UI automation.
Topics covered:
UI automation Introduction,
UI automation Sample
Desktop automation flow
Pradeep Chinnala, Senior Consultant Automation Developer @WonderBotz and UiPath MVP
Deepak Rai, Automation Practice Lead, Boundaryless Group and UiPath MVP
2. NMR screening exploits the fact that many NMR observ-
ables change even upon temporary binding because of
global and local effects (see Figures 1 and 2). The effects
of binding may be monitored either on the target or on the
ligand and are summarised in Table 1. The observed
molecule primarily dictates inherent limitations such as
sample availability (possibly with appropriate isotope
labelling), solubility, stability and molecular size.
Ligand-observed screening
Detection with these techniques generally takes place on the
dissociate, free form of the small ligand (implying a minimal
Kd of ca. 10–7 M), allowing the direct identification of bind-
ing species from mixtures. The unobserved target molecule
is not subject to size limitations and may even be immo-
bilised. In general, isotopic labelling is not required. Many of
these techniques rely on size-dependent effects, which can
be difficult to resolve for weak binding (implying a maximal
Kd of ca. 10–3 M) and small relative molecular size differ-
ences between ligand and target. These effects can be
selected for in 1D filtered spectra, in which tuning the filter
selects for different ligand affinities. Alternatively, the size of
the effect could be evolved in a second indirect dimension,
yielding pseudo-2D edited spectra. The information for
structure-based ligand optimisation obtainable through these
techniques is more limited than for target-observed tech-
niques. It may, however, reveal a ligand’s binding epitope, its
bound conformation, and even contacts to other ligands
binding at adjacent sites.
Use of molecular diffusion
Detection of transiently binding ligands by their decreased
diffusion coefficients is an established screening technique
(for exhaustive reviews see [16,17•]) Pseudo-2D diffusion-
edited experiments (diffusion-ordered spectroscopy,
DOSY), where molecules are separated according to their
individual diffusion coefficients, are particularly useful for
deconvoluting mixtures. Alternatively, 1D diffusion filters
can be combined with standard 2D correlation experi-
ments, such as in the diffusion-modulated gradient COSY
(correlation spectroscopy) [18] and diffusion-encoded total
correlation spectroscopy (TOCSY; DECODES) [19], to
elucidate spin systems and facilitate mixture analysis.
Diffusion filtering or editing might also be enhanced by
combination with other screening techniques, such as
NOE pumping [20,21] (see below). In a promising appli-
cation for combinatorial chemistry, diffusion filtering has
recently been used in solid-state MAS (magic-angle
spinning) NMR to identify and distinguish resin-bound
molecules from impurities [22].
Use of relaxation
Screening based on binding-induced slowing of global
dynamics, leading to a general decrease in NMR relaxation
times, is conceptually very similar to diffusion-based
screening. The diffusion filter is replaced by an inversion-
recovery delay, a spin-lock period or a CPMG echo to filter
for the different types of relaxation times, T1, T1ρ and T2,
respectively. One-dimensional relaxation-based screening
usually requires subtraction of a reference spectrum of the
target-free ligand mixture, and measures must be taken to
avoid artefacts due to binding-induced lineshape distor-
tions, chemical-shift changes and residual target signals
[23]. T2-based ligand screening may also be performed by
comparing spectral linewidths [24•] that increase upon
binding. Complications can arise from superposition of
line-broadening due to chemical exchange or slow
286 Combinatorial chemistry
Figure 1
Global NMR consequences of ligand–target
interactions. Observation is restricted to the
ligand where the effects are most
pronounced. Their strength depends on
molecular size differences and binding
kinetics.
kA
kD
+
Complex properties
similar to
target properties
• Slow Relaxation
• Fast diffusion
• Positive NOE
• Fast relaxation
• Slow diffusion
• Negative NOE
Small ligands ComplexLarge target
Current Opinion in Chemical Biology
Figure 2
Local NMR effects of complex formation may be observed on the
ligand and/or the target. Structural information is available as effects
are mostly localised to the binding site.
Surface protection
Current Opinion in Chemical Biology
Intermolecular
magnetisation
transfer
Chemical shift perturbation
Perturbation of local dynamics{
3. conformational motions (on the microsecond and
millisecond timescales), which are often relevant for
induced-fit during intermolecular interactions and damped
upon binding. Thus, a size-dependent decrease in T2
times might be compensated for by a flexibility-dependent
reduction in line-broadening.
In contrast, the recently proposed concept of transferred
T1 times [25] relies on the fact that relaxation rates
measured on a fast-exchanging ligand in the free state also
contain contributions from its local dynamics in the bound
state, as immobilisation of flexible regions of the ligand
generally increases relaxation rates. Transferred relaxation
rates may therefore be seen as a dynamic analogue of
transferred NOE (see below). Individual differences in
relaxation rates must be compared with their average in
order to distinguish local from global dynamic changes
[25]. Another complication arises from local relaxation
contributions because of molecular anisotropy.
Intramolecular magnetisation transfer: transfer-NOE
Transferred intramolecular NOEs (trNOEs) are well
established as unique sources of structural information on
the bound ligand (for a review see [26]) where spin-diffu-
sion should be suppressed to obtain optimal structural
precision [27,28]). The observation of trNOE has proved
to be equally useful in the fast screening of ligand
mixtures [24•], forming the basis of ‘bioaffinity NMR’
[29]. The technique relies on the size-dependence of the
intramolecular NOE, which shows slow NOE build-up
with weak positive maxima for free ligands and rapid
build-up with strong negative maxima for the bound state.
If dissociation of the ligand occurs quickly enough (i.e.
Kd > 10–7 M), a sufficient percentage of observable free
ligand will retain intense negative trNOE as a ‘memory’ of
the bound state and thus quickly indicate binding. If the
residence time is too short (i.e. Kd > 10–3 M), however,
trNOE will build up too weakly for detection [30].
Routinely, trNOE are recorded as 2D NOESY (NOE
spectroscopy) spectra with short mixing times favouring
build-up of trNOE over direct NOE. Relaxation filtering
suppresses residual target signals. Notable are applica-
tions of trNOE-based screening [30,31•,32] and recent
experimental improvements [33].
Screening by trNOE imposes upper limits on the ligands
(< 1 kDa), which have to fall into the positive NOE
regime. For borderline cases, this regime may be extend-
ed, for example, by increasing the temperature [31•].
Another problem is the possible cancellation of positive
direct and negative transferred NOE, which could be
resolved by more lengthy 3D experiments such as the 3D
TOCSY–trNOESY [34], or by resorting to other tech-
niques such as saturation transfer difference (see below).
Intermolecular magnetisation transfer
The intermolecular magnetisation transfer is always posi-
tive and cancellation cannot occur. For its measurement,
non-equilibrium magnetisation must first be created asym-
metrically (i.e. on one component only, usually the drug
target). It is then transferred to the binding partner during
a mixing period. The intermolecular magnetisation trans-
fer results in altered signal intensities for both components
and can finally be monitored on either the starting or the
receiving molecule (usually the ligand). Remaining target
resonances can be suppressed (e.g. by isotope filtering or
relaxation filtering). Subtraction of a reference spectrum
(recorded without initially disturbing the magnetisation) is
usually required to reveal these intensity changes and
entails the risk of subtraction artefacts.
Asymmetric non-equilibrium magnetisation on the target
molecule may generally be created by selective irradiation,
requiring the existence of a unique NMR frequency band.
Applications of NMR in drug discovery Diercks, Coles and Kessler 287
Table 1
NMR observable effects of ligand binding.
Global effects of binding Ligand observed Screening Applications References
Translational dynamics Diffusion rates Affinity NMR [17•,19]
Rotational dynamics Relaxation rates
Line broadening [23,24•]
Sign of transferred intramol. NOE Bioaffinity NMR (trNOE) [29,30,31•,32,34]
Molecular orientation Residual dipolar couplings *
Local effects of binding Ligand- or target-observed
Chemical shift perturbation Chemical shifts SAR-by-NMR (HSQC screening) [44•,48,49,51,53•]
Local dynamics perturbation Relaxation rates *
Surface protection Water exchange rates *
Paramagnetic surface probing *
Intermolecular magnetisation transfer Via scalar couplings *
(e.g. intermol. H-bonds)
Via nuclear dipolar couplings Bioaffinity NMR (STD) [32,35,37•]
(intermol. NOE) (Reverse) NOE pumping [20,21]
WaterLOGSY [38]
Via electron dipolar couplings
(paramagnetic relaxation) SLAPSTIC [39•,41•]
*No application to NMR screening reported yet. Intermol., intermolecular.
4. Initially localised, it is spread across the entire molecule by
spin diffusion such that magnetisation may be picked up
by a ligand binding anywhere on the surface. Alternatively,
non-equilibrium magnetisation may be created symmetri-
cally, followed by asymmetric suppression making use of
the large size difference between target and ligands. For
instance, a diffusion filter would suppress magnetisation
on the ligands, whereas a relaxation filter would suppress
magnetisation on the target.
Saturation transfer difference (STD) [35] as the special
case of intermolecular steady-state NOE is observed after
frequency-selective irradiation of the target and subtrac-
tion of a reference spectrum with off-resonance irradiation.
Although usually run as 1D spectra, STD can be coupled to
standard 2D correlation experiments such as TOCSY, COSY
and HMQC (heteronuclear multiple quantum correlation) to
alleviate spectral overlap [32,35]. The distance-dependence
of the observed signal intensities identifies those nuclei
closest to the target–ligand interface. This ‘epitope mapping’
[28,36] may be seen as a ligand-based complement to the
‘SAR-by-NMR’ approach. A remarkable application extends
STD screening to solid-state NMR [37•], in which the target
was immobilized on controlled-pearl glass, facilitating its
recovery. From a comparison of STD integrals, it was
possible to assess relative binding strengths of the dissolved
ligands. A reference spectrum with pure target-free resin
must rule out direct ligand–resin interactions.
NOE pumping [20] employs the scheme of symmetric
magnetisation inversion, followed by asymmetric suppres-
sion. Classically, the ligand magnetisation is suppressed via
diffusion filtering, and its recovery directly indicates inter-
molecular magnetisation transfer from the target, hence
binding. A suppression of remaining target magnetisation
prior to acquisition greatly facilitates identification, but the
diffusion filter employed may entail serious intensity losses
through concomitant relaxation of the target magnetisation.
This problem is alleviated in the reverse case (reverse
NOE pumping) [21], in which ligand magnetisation is
preserved while target magnetisation is suppressed via
relaxation or isotope filtering. The ligand magnetisation is
then reduced because of transfer onto the target, and signal
reductions observed on a ligand thus indicate binding.
These reductions, however, are only revealed after subtrac-
tion of a reference spectrum, which also takes account of
unspecific magnetisation losses through T1 relaxation.
WaterLOGSY [38] exploits the surface layer of target-
bound water molecules as a pool of selectively invertible
magnetisation that can be transferred onto binding ligands.
After simultaneous selective water suppression and
relaxation filtering (for target suppression), only signals of
binding ligands are detected.
Paramagnetic spin labels
Magnetic spin interactions can be enhanced drastically by
introducing paramagnetic spin labels. The induced NMR
effects — resonance shifts and line broadening — can
thus be observed over larger distances (up to some 20 Å),
shorter contact times, and at lower protein concentrations.
Soluble paramagnetic reagents have classically been
employed to search for metal ion binding sites and probe
solvent-accessible surfaces. Intermolecular binding, which
blocks part of the solvent-accessible surface, may be
detected and localised in a similar manner.
In a recent application to second-site screening, a
confirmed binding ligand was spin labelled and used to
search for ligands concomitantly binding to the target
protein at an adjacent site [39•]. The method is analogous
to the observation of transferred inter-ligand NOE
contacts [40], yet offers larger specificity and sensitivity,
establishing connectivity between more distant binding
sites. The need to synthesize spin-labelled ligands without
affecting their natural binding properties is, however, a
major drawback. A blind test must furthermore rule out
that ligands interact in the absence of the target.
In the SLAPSTIC method, the target protein is spin-
labelled to assist in identifying binding ligands [41•]. The
line-broadening of ligand signals upon binding is thus
strongly emphasized and allows for a reduction in protein
(and ligand) amounts by one or two orders of magnitude. It
is critical to spin-label the target near, but not within the
binding site, as this would invariably lead to altered
binding properties. Moreover, no structural information
can be derived from this method.
Use of residual dipolar couplings
Alignment of the target molecule also causes alignment of
the ligand upon temporary binding. RDC may thus be
transferred from the bound to the isotropic free state if
dissociation is fast enough (Kd in the millimolar range)
[42,43]. In contrast to NOE data, the structural information
available from RDC does not suffer from spin-diffusion
and is therefore precise even for large, immobilised targets.
RDC yield information on both the bound ligand’s
structure and its orientation relative to the target without
the need for assigning target resonances as demonstrated
in two recent applications [42,43]. This technique has not,
however, been applied to ligand screening yet.
Target-observed screening
Observation on the target, rather than on dissociated
ligands, is neither restricted by the ligands’ size nor by an
upper affinity limit. It also immediately reveals different
binding sites, enables direct distinction between specific
and unspecific binding and provides a wealth of structural
information for ligand optimisation. This information,
however, can only be extracted if the spectral assignments
for the target are known. Inherent limitations often
preclude observation on the large target molecule, although
relaxation-optimised techniques (TROSY) may be of
benefit. The amounts of protein required, which must be
isotope-labelled, can be reduced significantly by employing
288 Combinatorial chemistry
5. cryoprobe technology and screening larger mixtures of
compounds [44•]. Mixtures of ligands can not, however, be
directly deconvoluted.
Chemical-shift perturbations
Most target-observed screening applications rely on
chemical-shift changes as indicators for intermolecular
binding. After assignment, these changes can be mapped
upon the protein structure, revealing its binding sites
[45,46] and guiding structure-based ligand optimisation.
This forms the basis of the ‘SAR-by-NMR’ approach
[47,48], in which optimised ligands are constructed by
linking weakly binding fragments. The approach contin-
ues to be successfully used in drug development [49–52].
Standard screening using 2D 15N,1H correlation spectra
has recently been complemented by 13C,1H HSQC-based
screening on proteins bearing 13C-labeled methyl groups
[53•]. The selective labelling, using inexpensive starting
materials, avoids the detrimental effects of 13C–13C
homonuclear coupling while greatly reducing the cost of
sample preparation. This application benefits from the
higher proton multiplicity and slower transverse relaxation
of the methyl groups. Consequently, intensities were
reported to be on average three-times higher. On the other
hand, screening based on methyl groups suffers from
assignment difficulties and lower spectral dispersion. Note
that the distribution and accessibility of (polar) amide and
(non-polar) methyl groups are not equivalent, possibly
leading to different screening sensitivities.
Equating chemical-shift perturbations with spatial proximity
to the ligand may be misleading, especially in the presence
of large shielding anisotropies in a ligand or because of con-
formational changes in the target. Therefore, an alternative
has been proposed in which the binding site and the bound
ligand’s orientation can be determined more reliably [54]
without the need for a complete re-determination of the
complex structures. It is based on the comparison of chemi-
cal-shift changes in the proximity of the binding site
induced by a series of closely related ligands. This can be
seen as trading synthetic for spectroscopic efforts.
Intermolecular magnetisation transfer
The general scheme for intermolecular magnetisation
transfer experiments (see above) can also be employed for
target-detected screening. In one concept [55], target mag-
netisation is selectively suppressed by relaxation filtering
after symmetric excitation and then recovers by intermolec-
ular transfer from binding ligands. Prior to observation on the
target, the ligand signals are filtered out by selective multiple
frequency suppression. This, however, precludes the direct
screening of ligands with more complex 1H spectra.
Screening for macromolecular interactions
Screening for macromolecular interactions is a special case
in which the ‘ligand’ and ‘target’ have comparable molecu-
lar sizes, and size discrimination is impracticable. Other
than that, many of the techniques mentioned above can be
and have been applied to the problem. For instance, STD
was used to probe protein–nucleic-acid interactions by
saturating well-separated protons of the latter [56]. A varia-
tion of the scheme scans for protein–protein interactions
[57] with molecular distinction made by perdeuterating and
15N-labelling only one partner. The unlabelled protein
remains susceptible to saturation of the aliphatic protons,
while intermolecular STD is observed on the 1H,15N
moieties of the labelled protein. The low proton density in
this perdeuterated species precludes efficient spin-diffu-
sion and the STD is largely confined to the binding site.
Design of NMR screening libraries
In contrast to other screening techniques, NMR screening
requires relatively high sample concentrations and sampling
times. With its unprecedented sensitivity (detecting interac-
tions with Kd up to 10–2 M) and atomic resolution, however,
NMR is particularly suited for guiding the ‘bottom-up’
construction of complex ligands from small, weakly binding
molecular scaffolds. In line with this, the principles for com-
posing a dedicated ligand library are availability, good
solubility, low molecular mass (i.e. < 1–2 kDa), and limita-
tion to some 102–103 compounds of maximal diversity. For
mixture screening, batches should contain (non-reacting)
ligands with isolated NMR signals for easy monitoring.
Various attempts have been made to construct libraries using
consensus fragments identified from statistical analyses of
known drugs (e.g. see [24•,58,59]) and protein-binding
ligands [60], and from fragmenting an existing lead
compound [52]. Pre-selection of small ligands for NMR
screening can also be based on the scoring results obtained
from virtual docking [61].
Conclusions
NMR screening is now being recognised by industry as a
powerful technique for lead discovery. The underlying prin-
ciples have mostly been discovered and developed long ago
by academic scientists, such that the novelty of NMR
screening lies mostly with its applications. In this review, we
have offered a classification of the available NMR methods
and outlined possible future developments by a reduction to
basic NMR properties and principles. The distinction
between ligand- and target-observed screening techniques
is most important. In practice, a dual strategy that combines
the complementary strengths and detectable affinity ranges
of both techniques should prove advantageous. Most impor-
tantly, ligand-observed techniques may produce false
negatives in cases of very strong binding where exchange
between target-bound and detected free ligand is too slow.
These techniques nevertheless have clear advantages for
pre-screening large libraries as they implicitly deconvolute
ligand mixtures, require no isotope labelling and pose few
limitations on the target. Consecutive target-observed ‘fine-
’screening of the identified binding ligands can then rule out
unspecific binding, identify different binding sites and
modes, and guide structure-based ligand optimisation. By
Applications of NMR in drug discovery Diercks, Coles and Kessler 289
6. screening a carefully composed small library of basic
molecular scaffolds, the pre-screening step may be omitted
for amenable targets.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
••of outstanding interest
1. Moore JM: NMR screening in drug discovery. Curr Opin Biotechnol
1999, 10:54-58.
2. Moore JM: NMR techniques for characterization of ligand binding:
utility for lead generation and optimization in drug discovery.
Biopolymers 1999, 51:221-243.
3. Roberts GCK: NMR spectroscopy in structure-based drug design.
Curr Opin Biotechnol 1999, 10:42-47.
4. Roberts GCK: Applications of NMR in drug discovery. Drug Discov
Today 2000, 5:230-240.
5. Stockman BJ: NMR spectroscopy as a tool for structure-based
drug design. Progr NMR Spectrosc 1998, 33:109-151.
6. Keifer PA: NMR tools for biotechnology. Curr Opin Biotechnol
1999, 10:34-41.
7. Ross A, Schlotterbeck G, Klaus W, Senn H: Automation of NMR
•• measurements and data evaluation for systematically screening
interactions of small molecules with target proteins. J Biomol
NMR 2000, 16:139-146.
This reference gives valuable practical advice on performing industrial-scale
ligand screening by NMR, presenting a fully integrated hardware set-up with
robotized sample preparation, flow-through probehead and automated data
analysis of the acquired 15N,1H-HSQC. A pre-check of the protein–ligand
mixtures for aggregation and solubility is required to prevent clogging of the
flow-through probe. The authors conclude that screening individual ligands,
rather than mixtures, justifies the extra time by alleviating the need for char-
acterizing possible ligand–ligand interactions and deconvoluting mixtures.
The problem of using too low buffer concentrations, entailing artefactual
chemical shift changes, is also discussed.
8. Haner RL, Llanos W, Mueller L: Small volume flow probe for
automated direct-injection NMR analysis: design and
performance. J Mag Res 2000, 143:69-78.
9. Pervushin K, Riek R, Wider G, Wüthrich K: Attenuated T2 relaxation
by mutual cancellation of dipole-dipole coupling and chemical
shift anisotropy indicates an avenue to NMR structures of very
large biological macromolecules in solution. Proc Natl Acad Sci
USA 1997, 94:12366-12371.
10. Permi P, Annila A: Transverse relaxation optimised spin-state
selective NMR experiments for measurement of residual dipolar
couplings. J Biomol NMR 2000, 16:221-227.
11. Prestegard JH: New techniques in structural NMR — anisotropic
interactions. Nat Struct Biol 1998, (NMR suppl):517-522.
12. Rückert M, Otting G: Alignment of biological macromolecules in
novel nonionic liquid crystalline media for NMR experiments.
J Am Chem Soc 2000, 122:7793-7797.
13. Cordier F, Grzesiek S: Direct observation of hydrogen bonds in
proteins by interresidue h3JNC’ scalar couplings. J Am Chem Soc
1999, 121:1601-1602.
14. Goto NK, Kay LE: New developments in isotope labeling
strategies for protein solution NMR spectroscopy. Curr Opin
Struct Biol 2000, 10:585-592.
15. Fielding L: Determination of association constants (Ka) from
solution NMR data. Tetrahedron 2000, 56:6151-6170.
16. Johnson CS: Diffusion ordered nuclear magnetic resonance
spectroscopy: principles and applications. Progr NMR Spectrosc
1999, 34:203-256.
17. Chen A, Shapiro MJ: Affinity NMR. Anal Chem 1999,
• 71:669A-675A.
A review describing NMR screening, in particular diffusion-based affinity
NMR, in a modular way.
18. Gmeiner WH, Hudalla CJ, Soto AM, Marky L: Binding of ethidium to
DNA measured using a 2D diffusion-modulated gradient COSY
NMR experiment. FEBS Lett 2000, 465:148-152.
19. Bleicher K, Lin M, Shapiro MJ, Wareing JR: Diffusion edited NMR:
screening compound mixtures by affinity NMR to detect binding
ligands to vancomycin. J Org Chem 1998, 63:8486-8490.
20. Chen A, Shapiro MJ: NOE pumping: a novel NMR technique for
identification of compounds with binding affinity to
macromolecules. J Am Chem Soc 1998, 120:10258-10259.
21. Chen A, Shapiro MJ: NOE pumping. 2. A high-throughput method
to determine compounds with binding affinity to macromolecules
by NMR. J Am Chem Soc 2000, 122:414-415.
22. Shapiro MJ, Chin J, Chen A, Wareing JR, Tang Q, Tommasi RA,
Marepalli HR: Covalent or trapped? PFG diffusion MAS NMR for
combinatorial chemistry. Tetrahedron Lett 1999, 40:6141-6143.
23. Hajduk PJ, Olejniczak ET, Fesik SW: One-dimensional relaxation-
and diffusion-edited NMR methods for screening compounds that
bind to macromolecules. J Am Chem Soc 1997, 119:12257-12261.
24. Fejzo J, Lepre CA, Peng JW, Bemis GW, Ajay, Murcko MA, Moore JM:
• The SHAPES strategy: An NMR-based approach for lead
generation in drug discovery. Chem Biol 1999, 6:755-769.
Although no new techniques are described, a clear overview on the set-up
of NMR screening, including library design is provided.
25. LaPlante SR, Aubry N, Deziel R, Ni F, Xu P: Transferred 13C T1
relaxation at natural isotopic abundance: a practical method for
determining site-specific changes in ligand flexibility upon
binding to a macromolecule. J Am Chem Soc 2000,
122:12530-12535.
26. Ni F: Recent developments in transferred NOE methods. Progr
NMR Spectrosc 1994, 26:517-606.
27. Vincent SJF, Zwahlen C, Post CB, Burgner JW, Bodenhausen G: The
conformation of NAD++ bound to lactate dehydrogenase
determined by nuclear magnetic resonance with suppression of
spin-diffusion. Proc Natl Acad Sci USA 1997, 94:4383-4388.
28. Maaheimo H, Kosma P, Brade L, Brade H, Peters T: Mapping the
binding of synthetic disaccharides representing epitopes of
chlamydial lipopolysaccharide to antibodies with NMR.
Biochemistry 2000, 39:12778-12788.
29. Meyer B, Weimar T, Peters T: Screening mixtures for biological
activity by NMR. Eur J Biochem 1997, 246:705-709.
30. Mayer M, Meyer M: Mapping the active site of angiotensin-
converting enzyme by transferred NOE spectroscopy. J Med
Chem 2000, 43:2093-2099.
31. Henrichsen D, Ernst B, Magnani JL, Wang W-T, Meyer B, Peters T:
• Bioaffinity NMR spectroscopy: identification of an E-selectin
antagonist in a substance mixture by transfer NOE. Angew Chem
Int Ed Engl 1999, 38:98-102.
This article describes an application of trNOE-based screening to a mixture
of ligands, also discussing important details to be considered with this tech-
nique. Prior to screening, the library was tested for the positive NOE regime,
which was eventually reached only after increasing the temperature. The
binding ligand then identified by negative trNOE displayed strong spin-
diffusion, which in this case helped its identification, but generally blurs
structural information. Suppression of residual target protein signals by
means of a relaxation filter was largely complete, except for signals from
a highly flexible, attached carbohydrate chain.
32. Vogtherr M, Peters T: Application of NMR based binding assays to
identify key hydroxy groups for intermolecular recognition. J Am
Chem Soc 2000, 122:6093-6099.
33. Weimar T: Improving bioaffinity NMR spectra by means of zero-
quantum dephasing. Magn Reson Chem 2000, 38:315-318.
34. Herfurth L, Weimar T, Peters T: Application of 3D-TOCSY-tr-NOESY
for the assignment of bioactive ligands from mixtures. Angew
Chem Int Ed Engl 2000, 39:2097-2099.
35. Mayer M, Meyer B: Characterization of ligand binding by saturation
transfer difference NMR spectroscopy. Angew Chem Int Ed Engl
1999, 38:1784-1788.
36. Mayer M, Meyer B: Group epitope mapping (GEM) to identify
segments of a ligand in direct contact with a protein: STD NMR as
a tool to characterize binding to Ricinus communis agglutinin.
J Am Chem Soc 2001, in press.
290 Combinatorial chemistry
7. 37. Klein J, Meinecke R, Mayer M, Meyer B: Detecting binding affinity to
• immobilized receptor proteins in compound libraries by HR-MAS
STD NMR. J Am Chem Soc 1999, 121:5336-5337.
This article describes an exciting extension of the STD screening method to
high-resolution MAS NMR, using dissolved ligands and a target immobilized
on controlled pearl glass for easy protein recovery. A reference spectrum
with pure, target-free resin must be recorded to rule out ligand–resin inter-
actions. Relative binding strengths of competitive ligands can be assessed
from a comparison of STD integrals. Structural information, however, is
restricted to the binding epitope of the ligand.
38. Dalvit C, Pevarello P, Tato M, Veronesi M, Vulpetti A, Sundström M:
Identification of compounds with binding affinity to proteins via
magnetization transfer from bulk water. J Biomol NMR 2000, 18:65-68.
39. Jahnke W, Perez LB, Paris CG, Strauss A, Fendrich G, Nalin CM:
• Second-site NMR screening with a spin-labelled first ligand. J Am
Chem Soc 2000, 122:7394-7395.
This interesting alternative to the so-called ‘SAR-by-NMR’ approach uses a
spin-labelled known binding ligand to induce detectable signal broadening
or extinction on another ligand binding to an adjacent site on the target pro-
tein. This novel method for the exclusive observation of second-site binding
is highly sensitive and also provides some structural information.
40. Li D, DeRose EF, London RE: The inter-ligand Overhause effect: a
powerful new NMR approach for mapping structural relationships
of macromolecular ligands. J Biomol NMR 1999, 15:71-76.
41. Jahnke W, Rüdisser S, Zurini M: Spin label enhanced NMR
• screening. J Am Chem Soc 2001, 123:3149-315.
This article describes a novel technique, SLAPSTIC, which uses a modified,
spin-labelled target protein for ligand screening. Ligand binding is detected
by signal broadening and extinction on the free form of a temporarily binding
ligand. The strong magnetic marker allows a drastic reduction in the required
protein and ligand concentrations, which also translates into lower minimum
ligand solubilities and access to lower binding affinities.
42. Koenig BW, Mitchell DC, König S, Grzesiek S, Litman BJ, Bax A:
Measurement of dipolar couplings in a transducin peptide
fragment weakly bound to oriented photo-activated rhodopsin.
J Biomol NMR 2000, 16:121-125.
43. Thompson GS, Shimizu H, Homans SW, Donohue-Rolfe A:
Localization of the binding site for the oligosaccharide moiety of
GB3 on verotoxin 1 using NMR residual dipolar coupling
measurements. Biochemistry 2000, 39:13153-13156.
44. Hajduk PJ, Gerfin T, Boehlen J-M, Häberli M, Marek D, Fesik SW:
• High-throughput nuclear magnetic resonance-based screening.
J Med Chem 1999, 42:2315-2317.
This article presents a profound analysis of the benefits of cryoprobe tech-
nology to 15N-HSQC-based NMR screening. The merits alternatively trans-
late into lower detectable ligand affinities (with up to millimolar dissociation
rates), substantially reduced protein and ligand concentrations, or faster
screening rates. The authors claim that libraries with up to 200,000 ligands
(administered in mixtures of 100 compounds) could thus be screened effi-
ciently within less than a month.
45. Sakamoto T, Tanaka T, Ito Y, Rajesh S, Iwamoto-Sugai M, Kodera Y,
Tsuchida M, Shibata T, Kohno T: An NMR analysis of ubiquitin
recognition by yeast ubiquitin hydrolase: evidence for novel
substrate recognition by a cysteine protease. Biochemistry 1999,
38:11634-11642.
46. Sun C, Cai M, Gunasekera AH, Meadows RP, Wang H, Chen J, Zhang H,
Wu W, Xu N, Ng SC et al.: NMR structure and mutagenesis of the
inhibitor-of-apoptosis protein XIAP. Nature 1999, 401:818-822.
47. Hajduk PJ, Sheppard G, Nettesheim D, Olejniczak ET, Shuker SB,
Meadows RP, Steinman DH, Carrera GM, Marcotte PA, Severin J
et al.: Discovery of potent nonpeptide inhibitors of
stromelysin using SAR by NMR. J Am Chem Soc 1997,
119:5818-5827.
48. Shuker SB, Hajduk PJ, Meadows RP, Fesik SW: Discovering high-
affinity ligands for proteins: SAR by NMR. Science 1996,
274:1531-1534.
49. Hajduk PJ, Zhou M-M, Fesik SW: NMR-based discovery of
phosphotyrosine mimetics that bind to the Lck SH2 domain.
Bioorg Med Chem Lett 1999, 9:2403-2406.
50. Hajduk PJ, Dinges J, Schkeryantz JM, Janowick D, Kaminski M,
Tufano M, Augeri DJ, Petros A, Nienaber V, Zhong P et al.: Novel
inhibitors of Erm methyltransferases from NMR and parallel
synthesis. J Med Chem 1999, 42:3852-3859.
51. Hajduk PJ, Boyd S, Nettesheim D, Nienaber V, Severin J, Smith R,
Davidson D, Rockway T, Fesik SW: Identification of novel inhibitors
of urokinase via NMR-based screening. J Am Chem Soc 2000,
43:3862-3866.
52. Hajduk PJ, Gomtsyan A, Didomenico S, Cowart M, Bayburt EK,
Solomon L, Severin J, Smith R, Walter K, Holzman TF et al.: Design of
adenosine kinase inhibitors from the NMR-based screening of
fragments. J Med Chem 2000, 43:4781-4786.
53. Hajduk PJ, Augeri DJ, Mack J, Mendoza R, Yang J, Betz SF, Fesik SW:
• NMR-based screening of proteins containing 13C-labeled methyl
groups. J Am Chem Soc 2000, 122:7898-7904.
This article evaluates the advantages of 13C-HSQC-based screening on a
target protein selectively 13C-labelled at the methyl groups, including a com-
parison with 15N-HSQC- and 15N-TROSY-based screening.
54. Medek A, Hajduk PJ, Mack J, Fesik SW: The use of differential
chemical shifts for determining the binding site location and
orientation of protein-bound ligands. J Am Chem Soc 2000,
122:1241-1242.
55. Dalvit C, Floersheim P, Zurini M, Widmer A: Use of organic solvents
and small molecules for locating binding sites on proteins in
solution. J Biomol NMR 1999, 14:23-32.
56. Ramos A, Kelly G, Hollingworth D, Pastore A, Frenkiel T: Mapping the
interface of protein-nucleic acid complexes using cross-
saturation. J Am Chem Soc 2000, 122:11311-11314.
57. Takahashi H, Nakanishi T, Kami K, Arata Y, Shimada I: A novel NMR
method for determining the interfaces of large protein–protein
complexes. Nat Struct Biol 2000, 7:220-223.
58. Bemis GW, Murcko MA: The properties of known drugs. 1.
Molecular frameworks. J Med Chem 1996, 39:2887-2893.
59. Bemis GW, Murcko MA: Properties of known drugs. 2. Side chains.
J Med Chem 1999, 42:5095-5099.
60. Hajduk PJ, Bures M, Praestgaard J, Fesik SW: Privileged molecules
for protein binding identified from NMR-based screening. J Med
Chem 2000, 43:3443-3447.
61. Muegge I, Martin YC, Hajduk PJ, Fesik SW: Evaluation of PMF
scoring in docking weak ligands to the FK506 binding protein.
J Med Chem 1999, 42:2498-2503.
Applications of NMR in drug discovery Diercks, Coles and Kessler 291