Compound Aggregation in Drug Discovery: Implementing a Practical

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Compound Aggregation in Drug Discovery: Implementing a Practical NMR Assay for Medicinal Chemists Steven R. LaPlante, Rebekah J. Carson, James R. Gillard, Norman Aubry, René Coulombe, Sylvain Bordeleau, Pierre R Bonneau, Michael Little, Jeff Alan OMeara, and Pierre L. Beaulieu J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400535b • Publication Date (Web): 03 Jun 2013 Downloaded from http://pubs.acs.org on June 3, 2013

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Journal of Medicinal Chemistry

Compound Aggregation in Drug Discovery: Implementing a Practical NMR Assay for Medicinal Chemists

Steven R. LaPlante*, Rebekah Carson, James Gillard, Norman Aubry, René Coulombe, Sylvain Bordeleau, Pierre Bonneau, Michael Little, Jeff O’Meara and Pierre L. Beaulieu

Department of Chemistry, Boehringer Ingelheim (Canada) Ltd., 2100 Cunard St., Laval, Quebec, H7S2G5, CANADA

ACS Paragon Plus Environment


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Abstract: The pharmaceutical industry has recognized that many drug-like molecules can selfaggregate in aqueous media and have physicochemical properties that skew experimental results and decisions.

Herein, we introduce the use of a simple NMR strategy for detecting the

formation of aggregates using dilution experiments that can be performed on equipment prevalent in most synthetic chemistry departments. We show that 1H NMR resonances are sensitive to large molecular-size entities and to smaller multimers and mixtures of species. Practical details are provided for sample preparation, and for determining the concentrations of single molecule, aggregate entities and precipitate. The critical concentrations above which aggregation begins can be found, and were corroborated by comparisons with light scattering techniques. Disaggregation can also be monitored using detergents. This NMR assay should serve as a practical and readily available tool for medicinal chemists to better characterize how their compounds behave in aqueous media and influence drug design decisions.

Introduction Medicinal chemists almost exclusively synthesize and characterize their candidate drugs in organic solvents, then expedite the powders or stock solutions to multiple other laboratories for a broad range of pharmaceutical tests where the compounds are dissolved in or diluted with aqueous media. However, drugs behave much differently in organic solvents as compared to aqueous media, and thus the above workflow introduces an important and uncharacterized disconnect.


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This is of interest to medicinal chemists because the pharmaceutical community is recognizing that a compound’s physicochemical properties in aqueous media influence drug behavior.1-4 Compound self-aggregation has been implicated in giving rise to false-positive (promiscuous) hits in high-throughput inhibition screens.5-7

Compound lipophilicity has been

implicated in inhibitor potency, but it can also influence undesirable off-target inhibition and toxicity.8-18 Using a simple computational strategy, chemists can easily calculate lipophilicrelated parameters such as partition coefficients (cLogP)a and total polar surface area (TPSA) to assist in design efforts and predicting off-target promiscuity and toxicity.1-2 Complementary to this approach, we introduce here an empirical-based NMR assay for directly observing and screening for compound aggregation in aqueous media.19 Using this assay, a preliminary report noted an improved correlation compared to descriptor-based computational methods; compounds that were observed to aggregate in our NMR assay also were found to be promiscuous inhibitors in off-target in vitro pharmacology screens.19 The improvement is due to direct observation of molecular aggregation in relevant aqueous media. In this work, we show that the NMR assay clearly can distinguish between compounds that are known from the literature to self-aggregate versus those that do not. Interestingly, these control compounds also expose a variety of aggregate and resonance trends. Particular attention is made so that medicinal chemists could easily implement this method as a practical and readily available tool in their workflow. This should empower front-line scientists with a means to expose critical drug properties such as self-aggregation tendencies and solubility issues, and also to predict compound promiscuity during lead identification and optimization campaigns.



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Results and Discussion Concept of the NMR assay for detecting compound aggregation. Compounds in aqueous solution adopt equilibria between three broad states, namely soluble single molecules, soluble aggregate entities and solid forms. These states can be clearly identified and distinguished in 1H NMR spectra. For soluble drug-like compounds that behave as fast-tumbling single molecules, one expects to observe sharp 1H NMR resonances.

No resonances are expected for solid

precipitates because they tumble too slowly resulting in resonances that are too broad to be observed by solution NMR. Soluble compounds that assemble into aggregates may, however, appear to be homogeneous by the naked eye, but the concentration-dependence of these assemblies results in unusual 1H NMR spectral features, thus revealing their existence. The concept of the NMR dilution assay came from the notion that aggregates should be sensitive to changes in concentration, and the 1H NMR resonances should report structural changes in the aggregate and its environment. Based on our experience, we propose a simplistic and practical strategy for applying and interpreting the data. If unusual NMR features are noted as a function of concentration, then the compounds are flagged as aggregators. This manuscript provides examples, demonstrates the uses of centrifugation and addition of detergents, and presents optional corroborating methodologies (light scattering) and shows concentration determinations (NMR and HPLC). Detection of aggregation using

1

H NMR spectra as a function of compound

concentration. The NMR assay involves the acquisition and monitoring of 1H NMR spectra as a function of compound concentration as illustrated in Figure 1. In panel A, the expected behavior and NMR resonance read-outs for non-aggregating compounds are displayed. Single molecules


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are distal to one another and tumble freely, and are not affected by dilution. Sharp NMR resonances are expected at all concentrations, and the resonances do not shift left or right. There should be no changes in the number and shape of the resonances. The only changes expected are the resonance intensities as a function of concentration. Panel B of Figure 1 shows the expected behavior and NMR resonance read-outs for aggregating compounds. At higher concentrations, the molecules can self-associate, whereas upon dilution they become more distal and tumble more freely. As a result, unusual NMR features and changes are expected (see bottom inset) due to changes in local environments and magnetic fields.

Figure 1. Shown are illustrations of how non-aggregating and aggregating molecules may behave in solution as a function of dilution. (A) Non-aggregator compounds (i.e. shown is Etodolac – structure shown in Figure 6) are expected to remain distal, tumble rapidly and move independently at all concentrations. (B) Aggregator compounds are expected to have unusual NMR spectral attributes due to attractive tendencies at higher concentration that dissociate upon dilution. The buffer employed for these studies was 50 mM sodium phosphate pH 7.4 in 100% D2O solvent.



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The bottom of Figure 1 lists the NMR parameters that are sensitive to these concentration studies, such as resonance number, shape, shifts and intensities. First, resonance number implies that the same number of peaks exist at all concentrations. In the event that multiple aggregate species exist in solution, the number of peaks may change upon dilution. Second, any shifts in resonance (δ ppm) would be the result of local environmental changes in the magnetic field around the molecule; another indicator of an aggregate species. Third, the shape of a resonance (i.e. sharp or broad) is related to the size and tumbling rate of a species. A larger species would be expected to have a slower tumbling rate and thus a broader peak shape. The broadening of a resonance at higher concentrations is evidence for the existence of a multimeric species. Lastly, the peak intensities should correlate with the changes in nominal (or intended) concentration.

There are some possible reasons why the resonance intensities are not

representative of concentration. A compound can be solubility limited in the buffer used in the NMR assay. Also, a compound can exist in equilibrium between the single molecule and aggregate states, and at higher concentrations above the single molecule solubility limit, additional compound is incorporated into an NMR-invisible aggregate. In order to differentiate between the above two scenarios, concentration determinations and the use of surfactants were employed (vide infra). These expected spectral observations are exemplified in Figure 2 for known aggregators and non-aggregators.20-22 In Figure 2A, the drug Riluzole,20A which is a known non-aggregator as determined by dynamic light scattering (DLS), existed mainly in the single molecule state in solution (sharp resonances) at all concentrations. All resonances were well-aligned (vertically),


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they were sharp and did not shift left nor right as the compound was diluted. As expected, the resonance intensities decreased as the compound was diluted.

Figure 2. Shown are four examples of the NMR assay where a series of 1H NMR spectra are superimposed from various concentrations of compound (dilutions from 200 µM to 12 µM. (A) The spectra are from the known non-aggregator Riluzol. Also shown are the spectra of known aggregator compounds (B) Methylene Blue, (C) Evans Blue and (D) Pranlukast. The buffer employed for these studies was 50 mM sodium phosphate pH 7.4 in 100% D2O solvent.

Such NMR spectral trends were considered normal and distinct from those found for aggregating compounds (Figure 2B-D) which had unusual spectral trends. Figure 2B shows the data for the known aggregator Methylene Blue where the resonances shifted to the left upon



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dilution. Figure 2C had both broad and sharp resonances at higher concentration, and some resonances shifted left. Figure 2D exemplified an interesting case where low concentrations of free molecules (small sharp resonances) were observed at all dilutions. The first impression was that such a compound must be very insoluble, but the solution was clear and precipitate-free to the naked eye. However, as mentioned above, the resonances of very large aggregates may be too broad to observe. To address this, a strategy involving the addition of a detergent was introduced. Detergent effect to reveal large aggregates. To determine if large assemblies existed, the NMR assay included a sample for which detergent was added to purposely break up the assemblies into smaller entities that were observable by NMR. We refer to this as the “detergent effect” and applications are shown below. The idea that detergents may break up aggregates was based on an earlier report from Shoichet and coworkers.6 Addition of detergent Triton™ X-100 in a counter-screen of a high throughput assay, eliminated 95% of the hits as false positives, presumably due to the non-stoichiometric binding effects of compound aggregation. Several theories were proposed elsewhere for how aggregates may be implicated in influencing inhibition assays.28 We rationalized that if detergents are capable of breaking up aggregates, the resulting smaller entities should likely be observable by NMR. We therefore tested the effects of the addition of the detergents Triton™ X-100 and Tween® 80 on the aggregate assemblies. As control experiments, NMR spectra were acquired on free detergents (no drug) to evaluate if the detergents introduced any spectral interferences. The Supporting Information shows that Triton-X-100 results in the appearance of two broad resonances at 6.7 and 7.1 ppm. On the other hand, Tween 80 had no interfering resonances


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within the strategic aromatic region. Thus, Tween 80 was the preferred detergent and was incorporated in the NMR assay as described in the Materials and Methods. The NMR tests with both detergents indeed were useful for breaking up aggregate assemblies. This was exemplified with two compounds as shown in Figure 3. The spectra from the dilution samples of Pranlukast in Figure 3A all had low intensity resonances due to low concentrations of single molecules; there was no observable precipitate. However, the addition of Tween 80 resulted in the emergence of large resonances due to smaller compound entities arising from the breakup of large aggregates. Thus, application of the detergent effect helped to reveal the presence of previously undetected aggregates and resolves this NMR detection issue referred here as the “NMR blind spot”. Trends such as these were also noted for many other compounds, and Figure 3B showed similar results for Congo Red for which aggregates were reported earlier using DLS and electron microscopy (see insert in Figure 3B).20B The presence of aggregates for Pranlukast was further corroborated by HPLC experiments that determined the concentrations of free plus aggregated compound. Figure 3A showed that the nominal concentrations were similar to the experimental concentrations. Given that the NMR resonances of free molecules of Pranlukast existed at very low concentration (low intensities in 12-200 µM spectra), then Pranlukast must have existed almost exclusively in the aggregated form in solution. This was also corroborated by light scattering data (vide infra).



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Figure 3. Examples of the application of the “Detergent Effect”. Shown on the left are the 1H NMR spectra of Pranlukast in aqueous solution at various concentrations. A spectrum from an additional sample was acquired at 200 µM (after mild centrifugation, collection of the supernatant, then addition of Tween 80 detergent to the sample containing the supernatant as described in a later section of this work). The spectra on the right are of Congo Red. The insert is an electron micrograph of Congo Red in solution.20B The buffer employed for these studies was 50 mM sodium phosphate pH 7.4 in 100% D2O solvent.

Combining centrifugation then detergent to reveal large aggregates. The observation of aggregates was clear in the above two examples in Figure 3, given that the three-state equilibria were heavily populated toward the aggregate state. However, interpretation of the detergent effect also had the potential for ambiguity for compounds that also significantly populated the solid state. For example, perhaps the addition of detergent simply influenced the solubility of the compound. Visual inspection of the dilution samples can help alert one to the latter possibility, but some solid states were not necessarily visible to the naked eye (vide supra). We noted micro-particles for some compounds that were only detected with a light microscope. Also, one cannot necessarily rely on visual inspection for higher throughput aggregate screening of larger libraries of compounds.


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To help resolve this, the NMR assay included an additional 200 µM sample that was subjected to mild centrifugation (2,000 rpm, 10 minutes) to separate solid compound (if it existed), then removal of the supernatant, then addition of the detergent. Thus, the emergence of detergent-induced resonances should only be from breaking up of large aggregates, and not from detergent-induced increases in solubility. A set of control experiments were acquired and are provided in Figure 4 for example purposes. 1H NMR spectra were acquired for three compounds at 200 µM (nominal concentration) but prepared differently.

For some of the samples,

precipitate was left in the tube, and for others the precipitate was removed by mild centrifugation. In Figure 4A, the bottom spectrum was from the shown compound at 200 µM. The sample was not centrifuged and had no observable resonances. The addition of Tween 80 resulted in the emergence of large resonances suggestive of the break-up of aggregates (second spectrum from the bottom). To distinguish whether the resonances arose from the breakup of aggregates or from detergent-induced solubility, spectra were acquired on a similar set of samples but the precipitate was removed by centrifugation then the supernatants collected. The addition of Tween 80 again resulted in the emergence of large resonances (top spectrum in Figure 4A), confirming that the large resonances arose from the breakup of aggregates. The concentrations determined by NMR (also shown in Figure 4A) show that most of the compound behaved as aggregates (112 versus 183 µM); whereas 71 µM was removed as precipitate (difference from a total of 183 µM before centrifugation). The opposite observations and conclusion were noted for the non-aggregator compound from the same series (Figure 4B). After removal of the precipitate using mild centrifugation, the



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addition of Tween 80 did not result in the appearance of resonances (top two spectra in Figure 4B). In this case, the compound exhibited detergent-induced solubility.

The data in Figure 4C

indicate that this compound partially aggregates at 24 µM and the majority of the compound existed as a precipitate. No significant amount of single molecule was observed for all three samples. In summary, this method allows one to clearly estimate the amounts of compound in all three states. This type of data can help in the interpretation of many types of analytical and biological assays, where compound aggregation leads to ‘promiscuous’ hits in affinity screens, variable performance in toxicity screens and animal trials, problems in formulation, etc. Although the whole set of control experiments were informative for proof-of-principle purposes, the routine use of the NMR assay only required the top spectrum (+ centrifuge, + Tween) for practical purposes. This effectively reduced the number of samples, allowing for NMR data acquisition during a single overnight period on a low field 400 MHz NMR spectrometer equipped with a sample changer.


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Figure 4. Centrifugation and detergent experiments. Samples of three compounds of the same antiviral series were prepared in buffer at 200 µM and were either centrifuged or not, and were exposed to Tween 80 or not. The 1H NMR spectra are displayed for the three compounds. The buffer employed for these studies was 50 mM sodium phosphate pH 7.4 in 100% D2O solvent.

More examples of unusual trends in the NMR assay. Our experience in applying the NMR assay for screening hundreds of compounds led us to note that compound aggregates could give rise to a variety of NMR observations. This is likely reflective of the variety of aggregate tendencies and molecular attributes that drugs can assume. noteworthy for recognition purposes. molecule benzyl benzoate.

Two more examples were

Figure 5A displayed the dilution data for the small

At low concentrations, benzyl benzoate appeared to have the

expected 1H NMR spectrum for single molecules. However, at higher concentrations a new set of resonances emerged at higher field, reporting that another type of entity of benzyl benzoate formed. It was intriguing that molecules as small as benzyl benzoate could adopt alternate solution entities that were concentration dependent.20A



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Figure 5. (A) Shown is another example of unusual spectral trends from the NMR assay of the known aggregator benzyl benzoate. A series of 1H NMR spectra are superimposed from various concentrations of (dilutions from 200 µM to 12 µM). Refer to the Materials and Methods for experimental details. (B) Shown is an example of another example of unusual spectral trends from the NMR assay of the known aggregator Chlorpromazine. The buffer employed for these studies was 50 mM sodium phosphate pH 7.4 in 100% D2O solvent.

The spectral trends noted for Chlorpromazine in Figure 5B were less obvious but nonetheless significant.

A visual inspection suggested no immediately obvious anomaly.

However, resonance shifting was more visible upon closer inspection via zoomed-in views (see insets in Figure 5). Our experience has been that these less-noticeable trends are significant; Chlorpromazine was considered as a promiscuous drug and had antagonistic effects on several off-target receptors.26 Also, trends such as these were used by us to help correlate compound aggregation with higher probabilities of promiscuity in off-target in vitro pharmacology screens.19 The NMR assay sometimes exposed more details of the aggregate assemblies. During screens, we noted that some compounds exhibited a subset of resonances that shifted or broadened while others did not. Based on the hydrogen assignments of these resonances, it was possible to identify the parts of the molecules that were directly involved in the assembly scaffold or core, as compared to those parts that were solvent exposed and less structured. Also, optional NOESY data were employed to reveal the orientation and stacking of molecules within the core (data not shown). Critical aggregation concentrations (CAC) of compounds.

In colloid and surface

chemistry, the critical micelle concentration (CMC) is defined as being the concentration of a surfactant at which micelles form. Micelles are considered to be well-ordered aggregates, and as


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such, analogous considerations can be extended to some forms of compound aggregates. Reports have already employed the term critical aggregation concentration (CAC), and defined it as the concentration of compound at which molecular aggregates begin to form.22-23 In the event that a molecule is found to be an aggregator, the CAC may be an insightful measurement to assess whether the observed tendency to aggregate may be biologically relevant. For example, it has been reported that when aggregating anti-cancer drugs were dosed at concentrations above the CAC, their antiproliferative activities were greatly reduced.3 Compounds in the aggregate form were simply not getting into the cells, resulting in a diminished efficacy. In another report, the drug Clofazamine was able to penetrate the cell membrane, but aggregated within the cell and affected internal organelles.24 Also, Rilpivirine (TMC278) and analogues were found to be surprisingly orally bioavailable and this was attributed to its aggregation behavior.28,29 Until recently, the current literature method for CAC determinations made use of Flow Cytometry or using a BD Genentest Solubility Scanner.22 Another and more practical method is confocal Static Light Scattering (cSLS) which can be run on many DLS instruments.23 It is claimed that this method can also determine “monomeric solubility” in a robust and highthroughput manner. We evaluated and applied the cSLS method for determining the CAC values of known drug aggregators and non-aggregators. Clear examples are displayed in Figure 6. Etodolac was a control non-aggregator and no light scattering signal was noted above baseline. The other drugs Gefitnib, Pranlukast and Crizotinib are known aggregators and the CAC values are defined as the initial concentration at which increases in light scattering are noted. The increased light



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scattering was due to the formation of larger-sized entities such as aggregates. We also often noted that some drugs gave uninterpretable scattering of the data (i.e. had SLS intensity counts that did not demonstrate a clear baseline), thus presenting some application limitations.

.

Figure 6. Light scattering (cSLS) data on four drugs. Etodolac is the control non-aggregator, and Gefitnib , Pranlukast and Crizotinib are the control aggregators. CAC points shown above are where deviations from baseline initiate. The buffer employed for these studies was 50 mM sodium phosphate pH 7.4 in 100% D2O solvent.

We were also interested in learning how the cSLS method could be complementary to the NMR assay. Two examples of spectra from the NMR assay are shown for Gefitinib and Pranlukast in Figure 7. Visual inspection of the resonance shifts for Gefitinib suggested that the midpoint concentration for these changes (shifts and resonance lineshapes), as the resonance of single molecules shifts toward the resonance of the aggregates, was approximated in the 25 to 50


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µM range. These observed changes in the NMR resonances were corroborated by the cSLS data in Figure 6 where an increase in light scattering was noted at approximately this concentration of compound. Thus, both methods bring attractive and complementary insight into better detecting and understanding these types of compound aggregates. Comparative analyses of the two methods for Pranlukast was very insightful.

As

mentioned earlier, the NMR assay could not observe the large aggregates at all the dilution concentrations (Figure 7).

Breaking up the aggregates with Tween 80 was necessary for

detection purposes. On the other hand, the cSLS data in Figure 6 suggested that the CAC occurred at low µM concentrations which was below the lowest concentrations tested by NMR. This could explain why only the resonances of single molecules were observed whereas the large aggregate was too large and not directly visible by NMR. The cSLS data provided a direct corroboration of the data noted in the NMR assay and the detergent effect. An advantage of the cSLS and DLS data was the capability of monitoring properties of compounds at lower concentrations for these types of aggregates.

We further tested other compounds such as

Fulvestrant, Lapatinib, Nilotinib, Sorafenib, Vermurafenib, and Miconazole25 (data not shown) which all share a common property; their reported CAC values fall below the wall of this assay (95% pure as verified by HPLC. The suppliers and catalog numbers of the compounds are provided in the Supporting



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Information. The compounds used in Figure 4 were in-house antiviral inhibitors and analytical characterizations (>95% pure as verified by HPLC) are available in the Supporting Materials. Figure 8 provides a convenient summary of the procedure and materials required for preparing the samples for NMR aggregation assay. Briefly, the required supplies are the aggregation buffer (50 mM sodium phosphate pH 7.4 in 100% D2O), the Tween-80 stock (10 % vol/vol in above buffer), seven Eppendorf tubes, and two pipettes (2 – 20 µL and 200 – 1000 µL). Preparation of the 20 mM compound stock solution is described at the top right of Figure 8. The subsequent procedures are as follows and demonstrated in Figure 8. (A) Prepare 20 mM compound stock solution. (B) Portion out aggregation buffer, 1500 µL in tube 5 and 500 µL in each of tubes 1-4. (C) Add/mix 15 µL of above 20 mM compound stock to tube 5. This tube now becomes the 200 µM stock solution. If precipitate forms, take note and proceed. (D) Transfer 500 µL of the 200 µM stock solution to tube 6. (E) Take 500 µL of the 200 µM stock and add to tube 4. Mix with the 500 µL buffer already present. This becomes first 2x dilution. Transfer 500 µL from tube 4 to tube 3. Mix. Repeat dilution and mixing for tubes tubes 2 then 1. (F) Lightly centrifuge tube 6 (2,000 rpm 10 minutes). Transfer precipitate-free supernatant to a spare tube. (G) Add/Mix 8 µL of Tween 80 stock solution to the spare tube. solution from tubes 1-5 and the spare tube to separate NMR tubes.


(H) Transfer

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Figure 8. Diagram showing how to prepare the samples for the NMR aggregation assay.

Alternate ways of preparing the NMR samples were tested, i.e. keeping the 1% DMSO concentration constant during the dilution step, but so far no significant differences were noted from the method described above. All samples were visually inspected to note whether or not cloudiness or precipitates formed. The buffer employed for these studies was 50 mM sodium phosphate pH 7.4 in 100% D2O solvent, as it was considered to be a representative buffer consistent with those used in the off-target in vitro pharmacology assays. Other buffer conditions were surveyed for a limited set of compounds and similar NMR trends were observed. 100% D2O solvent was employed to



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facilitate NMR data acquisition (which can be acquired with or without solvent suppression if desired). When preparing the aggregation buffer, note that a pD of 7.8 corresponds to a pH of 7.4. The pulse programs for the NMR aggregation assay are the standard one-dimensional 1H NMR experiments available on all commercial spectrometers.

There are several optional

parameters that can be modified if desired. Given that the buffer consists of 100% D2O, one can choose to use a standard 1H NMR pulse program or one that includes solvent suppression. The latter may be desirable if large H2O resonance peaks exist due to the hygroscopic property of deuterium oxide. The experiments shown here were mostly run on a 400 MHz NMR equipped with a sample changer. The number of scans was typically 3282, with a relaxation delay plus acquisition time of 2 seconds, which ensured that all samples for one compound could be acquired overnight using a sample changer. Data visualization and interpretation are also simple.

For the work described here,

Bruker’s TOPSPIN software allows for the facile superposition of 1D NMR spectra along with zooming capabilities. Other software from ACD and other vendors also allow for spectral superpositions. The interpretation of the NMR data as unusual was based on an analysis of the superimposed spectra and the observation of major or minor unusual features in resonance shifts, shape or number.

Acknowledgements We thank the following colleagues for enlightening discussions and assistance: E. Beaulieu, M. Bertrand-Laperle. F. Bilodeau, C. Chabot, P. Edwards and P. Whitehead. We also appreciate valuable comments from the reviewers.


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Supporting Information Further data are available such as DOSY NMR data, other NMR assay examples of compound aggregators and non-aggregators, compound characterizations of primary structures, compound concentration studies, and more. This material is available free of charge via the Internet at http://pubs.acs.org.

* To whom correspondence should be addressed. Phone: 1-450-963-8501 E-mail: [email protected]

Keywords : Aggregation, colloid, drug, NMR, light scattering, medicinal chemistry, promiscuity, self-assembly

a

Abbreviations used: clogP, calculated partition coefficient;

CAC, critical aggregate

concentration; CMC, critical micelle concentration; cSLS, confocal static light scattering; DLS, dynamic light scattering; DMSO, dimethyl sulphoxide; HPLC, high-pressure liquid chromatography; NMR, nuclear magnetic resonance; NOESY, nuclear Overhauser exchange spectroscopy; SAR, structure-activity relationship; SPR, surface plasma resonance, T, temperature; TPSA, total polar surface area.



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Table of Contents graphic

Non-aggregator

Aggregator

NORMAL NMR

UNUSUAL NMR

dilution

dilution


Compound Aggregation in Drug Discovery: Implementing a Practical

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