LitCovid-sentences  

PMC:7594251 / 38062-103373 JSONTXT 11 Projects

Annnotations TAB TSV DIC JSON TextAE

Id Subject Object Predicate Lexical cue
T262 0-2 Sentence denotes 3.
T263 3-54 Sentence denotes NMR Methods for Drug Discovery and Drug Development
T264 55-152 Sentence denotes As stated, NMR spectroscopy can be fundamental in studying how drugs interact with their targets.
T265 153-316 Sentence denotes This has been done mainly via the Fragment Based Drug Design (FBDD) approach, which has two sub-approaches: target- (i.e., protein) based, or ligand- (drug) based.
T266 317-460 Sentence denotes Target based screening monitors how the target responds to binding molecules in a method called Structure Activity Relationship (“SAR”) by NMR.
T267 461-751 Sentence denotes Ligand (drug)-based screening methods provide ways to observe the binding/non-binding behavior of the drug in approaches such as Saturation Transfer Difference (STD) and other Nuclear Overhauser Effect (NOE) type methods, diffusion-based methods, relaxation-based methods (i.e., T1 and T2).
T268 752-867 Sentence denotes Target based screening, ligand (drug) based screening, and their respective methods, are discussed in detail below.
T269 869-873 Sentence denotes 3.1.
T270 874-914 Sentence denotes NMR in Fragment Based Drug Design (FBDD)
T271 915-1028 Sentence denotes NMR-based drug discovery can be broadly classified into two groups: chemical and biological (in-cell) categories.
T272 1029-1149 Sentence denotes One of the principal methods of drug discovery using NMR spectroscopy is called fragment-based drug design (FBDD) [194].
T273 1150-1246 Sentence denotes In-cell NMR (biological) based drug discovery techniques will be discussed later in this review.
T274 1247-1415 Sentence denotes FBDD was first reported in 1996 [195] and used throughout the late 1990s as evidenced by the use of keywords related to FBDD in papers published during this time [196].
T275 1416-1519 Sentence denotes The use of FBDD as a viable drug screening technique began to be widely adopted in the mid-2000s [197].
T276 1520-1609 Sentence denotes High Throughput Screening (HTS) is another technique widely used in drug discovery [198].
T277 1610-1727 Sentence denotes HTS analyzes molecules from a chemical library to see which ones are suitable leads [198,199,200,201] (see Figure 6).
T278 1728-2036 Sentence denotes FBDD techniques will screen against a carefully designed fragment library composed of a few thousand molecules (for details on the choice of compounds and design of fragment libraries, see [202,203]) and identified hits are further developed via fragment growing, fragment merging, or fragment linking [194].
T279 2037-2146 Sentence denotes For examples of drugs derived from the FBDD approach that are currently in clinical trials, refer to Table 2.
T280 2147-2339 Sentence denotes HTS has been productive in drug design [204,205], but the method is time and resource intensive [206] and expensive [206] because of the numerous molecules to be examined (~100 million) [207].
T281 2340-2412 Sentence denotes Furthermore, the success rate is only estimated to be at ~50% [204,208].
T282 2413-2659 Sentence denotes Unlike traditional HTS, which can survey a large number of molecules ranging from a few hundred thousand to a few million [209], FBDD usually surveys a few thousand molecules (~1000–15000) from libraries with greater chemical diversity [209,210].
T283 2660-2803 Sentence denotes FBDD is a main-stream screening technique for drug discovery [207,209,211,212,213,214,215,216] and NMR is standard for many FBDD studies [209].
T284 2804-2985 Sentence denotes Additional methods and techniques such as SPR, X-ray crystallography [209,217,218,219,220] etc. have also been used in FBDD studies, accompanied or unaccompanied by NMR experiments.
T285 2986-3065 Sentence denotes For examples of FBDD derived drugs using methods besides NMR, refer to Table 2.
T286 3066-3269 Sentence denotes At the time of writing, and to the best of our knowledge, there are three Food and Drug Administration (FDA)-approved drugs derived from the FBDD approach [221], and over 30 are in clinical trials [222].
T287 3270-3345 Sentence denotes The first marketed drug derived via the FBDD approach is vemurafenib [223].
T288 3346-3533 Sentence denotes Vemurafenib is also the first drug approved for treatment of BRAF-mutant cancer [224], and is reported to exhibit significant clinical benefit for patients with metastatic melanoma [224].
T289 3534-3752 Sentence denotes Venetoclax, a common drug used to treat patients with chronic lymphocytic leukemia [225], is considered the second drug to be discovered using the FBDD approach [221], and ribociclib, a CDK4 inhibitor, the third [221].
T290 3753-3896 Sentence denotes The names, structures, targets/applications, and clinical status of vemurafenib, venetoclax, ribociclib, and other drugs are listed in Table 2.
T291 3897-4044 Sentence denotes As mentioned, NMR spectroscopy can be used in FBDD in two different ways: (1) target (or receptor) based screening, and (2) ligand-based screening.
T292 4045-4150 Sentence denotes With the stated advantages and disadvantages, researchers must select based on their available compounds.
T293 4152-4158 Sentence denotes 3.1.1.
T294 4159-4181 Sentence denotes Target Based Screening
T295 4182-4320 Sentence denotes Target based screening typically utilizes the “SAR by NMR” (structure-activity-relationship by nuclear magnetic resonance) approach [246].
T296 4321-4405 Sentence denotes SAR is primarily used to identify and develop extremely tight-binding ligands [247].
T297 4406-4601 Sentence denotes The ligand to target binding is traditionally monitored via chemical shift changes [247] using a correlation spectroscopy such as 1H-15N HSQC starting with the target and no ligand present [248].
T298 4602-4686 Sentence denotes Multiple spectra for the target are recorded in the presence and absence of ligands.
T299 4687-4849 Sentence denotes The binding ligand will cause chemical shift perturbations in the target, and these perturbations are often easily visualized by overlaying the two spectra [247].
T300 4850-4984 Sentence denotes For example Hajduk et al. investigated the binding interactions of 2-phenylimidazole with the FKBP protein as shown in Figure 7 [249].
T301 4985-5194 Sentence denotes From the overlaid spectra, chemical shift changes are measured, and from the molecular location, extent, and rate of the chemical shift changes, the binding site and affinity of the ligand is calculated [250].
T302 5195-5438 Sentence denotes Then, by following a procedure completely analogous to that of FBDD (see Figure 6), a ligand developed from multiple fragments can be optimized for the binding site of interest, again by monitoring the changes in chemical shifts of the target.
T303 5439-5580 Sentence denotes Several examples of the successful applications of SAR by NMR in drug design research are replete in the scientific literature [204,251,252].
T304 5581-5761 Sentence denotes SAR by NMR spectroscopy allows researchers to observe directly ligand binding [247] in both solution state and solid-state spectra [253], increasing the method’s versatility [254].
T305 5762-5858 Sentence denotes It works particularly well for targeting proteins with adjacent “subpocket” binding sites [248].
T306 5859-5958 Sentence denotes Furthermore, SAR by NMR is cost-effective when combined with HTS (High Throughput Screening) [255].
T307 5959-6134 Sentence denotes SAR by NMR can also be used even when atomic peak assignments in spectra are unknown, though it is much more powerful when the resonance frequency of each atom is known [254].
T308 6135-6463 Sentence denotes The main limitation of SAR by NMR, however, is its inability to distinguish between multiple binding modes (i.e., cleavage of covalent bonds or allosteric changes), and if multiple binding modes are present, it can be difficult to pinpoint the “true” binding site of the ligand solely using data obtained using SAR by NMR [254].
T309 6465-6471 Sentence denotes 3.1.2.
T310 6472-6498 Sentence denotes NMR Ligand-Based Screening
T311 6499-6585 Sentence denotes Ligand-based screening, the second approach of NMR in FBDD, has three main categories:
T312 6586-6756 Sentence denotes 1) Saturation Transfer Difference (STD) and Nuclear Overhauser Effect (NOE) type methods, based on 2) diffusion methods, or 3) relaxation-based methods (i.e., T1 and T2).
T313 6758-6764 Sentence denotes 3.1.3.
T314 6765-6801 Sentence denotes Saturation Transfer Difference (STD)
T315 6802-6993 Sentence denotes Saturation Transfer Difference (STD) NMR depends on the Nuclear Overhauser Effect (NOE), which is often used to enhance the sensitivity of less sensitive nuclei such as 13C and 15N [256,257].
T316 6994-7123 Sentence denotes This increase in sensitivity is possible because of dipolar coupling (i.e., through space interactions of separate nuclei) [257].
T317 7124-7470 Sentence denotes The increase in sensitivity is actually brought about by applying a long, low power radiofrequency pulse that selectively saturates the magnetization [256] of a specific chemical group (i.e., the methyl groups on a protein), which is then given time to transfer to another chemical group via the NOE dipolar coupling within a few angstroms [258].
T318 7471-7631 Sentence denotes The transfer in magnetization is easily visualized on a NMR spectrum that takes the differences in the signal intensities from before and after the irradiation.
T319 7632-7778 Sentence denotes This new spectrum is called a “difference spectrum”, and it reveals what chemical groups interact with the irradiated signal [259] (see Figure 8).
T320 7779-7903 Sentence denotes STD NMR is an application of NOE used to probe the binding of ligands to a specific site within the targeted proteins [256].
T321 7904-7984 Sentence denotes A generic example of detecting ligand binding via STD is presented in Figure 9a.
T322 7985-8300 Sentence denotes The STD NMR method follows the same concepts as a normal NOE experiment: a spectrum of the ligand in the free, non-binding form is recorded, the ligand is allowed to bind to the protein, which has a functional group of interest (i.e., methyls) with a saturated signal from a previous selective radiofrequency pulse.
T323 8301-8505 Sentence denotes The saturated signal travels to the ligand, increasing the intensity of a signal on the ligand spectrum and finally a difference spectrum is used to determine precisely which sections of the ligands bind.
T324 8506-8585 Sentence denotes The difference in peak intensities proves the presence of ligand binding [260].
T325 8586-8691 Sentence denotes Water-Ligand Observed through Gradient Spectroscopy (WaterLOGSY) is a second type of STD (see Figure 9b).
T326 8692-8888 Sentence denotes The main difference with normal STD NMR is that water is the saturated signal [261], and instead of observing lower peak intensities, peak inversions indicate the presence of ligand binding [209].
T327 8889-9067 Sentence denotes For STD NMR to work properly, the ligand concentration must be in large excess (often 100–1000 fold) over the receptor so that effective saturation transfer can take place [260].
T328 9068-9190 Sentence denotes This means that for STD NMR, and WaterLOGSY, only small amounts (µg) of protein are required to get results [261,262,263].
T329 9191-9355 Sentence denotes This is advantageous for researchers, as they can perform STD NMR on a protein of interest, and preserve the rest of the unused sample for future/other experiments.
T330 9356-9420 Sentence denotes Also, the same sample can be used for multiple NMR measurements.
T331 9421-9630 Sentence denotes STD NMR facilitates the differentiation of binding ligands from non-binding ligands because the change in signal (as determined by the difference spectrum) is easy to measure and observe, as shown in Figure 9.
T332 9631-9712 Sentence denotes WaterLOGSY has been extended to study ligand interactions with DNA and RNA [261].
T333 9713-9904 Sentence denotes There are additional NOE-type experiments (trNOE, INPHARMA, SALMON, etc.) used for drug design, and specific details regarding individual methods are found in the scientific literature [264].
T334 9905-10051 Sentence denotes With the pressing search for new antiviral drugs, any techniques for identifying and characterizing novel leads has become increasingly important.
T335 10052-10287 Sentence denotes Benie et al. [265] described the use of saturation transfer difference (STD) NMR spectroscopy [262,266,267,268,269,270,271] to identify and characterize the binding of an antiviral compound to native human rhinovirus serotype 2 (HRV2).
T336 10288-10493 Sentence denotes The experiments demonstrated that it is possible to subject targets of the size and complexity of whole viruses (for a model of an HRV2 particle cut open, cf. the table of contents) to STD NMR experiments.
T337 10494-10736 Sentence denotes The principles of STD NMR have been known for many years [267,268] but it was only recently that the potential of this technique for screening libraries for compounds with binding activity toward protein receptors has been realized [262,266].
T338 10737-10829 Sentence denotes The technique also permitted the analysis of epitopes of ligands bound to receptor proteins.
T339 10830-10962 Sentence denotes Previous NMR studies of virus-ligand interactions used chemical shift titrations, which required very large quantities of the virus.
T340 10963-11025 Sentence denotes This approach was unworkable when studying pathogenic viruses.
T341 11026-11298 Sentence denotes Benie et al. [265] demonstrated that solution state STD methodology not only reduces the amount of virus required by approximately 2 orders of magnitude, but also allows for the identification and characterization of virus-ligand interactions with atomic resolution [272].
T342 11299-11512 Sentence denotes The very large size of viruses makes them particularly attractive for studies by STD NMR, as they inherently yield large line widths allowing for easy irradiation of the virus without affecting the ligand protons.
T343 11513-11683 Sentence denotes Furthermore, because of the larger correlation time of a virus in comparison to an average-sized protein, spin diffusion, and thus saturation transfer, is very efficient.
T344 11684-11924 Sentence denotes The large line width has additional benefits not just for STD-based NMR methods but also for transfer NOESY spectra, as protons from the virus capsid are invisible in the NMR spectra (for an example of a transfer NOESY spectrum, see [265]).
T345 11925-12029 Sentence denotes Moreover, competitive STD titration experiments can be used to determine the Kd value of a ligand [271].
T346 12030-12155 Sentence denotes Analysis of the STD spectra using the group epitope mapping method [271] allows for the determination of the binding epitope.
T347 12156-12278 Sentence denotes STD NMR methods can considerably speed up the determination of the binding epitope for potential antiviral lead compounds.
T348 12279-12481 Sentence denotes Simple STD NMR experiments provide substantial information on the binding of ligands to native viruses and require very small amounts of the virus with measurement times in the range of tens of minutes.
T349 12482-12728 Sentence denotes This allows for a high throughput of ligand samples without significant consumption of viral material because it remains unaffected by the experiments and is easily separated from the low molecular weight ligands by ultra-filtration subsequently.
T350 12729-12841 Sentence denotes In addition to the detection of binding, a complete mapping of the ligand-binding epitope can be achieved [265].
T351 12842-13012 Sentence denotes Noroviruses (NV) are non-enveloped, single-stranded, positive-sense RNA viruses that are the major cause of epidemic outbreaks of gastroenteritis worldwide [273,274,275].
T352 13013-13141 Sentence denotes The viral coat consists of a single protein, VP1, which assembles into a capsid with overall icosahedral symmetry [276,277,278].
T353 13142-13270 Sentence denotes Attachment of human noroviruses to histo-blood group antigens (HBGAs) is thought to be critical for the infection process [279].
T354 13271-13381 Sentence denotes The protruding domains of the VP1 proteins, called P-domains, harbor highly conserved binding sites for HBGAs.
T355 13382-13634 Sentence denotes STD NMR-based epitope mapping was used [262,271] to identify structural features of different core types critical for the binding of synthetic A- and B-tetrasaccharides [280] to virus-like particles (VLPs) of a highly homologous GII.4 strain (Ast6139).
T356 13635-13761 Sentence denotes STD NMR experiments provide a robust and straightforward technique for obtaining ligand binding epitopes at atomic resolution.
T357 13762-13896 Sentence denotes Comparing binding epitopes of related ligands then delivers critical information about structural requirements for ligand recognition.
T358 13897-14064 Sentence denotes Conversely, comparison of binding epitopes of a given ligand binding to wild type, and to mutant proteins reveals the importance of individual amino acids for binding.
T359 14065-14285 Sentence denotes STD NMR experiments with L-Fuc and B-trisaccharide in the presence of wild type and mutant VLPs yield virtually identical binding epitopes and suggest that these two mutations do not significantly alter HBGA recognition.
T360 14286-14555 Sentence denotes The STD NMR approach to characterize binding of HBGA ligands to noroviruses has employed VLPs as targets and thus taken advantage of the large size of VLPs yielding excellent signal-to-noise ratios of the corresponding STD NMR spectra, as demonstrated previously [281].
T361 14557-14563 Sentence denotes 3.1.4.
T362 14564-14614 Sentence denotes Transferred NOE (tr-NOE) in Ligand Based Screening
T363 14615-14713 Sentence denotes The application of the transferred NOE (Tr-NOE) effect was first demonstrated by Bothner-By [282].
T364 14714-14875 Sentence denotes The Tr-NOE is the nuclear Overhauser effect between ligand spins, which are in chemical exchange between the bound and unbound form with the protein or receptor.
T365 14876-14964 Sentence denotes Ligands, which are a mixture of target molecules, are small in size (below 500–1000 Da).
T366 14965-15145 Sentence denotes Since they are usually low molecular weight molecules, they exhibit much shorter correlation times when compared to the receptor and have slow NOE build-ups with no spin diffusion.
T367 15146-15212 Sentence denotes This is the reason they show small positive NOEs in the free form.
T368 15213-15365 Sentence denotes When binding to a protein receptor, the situation changes, where the ligand acquires large correlation times in the bound state with rapid NOE build-up.
T369 15366-15459 Sentence denotes Then they show spin diffusion and a strong negative NOE, which is termed the transferred NOE.
T370 15460-15630 Sentence denotes Signals arising from the protein are usually not observed for large proteins as they are generally kept low in concentration, with ligands in a high excess concentration.
T371 15631-15722 Sentence denotes In addition, most of the time protein signals are suppressed by their very short T2 period.
T372 15723-16091 Sentence denotes It is worthwhile to mention that ligands that are in fast exchange between the bound and the free form (dissociation constants ranging from μM to mM) get enough bound time to transfer the negative NOE from the protein complex to the population of the free molecules, yet usually retain the chemical shift of the free molecule along with the relaxation characteristics.
T373 16092-16276 Sentence denotes In order to observe tr-NOEs, the following condition have to be fulfilled:(3) |Nb∂b|≫|Nf∂f| where N and ∂ represent the number of molecules and the cross-relaxation rate, respectively.
T374 16277-16347 Sentence denotes The subscript b and f represent the bound and free form, respectively.
T375 16348-16449 Sentence denotes Therefore, to observe the tr-NOEs, a high excess concentration of ligands over protein is maintained.
T376 16450-16728 Sentence denotes On the other hand, if the ligand concentration is kept too high, the excess free ligand in solution will exhibit positive NOE, which can result in a significant reduction of the tr-NOESY enhancements due to negative NOE developed by the very small concentration of bound ligand.
T377 16729-16886 Sentence denotes Hence, the preparation of the sample becomes tricky and an optimum ratio between 10–30 to 1 is maintained while considering the dissociation constant values.
T378 16887-17001 Sentence denotes The binding of a ligand to a receptor protein can easily be identified by observing the sign and size of the NOEs.
T379 17002-17169 Sentence denotes There are some distinct experimental features for the discrimination between tr-NOEs from the bound state and NOEs of the ligand in free states like the build-up rate.
T380 17170-17265 Sentence denotes For tr-NOEs, this is in the range of 50 to 100 ms, whereas for small ligands it is much longer.
T381 17266-17394 Sentence denotes There have been various instances of experimental implementations to quickly determine the binding activity of ligand libraries.
T382 17395-17558 Sentence denotes One example was to find the ligand molecule among a library of 10 similar structure polysaccharides that is bioactive in binding with recombinant E-selectin [283].
T383 17559-17644 Sentence denotes This is a protein present in an IgG chimera with a molecular weight of about 220 kDa.
T384 17645-17694 Sentence denotes In this case, two 2D NOESY spectra were recorded.
T385 17695-17947 Sentence denotes The NOESY spectra for the ligand library was measured at several temperatures and it was found that most of the 10 compounds exhibited the weak positive NOEs at 310 K, which was then chosen to differentiate between trNOEs showing large negative values.
T386 17948-18100 Sentence denotes The trNOESY spectra of the ligand library in the presence of protein was recorded at different ratios, such as 5:1, 8:1, 12:1, 15:1, and 20:1, at 310 K.
T387 18101-18204 Sentence denotes In all the ratios, trNOEs were observed; however, the ratio of 15:1 represented the best-case scenario.
T388 18206-18212 Sentence denotes 3.1.5.
T389 18213-18258 Sentence denotes The INPHARMA Method for Pharmacophore Mapping
T390 18259-18477 Sentence denotes The INPHARMA method (see Figure 10) was designed to determine the relative orientation between two competitive ligands in the receptor-binding pocket through the observation of inter-ligand NOE between the two ligands.
T391 18478-18614 Sentence denotes It is a tr-NOE in nature as it is mediated by the bound conformation of the competing ligands and in exchange with the receptor protein.
T392 18615-18790 Sentence denotes The first example was competitive binding and observation of inter-ligand NOE between baccatin III and epothilone A in the presence of tubulin, which acts as a receptor [284].
T393 18791-18866 Sentence denotes Since the observation is on the ligand site, it provides unique advantages.
T394 18867-18958 Sentence denotes The detailed conformation of a ligand-protein complex can be addressed by conventional NMR.
T395 18959-19067 Sentence denotes However, it is time-consuming and demands full solving of the structure and there is also a size limitation.
T396 19068-19123 Sentence denotes From that aspect, ligand-based methods are more useful.
T397 19124-19320 Sentence denotes The only limiting fact is that it should fulfill all the conditions of tr-NOE explained previously in terms of dissociation constant (Kd), fast exchange regime, and proper ligand to protein ratio.
T398 19321-19428 Sentence denotes Then, information on the ligand structure can be derived from tr-NOE build up as a function of mixing time.
T399 19429-19506 Sentence denotes This can be readily explained using the originally proposed schematics [284].
T400 19507-19621 Sentence denotes The NOESY spectrum of a mixture of the two ligands A and B in the presence of the common receptor (T) is recorded.
T401 19622-19873 Sentence denotes Under the situation that each of A and B exhibit competitive binding in a fast exchange regime with the receptor T, intermolecular tr-NOE peaks between the two ligands A and B can then be observed in the NOESY spectrum due to extensive spin diffusion.
T402 19874-20017 Sentence denotes During the NOESY mixing time, the first proton of ligand A (HA) binds to receptor T, which results in transfers of magnetization from HA to HT.
T403 20018-20202 Sentence denotes Subsequently, the complex AT dissociates as they fulfill the dissociation constant range, which creates the opportunity for ligand B to bind to the receptor T at the same binding site.
T404 20203-20308 Sentence denotes This results in the transfer of the magnetization of HT, which had been originally coming from HA, to HB.
T405 20309-20454 Sentence denotes As a result, an inter-molecular correlation HA–HB can be seen, and this inter-molecular NOE will be a function of mixing time as described above.
T406 20455-20591 Sentence denotes The detailed analysis of such intermolecular NOE peaks helps in assessing the relative orientation of each ligand in the binding pocket.
T407 20593-20599 Sentence denotes 3.1.6.
T408 20600-20643 Sentence denotes Diffusion Based Spectroscopy in Drug Design
T409 20644-20762 Sentence denotes Diffusion is the random, translational motion of molecules in solution as a consequence of their thermal energy [285].
T410 20763-20926 Sentence denotes This type of motion is often referred to as “Brownian motion”, a motion that describes molecular movement induced by random collisions between the molecules [286].
T411 20927-21126 Sentence denotes In the presence of a concentration gradient, molecules will naturally move from places of higher concentration to places of lower concentration [287] after a period of time, t, as shown in Figure 11.
T412 21127-21187 Sentence denotes Fick’s Law can be used to model this type of movement [288].
T413 21188-21387 Sentence denotes The distribution of the diffusing molecules is accurately represented by a Gaussian curve, a normal distribution centered at a single point, which gradually “flattens” as t approaches infinity [213].
T414 21388-21483 Sentence denotes The extent to which a molecule diffuses is directly related to its shape, size, and mass [285].
T415 21484-21717 Sentence denotes In homogeneous isotropic solutions, the root mean square distance (zrms) traveled by a molecule is given by following equation [289,290]:zrms=(2Dt)12  where D is the diffusion coefficient of the molecule, and t is the diffusion time.
T416 21718-22072 Sentence denotes Making the assumption that the molecules are solid rigid spheres, the value of D can be calculated according to the famous Einstein-Stokes equation (Equation (2)):(4) D=kbT6πηrs  where kb is the Boltzmann’s constant (1.3807 × 10−23 J/K), T is the absolute temperature, η is the solution viscosity, and rs is the hydrodynamic radius of the molecule [290].
T417 22073-22315 Sentence denotes Equation (1) and Equation (2), however, are not universally applicable; they only apply to molecules that are freely diffusing in isotropic, homogeneous solutions, and importantly that can be accurately described as hard, rigid spheres [285].
T418 22316-22528 Sentence denotes Different molecular geometries and additional modes of diffusion (i.e., restricted and anisotropic) require more advanced mathematics and theory [291,292], but the essential concepts of diffusion remain the same.
T419 22529-22679 Sentence denotes The earliest pulse sequence used to measure diffusion in NMR spectroscopy is the gradient spin echo sequence (SE), developed by Stejskal et al. [293].
T420 22680-22724 Sentence denotes The SE pulse sequence is shown in Figure 12.
T421 22725-22911 Sentence denotes The SE pulse sequence uses a gradient (G) of the externally applied magnetic field, (pulsed field gradient), the first after the 90° pulse, and the other after the 180° refocusing pulse.
T422 22912-23043 Sentence denotes The first gradient pulse (G1) labels or gradient-encodes the NMR-active nuclei based on their physical position in the sample tube.
T423 23044-23190 Sentence denotes If the molecules diffuse during the time period they are not in the correct position to experience the second gradient which re-focuses the spins.
T424 23191-23247 Sentence denotes This is detected via NMR as a signal intensity decrease.
T425 23248-23440 Sentence denotes After a diffusion time (∆), the second gradient pulse is applied to decode the spatial labeling of NMR-active nuclei, obtaining a well-defined spectra of diffusing molecules in solution [294].
T426 23441-23595 Sentence denotes Additional NMR sequences are available for diffusion experiments [295], and are detailed in more comprehensive reviews dealing with the subject [296,297].
T427 23596-24032 Sentence denotes The signal intensity of the diffusing molecules depends on three factors, as described by Equation (3) [294]:(5) I=I0e−Dγ2g2δ2  where I is the observed intensity, I0 the reference intensity (unattenuated signal intensity), D is, of course, the diffusion coefficient referred to earlier, γ is the gyromagnetic ratio of the observed nucleus, g is the strength of the gradient, δ the length of the gradient, and ∆ the diffusion time [294].
T428 24033-24221 Sentence denotes From Equation (3), it is easy to see that the signal intensity decreases exponentially with time, so it is vital to optimize the values of g, δ, and ∆ for diffusion NMR measurements [294].
T429 24222-24388 Sentence denotes The drug design approach based on diffusion NMR is basically a screening technique used to differentiate the binding ligands (drug) from non-binding components [264].
T430 24389-24620 Sentence denotes Ligands able to bind should have significantly different diffusion coefficients (D) compared to non-binding ligands [297], i.e., the diffusion coefficients of binding ligands will be smaller than those of non-binding ligands [264].
T431 24621-24731 Sentence denotes Thus, diffusion-based NMR is a way of effectively “filtering” and identifying which ligands are binding [264].
T432 24732-24832 Sentence denotes Diffusion-based NMR spectroscopy has advantages in ligand based screening applied to drug discovery.
T433 24833-24967 Sentence denotes For example, Diffusion Ordered Spectroscopy (DOSY) does not require prior separation/purification of the ligand/target solution [298].
T434 24968-25380 Sentence denotes Diffusion based NMR allows simultaneous determination of diffusion coefficients in multicomponent systems containing large molecules (i.e., proteins) and possible binding partners (i.e., small drug compounds) [285], and no special labeling or contrasting agents are required, though their use is not exclusively inhibited (for an example of the use of labeled compounds in diffusion NMR spectroscopy, see [299]).
T435 25381-25499 Sentence denotes A problem occurs when there is significant chemical shift overlap between the binding molecule signals and the target.
T436 25500-25674 Sentence denotes This situation makes it hard to distinguish the NMR signals [300], and the calculations typically assign an intermediate value to the diffusion rate (i.e., one gets a smear).
T437 25675-25807 Sentence denotes Multidimensional diffusion NMR pulse sequences are available [301], which may help resolve spectral overlap in 1D experiments [300].
T438 25808-25906 Sentence denotes Another issue is that molecules in chemical databases may have generally low solubility [302,303].
T439 25907-26049 Sentence denotes Low solubility decreases the overall signal intensity and therefore makes accurately measuring diffusion experiments far more difficult [304].
T440 26050-26222 Sentence denotes There are many examples demonstrating the successful application of diffusion NMR in examining drugs of pharmaceutical interest [305], and ligand-target interactions [167].
T441 26223-26381 Sentence denotes Hajduk et al. [167] exploited the changes in diffusion rates to detect ligands that bind to the FK506 binding protein and the catalytic domain of stromelysin.
T442 26382-26647 Sentence denotes Nishimura et al. [306] utilized DOSY, in combination with NOESY to determine the orientation of two guest molecules, p-ethoxyiodobenzene and 2-iodo-6-methoxynaphthalene, within a host composed of a tetrakis(4-hydroxyphenyl)-cavitand and a tetra(4-pyridyl)-cavitand.
T443 26648-26879 Sentence denotes Furthermore, Matthias et al. [307] used 1H molecular diffusion and 19F spin diffusion to probe the drug loading properties of the Rf-PEG hydrogel for 5-fluorouracil (FU) and 1,3-dimethyl-5-fluorouracil (DMFU), two anticancer drugs.
T444 26880-27032 Sentence denotes DOSY can be combined with Saturation Transfer Difference (STD, discussed earlier in this review) to yield new insights about ligand-target interactions.
T445 27033-27185 Sentence denotes Kramer et al. [308] combined STD with DOSY to analyze a mixture composed of wheat germ agglutinin and two derivatives of N-acetyl glucosamine (ligands).
T446 27186-27290 Sentence denotes Using this new technique they were able to obtain high quality spectra of the components in the mixture.
T447 27291-27413 Sentence denotes Tanoli et al. [309] also combined STD and DOSY to explore the interactions of smaller molecules with bovine serum albumin.
T448 27414-27555 Sentence denotes These are just a few examples to show that diffusion NMR spectroscopy has played, and will continue to play, a prominent role in drug design.
T449 27557-27561 Sentence denotes 3.2.
T450 27562-27618 Sentence denotes NMR and In Silico Screening-Two Complementary Approaches
T451 27619-27829 Sentence denotes In silico (virtual) screening is now a standard technique in drug design and discovery [310] that has been in use since at least 1991 [311], though the exact origin of the phrase “in silico” is not clear [312].
T452 27830-28199 Sentence denotes The nearly ubiquitous use of virtual screening is due to its efficiency in searching massive chemical databases in order to generate lead molecules [313] that inhibit protein-protein interactions [314], and its ability to help identity ligand (drug) binding sites on the target of interest [310] to lend insight to the mechanisms of action for lead compounds [315,316].
T453 28200-28418 Sentence denotes Virtual screening is often accompanied by in vitro or in vivo techniques for pharmacology drug research [312], to increase drug throughput, helping to reduce the time and cost of developing novel drug candidates [317].
T454 28419-28533 Sentence denotes Virtual screening has also been used to identify candidates for anti-viral drugs [318] and anticancer drugs [319].
T455 28534-28614 Sentence denotes Several chemical databases are available both for public and academic use [320].
T456 28615-28795 Sentence denotes Virtual screening is properly identified as a high-throughput screening (HTS) technique [321], though using its full capacity as an HTS technique is not required for most purposes.
T457 28796-29076 Sentence denotes Virtual screening requires a minimum of two inputs, (1) a three-dimensional model of the ligand (drug), and (2) a three-dimensional model of the receptor (protein) [322], the latter generated from the atomic studies of proteins via X-ray crystallography or NMR spectroscopy [323].
T458 29077-29374 Sentence denotes Virtual screening is not a truly “stand-alone” technique and has often been combined with additional biophysical techniques besides NMR spectroscopy and/or X-ray crystallography [324], such as differential scanning fluorimetry [325], fluorescence polarization, and surface plasmon resonance [324].
T459 29375-29547 Sentence denotes In this section, we briefly introduce how virtual screening has been combined with NMR spectroscopy, and how they are complementary approaches to each other in drug design.
T460 29548-29742 Sentence denotes The complete details of how virtual screening works, and how it applies to drug design outside of its combination with NMR is well documented in additional reviews [310,322,326,327,328,329,330].
T461 29743-30010 Sentence denotes A prime example of the complementarity between NMR screening and virtual docking is found in the work of Chen et al. [331], in which the authors sought to target the A2A adenosine receptor (A2AAR) protein, a drug target for the treatment of Parkinson’s disease [332].
T462 30011-30176 Sentence denotes They used virtual screening and an NMR-based screening method against the same 500 molecules in a fragment library so they could compare the results of both methods.
T463 30177-30448 Sentence denotes The virtual screen successfully predicted (based on calculated binding affinities) four out of the five orthosteric ligands discovered by NMR that were within the top 5% of the fragment library, showing that the two separate methods can give similar and reliable results.
T464 30449-30753 Sentence denotes Later on, Chen et al. discovered that virtual screening picked up three additional fragments that remained undetected by the NMR-based method, and were, in fact, A2AAR ligands; this shows that though neither method is flawless, they are still perfectly complementary approaches for drug design [322,331].
T465 30754-30991 Sentence denotes In another scientific work that integrated NMR with virtual screening, Di Lello et al. [333] found small molecular inhibitors of the enzyme ubiquitin specific protease 7 (USP7), a key regulator of the tumor suppressor protein, p53 [334].
T466 30992-31136 Sentence denotes A fragment screen by NMR revealed a series of small molecules that bind in the active site of USP7 near the catalytic cysteine (amino acid 223).
T467 31137-31381 Sentence denotes A ligand-based virtual screen utilizing the fastROCS program identified ~30 hit molecules, several of which were further characterized by 1H-15N TROSY chemical shift perturbation and line broadening to probe the binding site of the active hits.
T468 31382-31599 Sentence denotes Di Lello. also tested the active compounds against EOL-1 cells to verify the hits as identified by virtual screening and further characterized by NMR, showing that the active compounds do indeed inhibit USP7 activity.
T469 31600-31846 Sentence denotes Through additional study of the active molecules and further optimization of their structures, they eventually discovered a series of ligands that bind in the “palm” region of the catalytic domain of USP7, inhibiting its catalytic activity [333].
T470 31847-31999 Sentence denotes This study clearly demonstrates that NMR screening-based techniques can be combined with virtual screening to find viable drugs for targets of interest.
T471 32000-32249 Sentence denotes Additional examples of the successful integration of NMR and virtual screening as applied to protein targets are also found in the literature, further demonstrating the practicality and complementarity of virtual screening and NMR [329,335,336,337].
T472 32250-32667 Sentence denotes For example, Li et al. [338] used virtual screening filtered by NMR to identify and characterize non-metal chelating metallo-β-lactamase (MBL) inhibitors, and in particular, Verona integron-encoded MBL (VIM)-2, when previously there were no clinically significant inhibitors of MBL, since MBL enzymes hydrolyse many, if not all, β-lactam antibacterials compounds specifically designed to inhibit their activity [339].
T473 32668-32789 Sentence denotes Furthermore, Shan et al. [340] and Bertini et al. [337] both used virtual screening and NMR, in their respective studies.
T474 32790-32984 Sentence denotes Through the combined use of NMR and virtual screening, Shan et al. was able to identify, design, and synthesize novel PDZ domain inhibitors, which are proteins implicated in tumorigenesis [340].
T475 32985-33156 Sentence denotes Bertini et al. was able to combine NMR to study the interaction of ligands with metalloproteinases, using known inhibitors of metalloproteinases as a starting point [337].
T476 33157-33348 Sentence denotes While HSQC NOESY NMR data provided structural and spatial constraints for the proposed 3D models, virtual screening was used to refine the models, and to probe the ligand-protein interaction.
T477 33349-33585 Sentence denotes In each case (i.e., ligand-protein interaction), Bertini et al. was able to obtain a well-defined ligand conformation in the protein binding site, thus offering a viable alternative to other approaches described in the literature [337].
T478 33586-33748 Sentence denotes Clearly, combining virtual screening with NMR-based methods is advantageous in studying how ligands (drugs) bind and interact with targets (proteins) of interest.
T479 33750-33754 Sentence denotes 3.3.
T480 33755-33795 Sentence denotes Paramagnetic Resonance in Drug Discovery
T481 33796-34010 Sentence denotes Paramagnetic NMR (PNMR) can also play a prominent role in drug discovery [341], as PNMR can provide key structural information in situations where crystal structures cannot due to the weak binding of ligands [341].
T482 34011-34128 Sentence denotes PNMR can be used to quantify the binding between ligands and large biomolecules such as proteins, DNA, and RNA [342].
T483 34129-34415 Sentence denotes PNMR depends on the presence of a group (called the paramagnetic center) with an unpaired electron [343], and since many naturally occurring biomolecules and organic compounds lack a paramagnetic center, one such as caged lanthanide (CLaNP) [344], must be introduced artificially [341].
T484 34416-34601 Sentence denotes Once the paramagnetic center (often a metal ion) is present, paramagnetic effects can be used to measure the distance and the relative orientation (i.e., angle) between molecules [345].
T485 34602-34691 Sentence denotes This information is crucial when it comes to determining how ligands and substrates bind.
T486 34692-34788 Sentence denotes Thus, PNMR is quite a useful technique for drug discovery when a paramagnetic center is present.
T487 34789-35022 Sentence denotes The most relevant consequence of PNMR for drug discovery is paramagnetic relaxation enhancement (PRE), although there are a number of studies demonstrating the use of pseudocontact shift (PCS) effect in drug discovery research [341].
T488 35023-35266 Sentence denotes Paramagnetic relaxation enhancement (PRE) is proportional to the inverse sixth power of the distance between the paramagnetic center and the nucleus of interest (i.e., 1H), although it does not reveal anything about relative orientation [341].
T489 35267-35343 Sentence denotes PRE can give quantitative information in the range of 10–25 Angstroms [346].
T490 35344-35531 Sentence denotes Several researchers have taken advantage of this outstanding property to study the structural and dynamic properties of complex biomolecular machineries in their native environment [347].
T491 35532-35887 Sentence denotes For example, Iwahara et al. (2003) demonstrated that a protein’s binding polarity to DNA can be determined by PRE, using EDTA-derivatized deoxythymidine (dT-EDTA) with a chelated metal ion (such as Cu2+ or Mn2+) as a probe. dT-EDTA with a chelated metal ion is a convenient choice, as it can be inserted into any position of a synthesized oligonucleotide.
T492 35888-36011 Sentence denotes With data derived from the PRE effect, one can easily determine the polarity of the protein (or drug) binding to DNA [348].
T493 36012-36319 Sentence denotes Several researchers have investigated DNA as a drug target [349], and the study of Iwahara et al. clearly demonstrates, and even indicates, that PRE can potentially be used to study the interactions between a drug and DNA [348], provided that a paramagnetic center such as dT-EDTA or a metal ion is present.
T494 36320-36428 Sentence denotes Brasuń et al. [350] also used PRE derived distances between a paramagnetic center and a nucleus of interest.
T495 36429-36609 Sentence denotes They replaced the Cys-S-S-Cys bridge found in oxytocin and vasopressin with the His-Cu2+-His motif to investigate if doing so would alter the stability of oxytocin and vasopressin.
T496 36610-36821 Sentence denotes They determined the distances between the Cu2+ ion and 1H nuclei (possible because of PRE), and used these values to generate three-dimensional models of the His-Cu2+-His motifs in both oxytocin and vasopressin.
T497 36822-37144 Sentence denotes In doing so, they indicated that such an approach using PRE can help in designing new biologically active compounds [350], and hence in drug discovery research, as many drug discovery studies require a reliable models for the successful generations of hit-lead molecules, especially in the case of in silico docking [351].
T498 37145-37240 Sentence denotes This study again proves the usefulness of PRE, and therefore, PNMR, in drug discovery research.
T499 37241-37382 Sentence denotes In two additional studies, Huang et al. [352,353] used PRE in their individual studies of protein binding and protein dynamics, respectively.
T500 37383-37546 Sentence denotes In the Huang et al. case [352], these authors used PRE to establish a model of the binding between the G-actin protein, and thymosin β4, an actin- binding protein.
T501 37547-37715 Sentence denotes Using PRE determined constraints (distances) and 1H-15N HSQC, they were able to establish a well-converging docking structure of the G-actin/thymonsin β4 complex [352].
T502 37716-37940 Sentence denotes On the other hand Huang et al. [353] did not measure protein binding, but studied the conformational changes and dynamics of select large membrane proteins utilizing 19F-NMR spectroscopy, and Ni2+ as the paramagnetic center.
T503 37941-38456 Sentence denotes Through a series of extensive experiments, they showed that conformational exchange rates of membrane proteins can be determined from measurements of the metal-enhanced longitudinal relaxation (i.e., PRE) of the 19F nuclei [353], thus yielding additional information (i.e., protein conformation dynamics) that could be utilized in drug discovery projects targeting proteins (i.e., understanding how the protein changes shape based on its environment can be used to find potential binding sites for drug candidates).
T504 38457-38720 Sentence denotes All these examples prove that PNMR is powerful approach in drug discovery research, given that PRE can aid in generating trustworthy models of interacting molecules, and that it can help researchers understand better how the molecules interact in the first place.
T505 38722-38726 Sentence denotes 3.4.
T506 38727-38760 Sentence denotes Solid State NMR in Drug Discovery
T507 38761-38911 Sentence denotes Since the late 1970s solid state NMR (ssNMR) has demonstrated its usefulness in complex biomolecular systems such as collagen or lipid bilayers [354].
T508 38912-39161 Sentence denotes However, over the past years ssNMR has gained attention in the field of drug design and is slowly becoming a commonly used technique as its proving to be a powerful tool for structural analysis of membrane proteins and amyloid fibrils [354,355,356].
T509 39162-39240 Sentence denotes ssNMR is becoming a more attractive alternative for several different reasons.
T510 39241-39386 Sentence denotes One of them is the fact that it enables the characterization of a chemical compound in a solid-state form such as in a tablet/pill [356,357,358].
T511 39387-40030 Sentence denotes Moreover, ssNMR is not only restricted to analyzing the chemical structure but it can also provide insight into the physical properties of a compound such as polymorphism (different crystalline structures of the same compound), disorder (crystal defects and amorphous solids in the compound) or the presence of cocrystals (multicomponent crystal made of a compound and one or more small organic molecules) [356,357]. ssNMR can also be used to quantify the amount of crystalline against the amount of amorphous material in the sample to establish phase purity (the amount of desired phase separated from other, undesirable phase) [356,357,358].
T512 40031-40111 Sentence denotes ssNMR differs from liquid state NMR by the presence of anisotropic interactions.
T513 40112-40207 Sentence denotes In liquids NMR these effects are averaged to zero as a consequence of rapid molecular tumbling.
T514 40208-40565 Sentence denotes In solid state however, the molecules are not tumbling rapidly and the residual effects of anisotropic (orientation depended) interactions such as anisotropic chemical shift, magnetic dipolar coupling, and quadrupolar coupling could be observed in the form of broad peaks, with could be much wider than the chemical shift range of the nucleus [355,358,359].
T515 40566-40751 Sentence denotes As a results, there has been a constant effort to improve the sensitivity and resolution of solid state NMR spectra, which increased the potential of ssNMR in future applications [360].
T516 40752-40857 Sentence denotes One of the methods that works for nuclei with spin value of I = 1/2 is called magic-angle spinning (MAS).
T517 40858-40976 Sentence denotes It increases the resolution by rapidly rotating the sample around a fixed (or so-called magic) angle of 54.736° [360].
T518 40977-41068 Sentence denotes This method can be combined with decoupling, to remove the dipolar couplings between spins.
T519 41069-41255 Sentence denotes This is done by applying radiofrequency pulses or cross-polarization (CP) transfer of magnetization from abundant and sensitive nuclei such as 1H to less sensitive such as 13C [328,333].
T520 41256-41333 Sentence denotes A broader comparison between ssNMR and liquid state NMR is provided in [361].
T521 41334-41423 Sentence denotes As mentioned before, ssNMR can provide information about membranes and membrane proteins.
T522 41424-41856 Sentence denotes For this reason, ssNMR can be used to detect interactions of ligands with receptors embedded to the membrane which enables the mapping of binding site of a receptor by utilizing CP-MAS (cross-polarization magic-angle spinning) NMR and site specific mutagenesis [355]. ssNMR can provide the conformation of ligands bound to the receptor which can then be used to optimize future drug in terms of better affinity and efficiency [355].
T523 41857-42014 Sentence denotes Since ssNMR is also applicable to amyloid research, it can be used for probing polypeptide structures of amyloid and intermolecular contacts between fibrils.
T524 42015-42130 Sentence denotes The potential is for the design a drug that will inhibit the process of aggregation of proteins and peptides [355].
T525 42131-42341 Sentence denotes Lastly, since ssNMR gains insight into physical properties of a chemical compound it can be used for control of the process of formulation and processing of a drug to help assess the purity of a compound [358].
T526 42342-42505 Sentence denotes An example of ssNMR application related to drug design is the work of Callari et al., who monitored the effect of drug loading on the properties of micelles [362].
T527 42506-42692 Sentence denotes Polymer micelles are widely used as nano-carries for drug delivery, but so far the effects of drug loading on the morphology of a drug carrier had not been thoroughly investigated [362].
T528 42693-42846 Sentence denotes They created a model consisting of a fructose hydrophilic block and a PMAA block (micelle), to which a different amount of platinum complex was anchored.
T529 42847-43004 Sentence denotes The results from this experiment showed that micelles loaded with a higher amount of platinum complex had reduced cellular uptake, release, and cytotoxicity.
T530 43005-43238 Sentence denotes The micelles with a lower load (LL) of platinum complex were more effective at targeting cancer cells (of cell lines MDA-MB-231 (breast cancer) and A549 (lung cancer) than the micelles with a higher load (HL) of the platinum complex.
T531 43239-43372 Sentence denotes This is evidenced by the lower IC50 (half maximal inhibitory concentration) values of the LL micelles as compared to the HL micelles.
T532 43373-43525 Sentence denotes Both of those results could be related to the micellar structure and their potential for interaction between the sugar moieties and the cell wall [362].
T533 43526-43731 Sentence denotes Another example of practical application of ssNMR is the work of Lee and colleagues [363] in which they investigated the structure of a designed zinc-binding amyloid fibril that catalyzed ester hydrolysis.
T534 43732-43865 Sentence denotes Metals ions such as zinc where found to affect the process of protein aggregation which resulted in arise of amyloid like structures.
T535 43866-44024 Sentence denotes Therefore, understanding the processes of aggregation and the factors related to them is crucial for creation of new drugs for amyloid related diseases [364].
T536 44025-44153 Sentence denotes In the experiment Lee et al. used Ac-IHVHLQI-CONH2 peptide (referred as HHQ) to form fibrils with varying Zn2+:HHQ molar ratios.
T537 44154-44384 Sentence denotes The results showed that Zn2+-bound HHQ fibrils form parallel-in-register form of packing β-strand in each sheet and His residues are coordinated to Zn2+ via Nδ1, while half of the His residues are also coordinated to Zn2+ via Nε2.
T538 44385-44443 Sentence denotes Additionally, Zn2+ binds in a 1:1 metal ion/peptide ratio.
T539 44444-44607 Sentence denotes After further analysis using structural bioinformatics, it was concluded that each zinc ion was coordinated by three histidine nitrogens from two adjacent strands.
T540 44608-44692 Sentence denotes Half of all histidines bridged to Zn2+ ions forming a metal–imidazolate chain [363].
T541 44694-44698 Sentence denotes 3.5.
T542 44699-44728 Sentence denotes NMR Validation in Drug Design
T543 44729-44912 Sentence denotes A “hit” is a molecule identified from a screening technique (HTS, FBDD, etc) as having a desirable effect (i.e., decreased cellular growth, high affinity score) on a target [365,366].
T544 44913-45094 Sentence denotes However, the question of whether the activity is related to actual binding to the target, or to interference with one of the components of the assay readout mechanism, is uncertain.
T545 45095-45131 Sentence denotes Thus, a validation step is required.
T546 45132-45295 Sentence denotes Hit-validation is therefore the process of confirming, or validating, that the molecule(s) identified previously have on target activity and selectivity [367,368].
T547 45296-45384 Sentence denotes One of the highest-impacts of NMR on drug discovery is the use as a hit-validation tool.
T548 45385-45557 Sentence denotes Though the hit-validation or confirmation of drugs is mostly limited to the solution state [369], this aspect of NMR truly is a “gold standard” technique in drug discovery.
T549 45558-45900 Sentence denotes NMR by itself is a powerful tool for drug validation as in the case of Sharma et al. (2012) [370] who sought to identify potential drug-like inhibitors against L-Aspartate α-Decarboxylase (ADC) an enzyme responsible for the decarboxylation of L-aspartate in order to generate β-alanine and carbon dioxide [371], in Mycobacterium tuberculosis.
T550 45901-46008 Sentence denotes They began with known inhibitors of ADC, and developed a protocol to measure the enzymatic activity of ADC.
T551 46009-46301 Sentence denotes Upon addition of ADC to a solution of L-aspartate, L-aspartate gradually disappeared because ADC was converted to L-aspartate to β-alanine; therefore the peak intensity of L-aspartate decreased, and the peak intensity of β-alanine increased in the presence of ADC (no inhibitor drug present).
T552 46302-46530 Sentence denotes Using this newly developed NMR-based protocol allowed direct measurement of ADC enzymatic activity, and Sharma et al. were able to confirm the enzymatic inhibiting activity of seven previously discovered inhibitors of ADC [370].
T553 46531-46668 Sentence denotes This study demonstrated that NMR can be an effective validation tool for known drugs and for new drugs generated by a screening approach.
T554 46669-46755 Sentence denotes NMR is also able to remove false positives that emerge from biochemical screens [372].
T555 46756-47092 Sentence denotes For example, an aptly named technique called A La Assay to detect Reactive Molecules by Nuclear Magnetic Resonance (ALARM NMR) is able to eliminate false positives from HTS methods [373], and in the presence of a test compound or mixture, measures dithiothreitol (DTT)-dependent 13C chemical shift changes of the human La antigen [373].
T556 47093-47320 Sentence denotes Dahlin et al. provided an updated protocol of ALARM NMR to aid researchers in the production of the 13C-labeled La antigen reporter protein, in testing compounds with the La protein, and in the analysis of obtained NMR spectra.
T557 47321-47390 Sentence denotes Using ALARM NMR prioritized hits identified from HTS screening [374].
T558 47391-47719 Sentence denotes An example of ALARM NMR is found in the work of Dahlin et al., where they used this technique to test molecules that were assumed to be inhibitors of histone acetyltransferase (HAT) inhibitors, and from their studies, actually discovered that 65% (15 out of 23) of the most commonly reported HAT inhibitors were actually faulty.
T559 47720-47832 Sentence denotes They were actually nonselective interference compounds, not necessarily specific to the inhibition of HAT [375].
T560 47833-48012 Sentence denotes Thus, ALARM NMR (and NMR in general) served as a useful validation method, especially for unvalidated hits identified from biochemical screens [372] or other screening techniques.
T561 48013-48152 Sentence denotes The last example highlights the need for cross validation, or the combination of two or more techniques to verify identified chemical hits.
T562 48153-48219 Sentence denotes Of course, NMR is not the sole technique used for drug validation.
T563 48220-48462 Sentence denotes Most often, NMR drug validation is coupled with additional methods [367] such as surface plasmon resonance (SPR) [376,377] X-ray crystallography [377,378,379], isothermal calorimetry (ITC) [379], UV-Vis and/or fluorescence spectroscopy [380].
T564 48463-48630 Sentence denotes The work of Goudreau et al. is an excellent example of combining NMR with another biophysical technique, in this case X-ray crystallography, for drug validation [378].
T565 48631-48804 Sentence denotes A series of benzodiazepine inhibitors of Human immunodeficiency virus 1 (HIV-1) was identified using an in vitro capsid assembly assay, and further characterized by 19F-NMR.
T566 48805-49001 Sentence denotes Analysis of the chemical shift perturbation and line broadening effect on the 19F-NMR spectra of the benzodiazepine inhibitors revealed the specificity and reversibility of the binding inhibitors.
T567 49002-49145 Sentence denotes The same set of 19F-NMR spectra were used to identify the N-terminal domain of the capsid as the binding site of the benzodiazepine inhibitors.
T568 49146-49309 Sentence denotes The specific amino acids involved in the binding of the benzodiazepine inhibitors were identified from the chemical shift perturbation of 1H,15N-TROSY NMR spectra.
T569 49310-49532 Sentence denotes Later, use of X-ray co-crystallography confirmed binding locations of the benzodiazepine inhibitors and their binding modes, which was useful for further development and optimization of the benzodiazepine inhibitors [378].
T570 49533-49667 Sentence denotes The work of Goudreau et al. therefore showed how NMR could be used as a co-validation technique with another biophysical method [378].
T571 49668-49815 Sentence denotes NMR can be also coupled with multiple biophysical techniques to validate a molecule’s ability to inhibit protein-protein interactions (PPIs) [367].
T572 49816-50121 Sentence denotes An example of the combination of NMR with SPR and X-ray crystallography can be found in the work of Fry et al., where the authors sought to understand how the nutlin molecule inhibits MDM2-p53, a protein-protein interaction that has been an important cancer therapy target for several years [381,382,383].
T573 50122-50366 Sentence denotes Fry et al. [377] gradually deconstructed RG7112, the first nutlin molecule to enter clinical trials [384], into 11 fragments so they could study the inhibitory effect of RG7112 on the MDM2-p53 interaction by SPR, NMR, and X-ray crystallography.
T574 50367-50536 Sentence denotes SPR was used to determine the Kd values of the RG7112 fragments and confirmed that RG7112 and some of its fragments do bind to MDM2, inhibiting the MDM2-p53 interaction.
T575 50537-50642 Sentence denotes 1H,15N-HSQC NMR chemical shift perturbation was also used to assess and verify binding identified by SPR.
T576 50643-50833 Sentence denotes Of the six fragments of RG7112 confirmed by 1H,15N-HSQC NMR as binding to MDM2, SPR showed binding for five of them; thus, the two separate techniques were in good agreement with each other.
T577 50834-51040 Sentence denotes The fragments of RG7112 that were confirmed to bind by both SPR and 1H,15N-HSQC NMR were further studied with X-ray crystallography, which can tell precisely where and how the molecules bind to the protein.
T578 51041-51258 Sentence denotes Using co-crystallization, Fry et al. were able to obtain structures for several of the verified binding fragments in complex with MDM2 and were able to visualize the binding of the fragments to the MDM2 protein [377].
T579 51259-51444 Sentence denotes NMR is obviously a powerful drug binding validation tool, but it becomes much more powerful when coupled with additional biophysical techniques, as seen in the work of Fry et al. [377].
T580 51445-51656 Sentence denotes Dias et al. [379] took a similar approach as Fry et al. [377] in that they took known inhibitors of a protein-protein interaction, and dissected them into individual fragments to assess a protein’s drug-ability.
T581 51657-51797 Sentence denotes The interaction studied was that between the proteins von Hippel–Lindau (VHL), and the alpha subunit of hypoxia-inducible factor 1 (HIF-1α).
T582 51798-51987 Sentence denotes Twelve compounds (known inhibitors and derived fragments) were developed using a crystal structure of HIF-1α peptide bound to the stable multiprotein complex pVHL-elongin C:elongin B (VCB).
T583 51988-52229 Sentence denotes Each of these compounds was screened using three separate NMR techniques, Saturation Transfer Difference (STD), Carr–Purcell–Meiboom–Gill (CPMG) relaxation experiments, and WaterLOGSY (to assess drug binding and to predict drug binding mode.
T584 52230-52473 Sentence denotes Each compound that was unambiguously detected (i.e., the molecule was identified as successfully binding by at least two of the three NMR methods of STD, CPMG, and WaterLOGSY) was subjected to further analysis by ITC and X-ray crystallography.
T585 52474-52641 Sentence denotes ITC was used to determine the dissociation constants of binding molecules, and X-ray crystallography was used to confirm the binding mode predicted by the NMR studies.
T586 52642-52891 Sentence denotes Generally speaking, the designed fragments had similar ligands efficacies compared to the parent molecules but had much higher dissociation constants (Kd values), meaning that the fragments bound less tightly than the original parent molecule [379].
T587 52892-53257 Sentence denotes With this example, it is possible to see the strength of using NMR as its own hit-validation tool (i.e., three different NMR techniques were used for screening compounds [379]), and yet, the follow-up of NMR studies with ITC and X-ray crystallography was useful in providing a basis for assessing the drug-ability of a protein-protein interaction [385,386,387,388].
T588 53258-53420 Sentence denotes Thus, it is clear to see that NMR is a prominent method of hit-validation in drug discovery research, especially in combination with other biophysical techniques.
T589 53422-53426 Sentence denotes 3.6.
T590 53427-53484 Sentence denotes Other Methods Used to Determine the Drug-Target Complexes
T591 53485-53639 Sentence denotes Substantial progress has been made in the NMR field over the past 5–10 years, and various methods were established to determine the drug-target complexes.
T592 53640-53781 Sentence denotes Most of them utilize either NOE or chemical shift perturbations (CSP) although in silico models/programs, using NMR-derivate data also exist.
T593 53783-53789 Sentence denotes 3.6.1.
T594 53790-53799 Sentence denotes DIRECTION
T595 53800-53987 Sentence denotes One of the methods called difference of inversion recovery rate with and without target irradiation (DIRECTION) is used to map pharmacophores and can be an alternative to STD experiments.
T596 53988-54144 Sentence denotes This method uses the difference between longitudinal relaxation rates of ligand protons with- and with-out irradiation of the protons of the target protein.
T597 54145-54239 Sentence denotes The DIRECTION approach, however cannot be used for slowly exchanging (strong binding) ligands.
T598 54240-54439 Sentence denotes The practical approach of this method was demonstrated on the experiment when analyzed the interactions between p38 MAPK (p38 a mitogen-activated protein kinase) and its inhibitor-SB203580 [389,390].
T599 54440-54626 Sentence denotes The results from this experiment showed that protons H1, H4, H5, and H6 of SB203580, are in close neighborhood with the protons of p38 MAPK when compared with H2, H3, and methyl protons.
T600 54627-54747 Sentence denotes It indicates that two aromatic rings (a pyridine ring and fluorophenyl ring) of SB203580 interact tightly with p38 MAPK.
T601 54748-54896 Sentence denotes The results were later confirmed with proton density map of each ligand’s proton, based on the crystal structure of SB203580–p38 MAPK complex [391].
T602 54897-55063 Sentence denotes Moreover, the same authors already created a new and improved protein–ligand docking method by combining the DIRECTION obtained NMR data with docking software. [392].
T603 55065-55071 Sentence denotes 3.6.2.
T604 55072-55076 Sentence denotes ILOE
T605 55077-55188 Sentence denotes A second method that can be used to map pharmacophores is called inter-ligand nuclear Overhauser effect (ILOE).
T606 55189-55414 Sentence denotes This 2D NMR experiment detects when two ligands bind simultaneously to adjacent sites on a protein surface although both of the ligands do not have to bind to the same binding pocket (opposite to INPHARMA, see above) [5,393].
T607 55415-55615 Sentence denotes A negative ligand−ligand NOE signal will be created when ligands bind in close proximity to each other whereas ligands that do not bind will show no NOEs, or at most very weak positive ones [372,394].
T608 55616-55709 Sentence denotes ILOE also enables determination of the ligand orientations with respect to one another [393].
T609 55710-55834 Sentence denotes As in the case of INPHARMA, ILOE can be utilized even in the absence of a 3D protein structure and used with large proteins.
T610 55835-55965 Sentence denotes Additionally, ILOE differs from INPHARMA in mixing times—for ILOE the mixing times are typically in the range of 600–800 ms [345].
T611 55966-56234 Sentence denotes Application of ILOE was first shown on glycolate+NAD+ in the presence of porcine heart lactatedehydrogenase, and by glucose-6-phosphate+NADPH in the presence of L. mesenteroides glucose-6-phosphatedehydrogenase and from that time it has been widely used [393,395,396].
T612 56236-56242 Sentence denotes 3.6.3.
T613 56243-56250 Sentence denotes SOS-NMR
T614 56251-56514 Sentence denotes A third method called structural information using Overhauser effects and selective labeling (SOS-NMR), relies of STD experiments performed on ligand complexes with different protein samples that have been fully deuterated excluding a specific type of amino acid.
T615 56515-56757 Sentence denotes In other words, the data obtained by SOS-NMR gives insight into the ligand-binding amino acid composition and when taken into consideration the 3D structure of targeted protein can be used to establish the structure of protein-ligand complex.
T616 56758-56935 Sentence denotes This approach has been demonstrated using two complexes—FKBP complexed to 2-(3′-pyridyl)-benzimidazole and MurA complexed to uridine diphosphateN-acetylglucosamine (UDP-GlcNAc).
T617 56936-57017 Sentence denotes The results showed that for FKBP and MurA, only four and three amino acids (FKBP:
T618 57018-57043 Sentence denotes Ile, Val, Leu, Met; MurA:
T619 57044-57162 Sentence denotes Trp, Phe, His) were needed to be selectively protonated in perdeuterated samples to establish the ligand-binding site.
T620 57163-57275 Sentence denotes Additionally, on average only 6 amino acids were required for accurate identification of ligand-binding surface.
T621 57276-57378 Sentence denotes According to authors SOS-NMR can greatly improve the early stages of the drug discovery process [397].
T622 57379-57506 Sentence denotes Moreover, combining SOS-NMR with other methods can even further increase chances for a positive outcome of an experiment [398].
T623 57508-57514 Sentence denotes 3.6.4.
T624 57515-57535 Sentence denotes Tert-butyl Labelling
T625 57536-57617 Sentence denotes A completely different approach to this topic was taken by Chen et al. [399,400].
T626 57618-57798 Sentence denotes Instead of using isotope labeling, Chen’s group decided to use a tert-butyl group contained within ligand-1 to obtain structural information about the protein-ligand complex [400].
T627 57799-57945 Sentence denotes The tert-butyl group formed an intense singlet in 1.0 to 1.5 ppm range thanks to rapid methyl rotation and methyl reorientation within that group.
T628 57946-58229 Sentence denotes When compared with the protein’s 1H-NMR signal, the tert-butyl signal tended to be much narrower and resulted in easy detection without the need for isotopic enrichment even in protein complexes of high molecular mass such as Bacillus stearothermophilus DnaB hexamer (320 kDa) [399].
T629 58230-58409 Sentence denotes Additionally, the tert-butyl group produces intense NOESY cross peaks that can be observed even in the situations where normally cross-peaks of the proteins are barely detectable.
T630 58410-58500 Sentence denotes This is partially because the signal corresponded to nine protons within tert-butyl group.
T631 58501-58615 Sentence denotes Those aspects enable measurements of pseudo-contact shifts generated by paramagnetic tags attached to the protein.
T632 58616-58680 Sentence denotes As a result, it allows positioning of the ligand on the protein.
T633 58681-58836 Sentence denotes An example of this approach, is dengue virus NS2B-NS3 protease from serotype 2 (referred as DENpro) in complexed with ligand containing a tert-butyl group.
T634 58837-58957 Sentence denotes The result of this experiment showed NOEs between the tert-butyl group of ligand-1 and residue Val155 from DENpro [400].
T635 58959-58965 Sentence denotes 3.6.5.
T636 58966-58972 Sentence denotes SALMON
T637 58973-59150 Sentence denotes Solvent accessibility, ligand binding, and mapping of ligand orientation by NMR spectroscopy (SALMON) is another method based on the data obtained via nuclear Overhauser effect.
T638 59151-59409 Sentence denotes This method utilizes WaterLOGSY [401] to probe for solvent accessibility to the ligand and determine the orientation of the ligand by analyzing signal changes in WaterLOGSY spectra (positive signal from unbound ligand vs. negative for protein-bound ligands).
T639 59410-59601 Sentence denotes This method was first used to determine the orientation of prodrug called tretazicar ((5-(aziridin-1-yl)-2,4-dinitrobenzamide) known as CB1954 in NQO2 (quinone oxidoreductase 2) binding site.
T640 59602-59817 Sentence denotes Previous attempts had been made to obtain the orientation of tretazicar bounded to NQO2, however the results obtained from X-ray crystallography were inconclusive as two orientations of tretazicar could be possible.
T641 59818-60044 Sentence denotes The information obtained via SALMON showed that the side chain of asparagine at position 161 formed a hydrogen bond with 2-nitrogroup of tretazicar, and that the aziridine moiety of tretazicar pointed toward the solvent [401].
T642 60046-60052 Sentence denotes 3.6.6.
T643 60053-60068 Sentence denotes LOGSY Titration
T644 60069-60187 Sentence denotes Another variant of WaterLOGSY method called LOGSY utilizes the titration slopes as a measure of solvent accessibility.
T645 60188-60270 Sentence denotes The titration slopes are created by a constant increase of protein concentrations.
T646 60271-60415 Sentence denotes This method also provides more insight into the process of ligand solvation by checking the influence of protein concentration onto the process.
T647 60416-60577 Sentence denotes This approach was used on the bromodomain 1 of protein 4 (Brd4-BD1) by mapping epitopes of two ligands interacting with Brd4-BD1 and predicting ligands position.
T648 60578-60698 Sentence denotes The results showed that the triazolopyridazine moiety of both ligands was implanted into the binding pocket of the Brd4.
T649 60699-60875 Sentence denotes Additionally, the results from LOGSY titration showed that methyl-group 1 of ligand 1, aromatic proton 8 of ligand 2 and aromatic proton 8 of ligand 1 exhibit strong water NOE.
T650 60876-61105 Sentence denotes This information enabled researchers to utilize a chemical replacement strategy (substitute bound water molecules by suitable functional groups) for aromatic proton 8 in a series of ligands containing the triazolopyridazine ring.
T651 61106-61244 Sentence denotes Those protons were replaced with an amino or aminomethyl groups and as a result, the binding affinity of those ligands increased 100-fold.
T652 61245-61566 Sentence denotes Finally, the results obtained from X-ray crystallography for ligands with such modifications allowed to find the binding mode of the triazolopyridazine ring of ligand 1 (with methyl group pointing internally) and the substituted amino group was found to create hydrogen bond to the side chain of Asn140 of Brd4-BD1 [402].
T653 61568-61574 Sentence denotes 3.6.7.
T654 61575-61630 Sentence denotes Nuclear Magnetic Resonance Molecular Replacement (NMR2)
T655 61631-61838 Sentence denotes The most recent approach called Nuclear Magnetic Resonance Molecular Replacement (NMR2) utilizes spatial data obtained through solution-state NMR in order to locate the binding pocket of a complex structure.
T656 61839-62009 Sentence denotes For that, it uses a receptor model, e.g., a X-ray structure of a homolog, to conduct an analysis and at the same time excluding the need for protein resonance assignment.
T657 62010-62079 Sentence denotes To conduct an experiment using such an approach requires a few steps.
T658 62080-62174 Sentence denotes First, either the protein or ligand used in the complex must be uniformly 13C and 15N labeled.
T659 62175-62264 Sentence denotes Then, an experiment to assign the ligand is needed such as 2D 13C,1H-HMQC or 13C,1H-HMBC.
T660 62265-62428 Sentence denotes The next step is the evaluation of ligand intra- and ligand–protein intermolecular distances through NOE cross peaks obtained from F1-15N,13C-filtered 1H,1H-NOESY.
T661 62429-62607 Sentence denotes Lastly, choosing a proper input structure is required which can be either X-ray or NMR structures in apo form, with another bound ligand, or a homolog to the protein of interest.
T662 62608-62777 Sentence denotes Then the NMR2 program analyzes for all possible partial assignments (such as methyl groups of a protein) and calculates the complex structures for all options [403,404].
T663 62778-62910 Sentence denotes This method was already successfully used to resolve complex structures in case of slow and fast exchange ligands [403,404,405,406].
T664 62912-62918 Sentence denotes 3.6.8.
T665 62919-62924 Sentence denotes HECSP
T666 62925-63042 Sentence denotes In silico methods combined with NMR derived information can also be used to determine accurate drug-target complexes.
T667 63043-63178 Sentence denotes 1H empirical chemical shift perturbation (HECSP) is an empirical model that is based on chemical shift perturbation (CSP) of a protein.
T668 63179-63322 Sentence denotes CSP represents the change in chemical shifts in a protein due to alteration of its chemical environment (which can happen upon ligand binding).
T669 63323-63482 Sentence denotes The CSP of a target protein is obtained by a series of 2D HSQC experiments with a set of ligand titrations involving samples that contain 15N-labelled protein.
T670 63483-63560 Sentence denotes The calculation of 1H-CSPs inside the protein are based on four contributors:
T671 63561-63650 Sentence denotes 1) ring current, 2) electric field, 3) hydrogen bonding, and last 4) magnetic anisotropy.
T672 63651-63851 Sentence denotes To show the value of the HECSP model two CSP examples were used: apo-neocarzinostatin (apoNCS)-naphthoate ester complex, and human intestinal fatty acid binding protein (hIFABP)-ketorolac-ANS complex.
T673 63852-64043 Sentence denotes The results from the experiment showed that HECSP model can distinguish native ligand from decoys and more clearly define protein-ligand complex structures with NMR derived information [407].
T674 64045-64051 Sentence denotes 3.6.9.
T675 64052-64059 Sentence denotes SAMPLEX
T676 64060-64241 Sentence denotes Another program that can utilize CSP called Smoothed Automatic Mapping of Protein from Listed Extremes (SAMPLEX) can help to determine the interaction surface of proteins complexes.
T677 64242-64399 Sentence denotes SAMPLEX takes the chemical shifts of the protein of interests in both the free and bound state and corresponding 3D structure of a protein in the free state.
T678 64400-64583 Sentence denotes The programs returns a confidence value for each residue to be in a perturbed or unperturbed state (0.05 as being in a perturbed state, −0.05 as remaining in their unperturbed state).
T679 64584-64732 Sentence denotes This approach was tested on five examples, one of which was Subtilisin BPN’ (serine protease) complexed with its inhibitor–chymotrypsin inhibitor 2.
T680 64733-64871 Sentence denotes The results showed that residue 2, and residues 56–62 of chymotrypsin inhibitor-2 were perturbed and residue 63 was in an ambiguous state.
T681 64872-64978 Sentence denotes To compare, the X-ray crystallography data showed residues 50 and 54–61 to be involved in the interaction.
T682 64979-65155 Sentence denotes For subtilisin BPN’ the program predicted residues 33, 97, 99–109, 126-128, 141, 154–156, 167–171 and 218–219 to perturbed and residues 65, 98 and 220 to be in ambiguous state.
T683 65156-65311 Sentence denotes That information was also confronted with the X-ray crystallography data which shown residues 99–104, 125–128, 154–157, 167, 218–221 to be perturbed [408].