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LC/MS/MS Analysis of N-Terminal Protein Adducts with Improved Sensitivity: A Comparison of Selected Edman Isothiocyanate Reagents

This study provides a basis for a new and straightforward method for LC/MS/MS-based screening of N-terminal protein adducts. This procedure is denoted the “FIRE procedure” as fluorescein isothiocyanate (FITC) gave superior sensitivity by LC/MS/MS when measuring adducts (R) of electrophilic compounds with a modified Edman procedure. The principles of the FIRE-procedure are that adducts to N-terminal amino acids selectively are detached and measured from of proteins after derivatisation by isothiocyanate Edman reagents. In this study, FITC, 4-N,N-dimethylaminoazobenzene 4′-isothiocyanate (DABITC) and 4-dimethylamino-1-naphthyl isothiocyanate (DNITC) were used to synthesize thiohydantoin analytes from valine and N-methylvaline. The sensitivity by LC/MS/MS was enhanced by up to three orders of magnitude as compared to phenyl isothiocyanate and higher as compared to pentafluorophenyl isothiocyanate. The FITC reagent will enable measurements of low background adduct levels. Synthesized analytes were characterised with, for example, 1H NMR, 13C NMR, LC/MS/MS, and UV.

Introduction
Electrophilic chemicals/metabolites presented in vivo can be monitored by measuring the products (adducts) of their reaction with proteins, in particular hemoglobin (Hb) [1][2][3]. For this purpose methods such as the GC-MS/MS optimized N-alkyl Edman-procedure have been developed for measurement of adducts to N-terminal valine in Hb [4].
Although the N-alkyl Edman-procedure has become an established method the method has its limitations, for example, the range of adducts that can be analyzed by GC-MS is limited. Small adducts, for example, ethylene oxide [5], propylene oxide [6], and acrylamide [7,8], can be quantified at the pmol/g globin level, which is sensitive enough for measurement of background adduct levels (levels found in the general population). However, polar adducts are more difficult to analyse, due to the limitations imposed by the GC separation system prior to MS detection. Some of these obstacles can be solved, for example, by shielding polar groups through additional derivatisation, which for instance was undertaken for measurement of glycidamide adducts in Hb [9]. However, derivatisation of polar groups is both time-consuming and demands the development of new procedures for each specific adduct. Furthermore, adducts of high molecular weight (>500 mass units, mu) and thermolabile adducts are extremely difficult to analyze using the GC-MS based N-alkyl Edman-procedure.
More recently modifications to the N-alkyl Edmanprocedure have been published which instead utilize LC-MS/MS for measurement of adducts after derivatization with phenyl isothiocyanate (PITC) [10,11] or pentafluorophenyl isothiocyanate (PFPITC) [12] which are the reagents that have been used in this procedure. These modified methods presented by Fennell et al. and Ospina et al. [10,12,13] have a much higher sample throughput compared with the analysis by GC/MS. The main reason for this higher throughput is that the clean-up procedure is performed on solid phase extraction (SPE) columns, which can be readily automated. Additional benefits are that further derivatization steps of certain analytes (e.g., from polar adducts such as N- [2hydroxycarbamoylethyl]valine) can be excluded.
Although the existing methods for adduct measurement have been developed and also partly automated, globin [14] and more recently shown the erythrocytes [15] must still be isolated from whole blood and the purification of analytes includes several steps in order to selectively enrich the desired analytes. Furthermore, it is difficult to develop a multianalyte screening procedure with analytes having different polarities as this normally will demand additional purification steps.
Aiming to overcome such problems, this study was designed to evaluate the role of the utilized isothiocyanate reagent regarding mainly the sensitivity when the formed analytes are measured with LC/MS ion trap and LC/MS triple quadrupole instrument. The long-range aim has been to present a more straightforward and general method for adduct measurement. As the utilization of isothiocyanate reagents, for example, FITC leads to formation of analytes with fluorescent properties an alternative route to LC-MS/MS was tested with separation by capillary electrophoresis and spectroscopic measurements.
Three new isothiocyanate reagents were selected and compared with the reagents previously utilized in the N-alkyl Edman procedure. The studied isothiocyanate reagents and reactions are given in Figure 1. The thiohydantoin analytes and the studied reference compounds are given in Table 1. The selected isothiocyanate reagents contained the following ionisable groups, a carboxylic acid group in fluorescein isothiocyanate (FITC) and tertiary amines in both 4-dimethylaminoazobenzene-4isothiocyanate (DABITC) and 4-dimethylamino-1-naphthyl isothiocyanate (DNITC). These three reagents were reacted with valine or N-methylvaline, which are models for methylated and normal valyl terminals in Hb, to form methylated and nonmethylated valyl thiohydantoins 1-6 (see Figures 1 and 2). These analytes were characterised and relative responses were measured as LOD on two different LC/MS/MS systems under various analytical conditions. The obtained data was then compared with results from analytes formed in the N-alkyl Edman-procedure, mainly PTH-MeVal (7), but also with PFPTH-MeVal (8) and PFPTH-HOEtVal (9).

TLC Analysis.
TLC was performed using silica gel 60 f-254 plates (SiO 2 , Merck) and the spots were visualized with both UV (254 nm) and at long wavelength (378 nm). Melting points were determined using a Büchi 535 instrument. Measurements of pH were carried out using an Orion EA 920 pH-meter equipped with a Ross 8130 glass electrode.

LC/MS/MS Analysis.
Two different LC/MS systems were used. LC/MS-system 1 consisted of a Rheos 4000LC pump (Flux Instruments, Basel, Switzerland) interfaced with an LCQ MS (ThermoQuest, CA, USA). Both electrospray ionization (ESI) and atmospheric pressure-chemical ionization (APCI) were used in a positive and negative mode. 0.1% TFA, 0.1% ammonium acetate, and 0.3 mM aqueous ammonia, were used as pH modifiers. Instrument settings (ESI): Isocratic flow of aqueous buffer/ACN [1 : 1 (v/v)] of 200 μL/min, sheat gas flow 80(N 2 ), auxiliary gas flow 10(N 2 ), spray voltage 5000 versus −5000 V, capillary temperature 250 • C, capillary voltage 10 versus −10 V. Instrument settings (APCI): Isocratic flow of aqueous buffer/ACN [1 : 1 (v/v)] of 500 μL/min, sheat gas flow 70(N 2 ), auxiliary gas flow 15(N 2 ), capillary temperature 150 • C, capillary voltage 5 versus −5 V, evaporation temperature 500 • C, discharge current 5 μA. The injection volume was 5 μL and the analytes were dissolved in given solvent mixture at concentrations ranging from 10 μg/mL down to 1 ng/mL, depending on their response and the operating conditions. Determinations in the MS-MS mode were performed by collision-induced dissociation (CID) of the [M+H] + ion. The software Xcalibur (version 1.1) was used for data acquisition, LOD was determined by signal to noise (S/N) of 3, samples were injected in triplicates for each compound and concentration. LC/MS data presented for the studied compounds (1-9) have been obtained from this system unless otherwise specified. LC/MS system 2 consisted of an Agilent 1100 series LC system (Agilent technologies, Santa Clara, CA, USA) interfaced with an API365 triple-quadrupole (MS/MS) detector with a turbo electrospray-interface (ESI) obtained from Sciex, Concord, ON, Canada. An Xterra column C18 (3.5 μm, 1.0 × 100 mm) was used with an isocratic flow at 50 μL/min for determination of relative response (LOD). The injection valve was a six-way standard valve from Valco (VICI International, Switzerland). In positive ion mode the mobile phase was a mixture of H 2 O/ACN [1 : 1 (v/v)] acidified with 0.1% trifluoroacetic acid (TFA). In negative ion mode the mobile phase was water/ACN [1 : 1 (v/v)] buffered with triethylamine acetate (TEAOAc, pH ∼7). Instrument

Capillary Electrophoresis (CE).
Separation was performed on an HP 3D CE column (Agilent, CA, USA) with a five-channel diode array UV detector. A fused silica capillary (i.d. 50 μm, o.d. 375 μm) with a total length of 64 cm and an effective length of 56 cm was used. The separation voltage was +30 kV, resulting in a separation current of 32 μA. The buffer system consisted of 17 mM phosphate buffer (adjusted to pH 7) containing 20 mM SDS and the injection volume was 1 nL.

Fluorescence Measurements.
Excitation and emission spectra were recorded in water : acetonitrile solutions (9 : 1) buffered at pH 1, 3, 5, 7, and 9 with 0.1 M standard buffers (exact composition presented for shown data) using a Shimadzu RF5000 fluorescence spectrophotometer. Emission was scanned from 10 nm above the excitation wavelength, locked at the optimal absorbance wavelength determined from the UV absorbance measurements described above, to 600 nm. Excitation was then scanned from 200 nm to 10 nm below the emission wavelength locked at the optimal wavelength obtained during the emission scan. Fluoranthene was used as reference. In this study introduced Edman reagent (FITC is dianionic at pH 7) Previously used Edman reagents N-methylvaline or valine (H) * * pH ∼ 7 * The reaction between valine with FITC, DABITC, and DNITC was also studied, the conversion to thiohydantoins requires an additional acidification step for these analytes.   . The chemical shifts of five of the carbon atoms are not given (impossible to separate from the background noise). However, the presented carbons were in accordance with the predicted shifts of compound 1 in its assumed spiroconformation (cf. IIIb in Figure [17] and fluorescein in [18]. developed with both UV and long wavelength light) and after 30 minutes all the DABITC had been consumed. The subsequent extraction was performed as described above.

General Experimental Setup.
In order to investigate if the original N-alkyl Edman-procedure could be better adapted for measurement by LC-based analytical systems, fluorescent and ionisable isothiocyanate reagents were explored. The selected reagents were reacted with N-methylvaline and valine which are models for methylated and normal valyl terminals in Hb, to form the corresponding methylated and nonmethylated valyl thiohydantoin (compounds 1-6, Table 1). These compounds were purified and compared with regards to LOD on LC/MS/MS with the phenylthiohydantoin of methylvaline (PTH-MeVal, 7) and, in few experiments, with the pentafluorophenylthiohydantoins of methylvaline and 2-hydroxyethylvaline (PFPTH-MeVal, 8 and PFPTH-HOEtVal, 9, resp.). Compound 7 is formed from phenyl isothiocyanate (PITC) and compounds 8 and 9 are formed from pentafluorophenyl isothiocyanate (PFPITC); both reagents are utilized in the N-alkyl Edman-procedure (see Table 1). The analytes were analysed by 1 H NMR (compounds 1-6), 13 C NMR (1, 2, 5, and 6), LC/MS/MS (1-9), CE-DAD (1-2), and UV/vis spectroscopy (1-7). Compounds 1-2 were also characterized by fluorescence spectroscopy. In general, for all these measurements and determinations, the pH was 8 International Journal of Analytical Chemistry below or above pKa for the ionisable group of each respective analyte (1-6).

Coupling and Cyclisation
Reaction. TLC was used to follow the coupling reaction (compounds 1-6). In general terms, the coupling/cyclisation reaction was complete within 15 minutes when reacting FITC, DABITC, and DNITC with N-methylvaline (2, 4, and 6), whereas the cyclisation reaction for nonmethylated valine analytes (1, 3, and 5) took place first after an additional acidification step. This is in accordance with the traditional Edman degradation procedure [17] and the previous studies [15,17]. The coupling/cyclisation reaction was also studied using model peptides, N-methylvalylleucylanilide, and ValLeuSer derivatised with FITC. The results were in accordance with the studies on model amino acids; FTH-MeVal was formed directly during the conditions for the coupling reaction (pH ∼8) while FTH-Val was formed first after an additional acidification step. All reactions could easily be followed on TLC as the spots were visible to the naked eye. Intermediates and products formed from FITC, DABITC, and DNITC could also be visualized with the wavelengths 366 and 245 nm, respectively. (1)(2)(3)(4)(5)(6). It could also be established that the analytes (1-6) were in the cyclic, thiohydantoin, conformation, which was seen by the well separated shifts for the protons assigned to the γ, γ -methyls (dd) in the valine spin residue. For ring-open analytes, in the thiocarbamoyl conformation, the γ, γ -methyls are only slightly separated (c.f. [18]).

LC Separation of Standards.
Full baseline separation of the methylated and nonmodified tiohydantoins (compounds 1-7 and PTH-Val) was achieved with a reversed phase C18 column, using gradients of water and acetonitrile acidified with 0.1% TFA.

MS-Analysis, Comparison of Relative Response (LOD).
Mass spectrometry was used for identification of the studied analytes and for measurement of their relative response under a number of selected conditions to optimise sensitivity. The measurement of LOD by LC/MS/MS was performed in order to compare the relative sensitivity between the analytes formed from the five isothiocyanate reagents studied, reacted with MeVal. Besides the N-methylated analytes (2, 4, 6-8) FTH-Val (1) was also included in order to study possible differences in LOD of the corresponding normal (unsubstituted) valine analyte as compared to FTH-MeVal. The LOD studies were performed by LC/MS system 1 after optimization of cone voltage and collision energy for the analytes at each given condition. The results from this system were confirmed and further improved with LC/MS/MS system no. 2. In this study all instrumental parameters were fully optimized which gave a general increase of the sensitivity by a factor of 25, when, for example, comparing the LOD's of FTH-MeVal by LC/MS system 1 with the LOD's by LC/MS/MS system 2 (obtained from values given in Tables 2  and 3). An example of the LC/MS/MS separation/analysis of FTH-Val and FTH-MeVal is presented in Figure 4, showing an expanded view of an LC-MS (TIC) chromatogram of the standards 1 and 2 and an LC-MS/MS spectrum of m/z = 503 (FTH-MeVal).
FTH-MeVal (2) gave the highest response on both systems compared to all other N-substituted analytes (4, 6, 7) with one minor exception at one condition (DABTH-MeVal, unmodified condition, ESI+) (see Tables 2 and 3). Using the conditions stated for LC/MS system no. 1 the lowest LOD was 2.6 nM for FTH-MeVal under optimal conditions and 0.5 nM when measured with LC-system 2 (ESI, negative ions, ammonia).
The difference in LOD between FTH-Val and FTH-MeVal was measured in order to see the difference between N-methylated analytes (2) and normal valine analytes (1) (see Table 2). As this difference was relatively small, for example, as compared to the much larger difference between the five isothiocyanate reagents tested, further studies on normal valine analytes were excluded.
Initially the LODs were measured and compared for FTH-Val, FTH-MeVal, DABTH-MeVal, DNTH-MeVal, and PTH- MeVal (1, 2, 4, 6, and 7). The results from these studies are given in Tables 2 and 3. When it became clear that PTH-MeVal (7) gave a much lower relative response by LC/MS (ESI positive and negative ion modes) than the other compounds (1, 2, 4, and 6), two additional analytes, PFPTH-MeVal (8) and PFPTH-HOEtVal (9), were also included in this study for a few comparative measurements. The results for compounds 8 and 9 gave an even lower relative response than those for PTH-MeVal (7); that is, PFPTH-MeVal (8) showed an LOD of 77,000 nM when measured with ESI (negative ions, unbuffered solvent system). An attempt to measure positive ions failed as product ions m/z 311 and m/z 266 were too weak to be measured. However, compound 8 could be measured when ionized with atmospheric pressure chemical ionization (APCI) and gave an LOD of 82,600 nM (0.1% TFA, H 2 O/ACN 1 : 1, v/v, negative ions measured) which was just above ten times lower compared to compound 9 (LOD = 950,000 nM) at this specific condition. When comparing the relative sensitivity between compounds 1, 2, 4, and 6 (see Table 2) with compounds 7-9, at the same condition, the relative differences were very large, that is, 2,200 times lower LOD for compound 2 as compared with 7, 18,000 times lower LOD for 2 as compared to 9, and 210,000 times lower LOD for 2 as compared to 8. PFPTH-HOEtVal (9) was also measured with APCI (buffered with NH 4 OAc, negative ions measured) which gave an LOD of 4,400 nM which can be compared to the LOD of 790 nM for compound 2 at this condition.
3.6. Fluorescence. The fluorescence properties of FTH-Val (1) and FTH-MeVal (2) were measured by recording excitation and emission spectra. Measurements of the dianions of 1 and 2 (Figure 3) which were obtained above pH 5, that is, at pH 7 and 9, gave maximum excitation wavelength of 492 nm and maximum emission at 515 nm with almost identical spectra for 1 and 2; see Figure 2 recorded at pH 7. This means that   the adduct, that is, the methyl group in FTH-MeVal, does not affect the spectroscopic properties. The relative fluorescence intensities of the dianions of 1 and 2 were around 360-fold higher as compared to the reference fluoranthene.

Capillary Electrophoresis (CE).
The separation of FTH-MeVal from FTH-Val using a capillary electrophoresisdiode array detection (CE-DAD) system was evaluated in order to assess the possibility of determining N-terminal adducts using separation techniques other than LC as well as to be able to utilize and benefit from such sensitive techniques as capillary electrophoresis-laser-induced fluorescence detection (CE-LIF). This separation was performed using 17 mM phosphate buffer (adjusted to pH 7) containing 20 mM SDS. A complete baseline separation was obtained; FTH-Val eluted after 7.44 minutes and FTH MeVal after 8.91 minutes, both with narrow well-defined peaks (see Figure 5).

4.1.
Since the N-alkyl Edman-procedure was introduced 25 years ago [19] only small modifications of the originally presented procedure have been introduced. Initially, PITC was utilized as the Edman reagent. Somewhat later PFPITC was introduced, which gave a much higher sensitivity when measuring the analytes by GC/MS in the negative ion chemical ionization (NICI) mode [20]. The N-alkyl Edmanprocedure using PITC or PFPITC has lately been adapted in order to perform the analysis by LC/MS/MS [10][11][12][13][14]. However, these modifications have so far not included studies of different Edman reagents to optimise the method for LC/MS analysis. The change from GC/MS to LC/MS has important implications for principles concerning separation and methods for the ionization of analytes. The introduction of isothiocyanate reagents containing ionisable groups (i.e., FITC, DABITC, and DNITC) was demonstrated to be a valuable approach which gave several improvements, for example, increased solubility in blood at biological pH (regards FITC), and enhanced sensitivity when measuring by LC/MS/MS as compared to the PITC and PFPITC reagents (regards all three reagents). The ionic group and fluorescent properties of the analyte also provides new alternatives such as CE separation and spectroscopic detection, for example, with sensitive fluorescence detection. Ireland et al. showed, for example, that they could measure FTH-amino acids with a limit of detection on a low zeptomol (10 −21 ) level per injection by using CE separation with laser induced fluorescence detection [21].
Although analysis with other methods for detection is possible when utilizing reagents such as FITC, MS/MS procedures are normally preferred due to the combination of high sensitivity, selectivity, and accuracy. This fundamental work was done in order to develop an LC/MS/MS procedure capable to deliver high sensitivity, throughput, and a method which is less restrictive regarding the types of adducts which can be measured.

Coupling and Detachment Reactions.
One crucial question for the success of this procedure was if the coupling reaction as well as the detachment reaction (formation of the analytes already in the coupling reaction) would be hampered by the introduction of much bulkier reagents than used earlier. When using FITC, the largest Edman reagent tested, it was shown (e.g., on TLC) that Nsubstituted valines or N-substituted Val-Leu-anilides reacted readily and formed FTH-MeVal at the conditions for the coupling (pH ∼ 8). The same result was obtained for DABITC forming DABTH-MeVal, and DNITC forming DNTH-MeVal. When thiocarbamoylating normal valine or ValLeuSer with FITC, DABITC and DNITC no rinclosure occurred as expected. To convert these products to thiohydantoin analytes an acidification step was demanded, which is in agreement with earlier studies with PITC and PFPITC [15,17].
A bonus with FITC, DABITC, and DNITC is that the analytes formed are coloured. All steps could therefore easily be followed in real time by the naked eye, which was beneficial throughout the whole development work of the procedure as well as for preparation of synthetic standards.

LC-Separation Prior
Analysis. All pairs of analytes, that is, FTH-Val and FTH-MeVal, 3, and 4, 5 and 6 could easily be separated with full baseline separation by using water/acetonitrile gradients on a standard reversed phase HPLC column. When using volatile pH modifiers such as 0.1% TFA, the solvent system is compatible with LC/MS/MS.   Tables 2 and 3). This high relative response obtained with the FTH analytes 1 and 2 was interpreted to be favoured by the readily ionisable groups (e.g., phenolic groups) in the fluorescein body, which can form both negative and positive ions. Another factor, which also increases the signal-to-noise ratio, is that the FTH-analytes had the highest molecular weights, which is beneficial as the chemical background noise decreases with higher m/z. The order of relative sensitivity for the studied analytes was (lowest LOD first); FTH-MeVal ∼ FTH-Val > DABTH-MeVal > DNTH-MeVal > PTH-MeVal > PFPTH-HOEtVal (9) > PFPTH-MeVal (8). Interestingly the LODs for 9 were a factor 10 lower than for 8 which must be assigned to the hydroxyl group on 9 which has higher ion affinity and is more prone to be ionized, giving lower LOD. This result indicates that the relative sensitivity of a specific analyte will vary more when PITC or PFPITC is used as the derivatisation reagent compared to, for example, FITC. For the PITC/PFPITC reagents the ion affinity of the adduct will determine the ion affinity in contrast to ionisable isothiocyanate reagents which are more prone to be ionised. Reagents such as FITC will therefore give less difference in obtained relative response as long as the ion affinity of the adduct is lower than the isothiocyanate body (under investigation). The sensitivity obtained for especially the FITC reagent should be sufficient for measurement of background adduct levels of, for example, 30 pmol/g globin, common for ethylene oxide. From a sample containing 50 mg globin there will be 1.5 pmol ethylene oxide adduct. If this is quantitatively detached, purified, and concentrated to a volume of 100 μL we will have a final concentration of 15 nM which is about 10 times over LOQ. Naturally there will be losses in the analytical chain and lower sensitivity with real samples due to, for example, ion suppression, but there will still be a good marginal to measure low background adduct levels. This marginal will also be enough for using TFA as pH modifier which gives better retention and sharper peaks when separated on a C 18 LC column as the analytes then are neutral and much more lipophilic. Studies on real samples are ongoing and will be published soon.

12
International Journal of Analytical Chemistry This study was conducted on two different MS/MS systems to verify the rather drastic difference in sensitivity between the used Edman reagents seen on system no 1. Comparison of obtained relative sensitivity (LODs) of studied analytes gave rather constant ratios where system 2 gave 25 times lower LODs than system 1 (compare Table 3 with Table 2). It was expected that system 2 which is a triple quad instrument operating in mrm mode should give lower LODs than the LCQ iontrap instrument (system 1). This relative difference between the two systems could in fact be even higher as system 1 was used with direct injections of the analytes, giving no dilution at all, while system 2 used an LC column with a small i.d. (1.0 × 100 mm) operating at 50 μL/min which definitively diluted the analytes as compared to direct injections. However, much effort was made to optimize system 2 to maximize the sensitivity. On more modern and sophisticated instruments we will expect even lower LODs and we will also expect that the relative difference between the analytes will be rather constant in accordance to this study. Finally, triple quad instruments such as system 2 operating in MRM mode mode are the instrument of choice for selectivity and sensitivity in quantitative measurements.

Capillary Electrophoresis and Fluorescence.
Ireland et al. [21] showed that amino acids in proteins could be sequenced with FITC as Edman reagent and measured by CE-LIF with outstanding sensitivity. In order to test the same principle for adduct measurements without having access to a CE-LIF system we used CE-DAD to study the separation between FTH-Val and FTH-MeVal and measured the fluorescence properties separately. The conclusion of this study and Irelands work on FTH-amino acids is that the sensitivity of CE-LIF should be high enough for low adduct level measurements. A probable disadvantage with CE and fluorescence measurements is that by-products always are formed in huge excess when analyzing real samples. In spite of the high separation capacity of CE, byproducts might interfere with the low adduct levels that are commonly found (pmol/g globin level) which corresponds to about one adduct per 1-10 million normal valines [22]. However, when measuring adducts levels that occurs at much higher levels such as aldehydes, CE separation with some kind of fluorescence measurement might be a valuable alternative to LC-MS/MS analysis. To exemplify this, Kautiainen et al. [23] recorded 1000 times higher levels of 2-hydroxyethylvaline (13-17 nmol/g globin) on reduced globin samples (probably source is the reduced Schiff base from acetaldehyde) as compared to levels between 16-23 pmol/g globin for unreduced samples (adducts formed directly from ethylene oxide). Finally, to combine powerful separation techniques (which are improved continuously), with fluorescence measurements and MS/MS analysis might be an interesting alternative for future work. As shown in this study, the spectroscopic properties were not affected by the adduct. The fluorescence properties could thus be used for quantification and MS/MS for structural characterisation. This combination of techniques would be powerful for analysis of today unknown adducts.

Conclusion
This study demonstrated that the N-alkyl Edman-procedure could be successfully adapted towards LC conditions by exchanging the isothiocyanate reagents with ionisable reagents such as FITC, DABITC, or DNITC. FITC was superior compared to the other reagents tested. The use of FITC as a reagent opens up for new possibilities for the analysis of haemoglobin adducts. The present study provides a basis for the development of the Adduct FIRE procedure [24] facilitating application of Hb adduct measurement in experimental and epidemiological studies.


Adsorption and Removal Reactions of (CH3)2S on Rh/Al2O3/NiAl(100): Structural and Spectroscopic Study

We have studied the adsorption reaction of (CH3)2S on Rh/Al2O3/NiAl(100) and sulfur removal reaction depending upon heat-treatment using X-ray Photoelectron Spectroscopy and Atomic Force Microscopy techniques. The results show a difference in the adsorption reaction between the as-deposited Rh/Al2O3/NiAl(100) and that heat-treated at 1000 K. (CH3)2S dissociates on the Rh/Al2O3/NiAl(100) as-deposited surface at 300 K, and the sulfur removal reaction occurs at 523 K induced by O2 dosing. In contrast, the heat-treated surface is quite inert against the dissociative reaction of (CH3)2S, because the Rh atoms dissolute into the NiAl(100) substrate through the Al2O3 layer by the high temperature treatment (1000 K). The AFM results also show the morphological changes in these systems, such as many stripe structures for the Al2O3/NiAl(100), nanoclustered surface for the Rh/Al2O3/NiAl(100) as-deposited surface, and nano-hole structures for the Rh/Al2O3/NiAl(100) heated at 1000 K. [DOI: 10.1380/ejssnt.2009.239]

I. INTRODUCTION
Platinum group metals supported on oxides, such as Al 2 O 3 , CeO 2 and ZrO 2 , are widely used as the typical catalysts in the purification process of the automobile exhaust gas. For example, Rh/Al 2 O 3 catalyst is noticeable in the reduction of NO x , and Pd/Al 2 O 3 is available in the oxidation of CO. Those performances are frequently degraded by some deactivation factors because of the use in the severe environment. Especially, the poisoning of catalyst surface and the aggregation of catalyst metals under high temperature are regarded as the main problems [1].
The sulfuric atmosphere as low as few ppm causes the sulfur adsorption on the catalyst surface, which disturbs the other purification reactions; the so-called "Sulfur-Poisoning" is one of the most important deactivation reaction for metal catalysts [2,3]. Therefore not only the resistance against poisoning but also the removal of adsorbed sulfur is required for those catalysts to apply into the purification of the automobile and industrial exhaust gases. In this regard, many works have investigated the interactions between the sulfur-containing molecules and the transition metal surfaces [4][5][6][7]. These studies show that the sulfur poisoning is mainly caused by the adsorption of the atomic sulfur generated by the dissociation of the chemical bonds around sulfur atom. On the other hand, some studies take notice of the removal reaction of sulfur atoms from platinum group metal surfaces [8]. For example, Dohmae reveals that the adsorbed sulfur atoms on Rh polycrystalline surface desorb as SO 2 at around 500 K under O 2 environment [2]. This work also com-pares with Pt and Pd surfaces, and shows the excellent performance of Rh for the sulfur removal reaction. In recent years, the collection of those poisoned metals is difficult because of the low cost performance. Thus, the easy recycle of the catalyst metals can be expected by means of the sulfur removal reaction.
A high temperature environment (∼1000 K) also gives damages to the catalyst metals supported on Al 2 O 3 . One of the problems is the migration of metal atoms under high temperature, and it leads to the aggregation of catalyst metals. The metal dissolution into Al 2 O 3 is the second problem. It was reported that a part of Rh atoms was dissolved into Al 2 O 3 support material by oxidation at 873 K [9]. Both of these two problems are related to the decrease in the reaction area.
In our previous studies, the adsorption system of (CH 3 ) 2 S (dimethyl sulfide: DMS) on Rh(100) single crystal surface have been revealed, where we have investigated the temperature dependent dissociative reaction and the sulfur removal reaction [8,[10][11][12]. However, these systems are too fundamental to apply into practical use. Therefore, we are required to obtain the more actual findings of the sulfur poisoning by clarifying the adsorption behavior of DMS on Rh/Al 2 O 3 system. The aim of this work is firstly to study the growth of Rh on Al 2 O 3 /NiAl(100) depending upon heat treatment, and secondary to reveal the adsorption reaction of DMS on Rh/Al 2 O 3 /NiAl(100) surface and the sulfur removal reaction by means of Xray Photoelectron Spectroscopy (XPS) and Atomic Force Microscopy (AFM) techniques.

II. EXPERIMENTAL
A commercially available NiAl(100) single crystal (10 mm φ diameter, 2 mm thickness, Surface Preparation Laboratory Inc.) was mechanically polished with 0.05 µm Al 2 O 3 to a mirror finish. The NiAl(100) crystal was cleaned by the alternating cycles of Ar + ion sputtering (3 keV, 2 µA, 30 min) to remove sulfur, carbon and oxygen impurities and annealing at 1100 K for 30 min by an electron bombardment in an ultrahigh vacuum (UHV) chamber. The base pressure was better than 4×10 −8 Pa. The cleanliness of NiAl(100) surface was verified by XPS measurements (S 2p, C 1s and O 1s).
A well-ordered ultrathin θ-Al 2 O 3 layer was grown on exposing the cleaned NiAl(100) to O 2 (∼1000 L) at 500 K and subsequently annealing at 1000 K for 5 min [13]. Rhodium was deposited on the Al 2 O 3 thin layer in-situ at room temperature with Rh vapor from an ultra-pure Rh wire (99.9%) set in an electron beam evaporator. The Rh deposition rate and time were 0.025 ML/min and 40 min, and then the total coverage was about 1.0 ML. After deposition, the sample was heated at 1000 K for 5 min. Three types of samples were prepared in this work, which were Rh/Al 2 O 3 /NiAl(100) as Rh deposited, Rh/Al 2 O 3 /NiAl(100) heated at 1000 K, and Al 2 O 3 /NiAl(100) surface.
The research grade DMS was purified by means of a few cycles of freezing with liquid N 2 under high vacuum condition and melting at ambient temperature. DMS gas was admitted via the variable leak valve into the UHV chamber in this procedure, as the sample surface was cooled down to 90 K with liquid N 2 . The dosage was set at 0.3 L. The temperature of substrate (90-300 K) was controlled by resistive heating with a tungsten-filament, which was set behind the crystal. The sulfur removal reaction was promoted under O 2 environment (2.7×10 −5 Pa for 1h) at 423, 473 and 523 K.
The surface morphology was obtained by ex-situ AFM measurement with NanoScope III-a (Veeco Instruments) with tapping mode at Innovation Plaza Hiroshima JST. XPS measurements were recorded by use of the concentric hemispherical electron energy analyzer (PHOIBOS 100-5ch, SPECS) with MgKα X-ray (1253.6 eV). All binding energies were referenced to Ni 3p 3/2 peak position of NiAl at 853.0 eV [14]. The initial thickness of Al 2 O 3 layer was 4-5Å estimated from the signal ratio of I Al2p Al2O3 and I Al2p NiAl were the Al 2p peak intensities of Al 2 O 3 and NiAl as shown in Fig. 3 [15]. The coverages (ML) of Rh and sulfur were obtained with their XPS peak intensities in Rh 3d and S 2p regions (1ML corresponds to the area density of fcc Rh(100) surface atoms, 1.37×10 13 atoms/mm 2 ).

III. RESULTS AND DISCUSSIONS
A. S 2p XPS Figure 1 shows the temperature dependent S 2p XPS spectra for DMS on Rh(1.0 ML)/Al 2 O 3 /NiAl(100) asdeposited, Rh(1.0 ML)/Al 2 O 3 /NiAl(100) heated at 1000 K, and Al 2 O 3 /NiAl(100) surfaces. The initial DMS coverages are estimated at 0.34, 0.34 and 0.26 ML for each sample, respectively. Those amounts mean almost monolayer adsorption. There is no sulfur contamination on each initial surface. On the Al 2 O 3 /NiAl(100) surface, most of DMS adsorb on Al 2 O 3 layer without dissociation into CH 3 S − and atomic S at 90 K. It is found that the sulfur in DMS has an interaction with the substrate and is picked some electrons up by the Al 2 O 3 layer, because the main peak position located at 164.3 eV undergoes a higher energy shift from that of the multilayer DMS at 163.8 eV [10]. Almost all of the adsorbates desorb from the Al 2 O 3 /NiAl(100) surface at 200 K, indicating no catalytic performance for DMS dissociative reaction. Dissociation of DMS also hardly occurs on the Rh/Al 2 O 3 /NiAl(100) as-deposited surface at 90 K. The peak position shifts to the lower binding energy side from that of the Al 2 O 3 /NiAl(100) surface. This shift gives the interaction between DMS and Rh layer. More than half the molecules (0.18 ML) remain on the surface at 200 K without desorption. Some of those molecules undergo the scission in the S-C bonds. The dissociation is promoted as the temperature rises, and most of the S-C bonds are cleaved at 300 K. Thus the Rh over-layer possesses a catalytic performance for the dissociative reaction of DMS.
In the case of the Rh/Al 2 O 3 /NiAl(100) heated at 1000 K, the dissociative reaction and the desorption behavior are quite equivalent to that on the Al 2 O 3 /NiAl(100) surface meaning inert against the dissociation process of DMS.
The sulfur-poisoned Rh/Al 2 O 3 /NiAl(100) as-deposited surface was heated under O 2 environment to remove the sulfur atoms. The main peak is located at 162.0 eV showing the adsorption of the atomic sulfur. One can find the broad peak at 166 eV to 169 eV, when the sample is annealed at 423 K and 473 K. This chemical state is assigned to SO x species [2]. The broad peak disappears at 523 K, and simultaneously the total coverage of sulfur decreases. Those changes indicate the oxidation of sulfur and the desorption of SO x from the Rh/Al 2 O 3 /NiAl(100) as-deposited surface. The desorption onset temperature is higher than that reported with polycrystalline Rh and Rh(100) surfaces, on which the sulfur atoms desorb at 423 K [2,8]. Therefore, the performance of Rh in the sulfur removal reaction is weakened on the Al 2 O 3 layer.
B. Rh 3d XPS Figure 2 shows the Rh 3d 5/2 peaks for the Rh/Al 2 O 3 /NiAl(100) systems before DMS adsorption. The peak shape and the FWHM were estimated using a Shirley background and a mixed Gaussian-Lorentzian function with an asymmetric parameter. Table I shows these intensities, peak positions and FWHM of the Rh 3d 5/2 peak. Metallic Rh state has a single peak at 307.2 eV, shown as the spectrum of Rh(111) bulk [16]. The Rh 3d 5/2 peak of Rh/Al 2 O 3 /NiAl(100) as-deposited becomes broader than that of Rh(111) bulk. The peak position also shifts from that of Rh(111) bulk to 0.25 eV higher binding energy side. Therefore, the spectrum for Rh/Al 2 O 3 /NiAl(100) as-deposited seems to contain other components at the higher binding energy side in addition to the peak for metallic Rh state. The Rh atoms in second layer or more shows their peak at the corresponding position with metallic Rh state at 307.2 eV, because the spectrum becomes similar shape to the Rh(111) bulk spectrum as Rh coverage increases. On the other hand, the additional components shift from the metallic Rh state to the higher energy side, indicating the oxidation or the interaction with the Al 2 O 3 layer. The oxidized Rh state is reported to represent the Rh 3d 5/2 peak at 308.0 eV or more; for Rh 2 O 3 at 308.35 eV and for RhOOH at 308.55 eV [16]. Thus, these components are confirmed the first layer Rh atoms on the surface which interact with Al 2 O 3 layer. These Rh atoms are squeezed some electrons by the Al 2 O 3 layer, which causes the Rh 3d 5/2 peak shift to the higher binding energy side. We can also find the reverse shift of the Al 2 O 3 peak in Al 2p and O 1s spectra shown in Figs. 3 and 4. These results indicate multilayer or nanocluster structure composed with Rh more than two layers.
In contrast, the peak shape of the Rh/Al 2 O 3 /NiAl(100) heated at 1000 K can be described with a single state at 307.50 eV. The state extremely stabilizes against various treatments, such as the DMS adsorption, the heating and the O 2 exposure. The integral intensity decays in comparison with that of the Rh as-deposited sample by 22%. Hence, two possible processes can engender the stability and the decrease in the Rh 3d 5/2 peak intensity; the morphology of the Rh over-layer changes from a flat structure to a cluster structure; and the Rh atom dissolves into the substrate. We confirm the dissolution of Rh into the substrate under high temperature treatments because of the low reactivity to DMS dissociation. Al 2p and O 1s XPS results also agree with that standpoint in next chapter. Rh 3d 5/2 XPS result reveals the significantly different environment between Rh atoms on the as-deposited surface and that on the heat-treated surface (1000 K).  interaction between the Al 2 O 3 layer and the deposited Rh atoms. The lower shifted peak position does not move any longer in the adsorption system of DMS on Rh/Al 2 O 3 /NiAl(100) as-deposited surface, indicating that those adsorption reaction is exclusive on the Rh surface. A dramatic change is observed in the signal ratio of I Al2p Al2O3 /I Al2p NiAl after O 2 treatment at 423K, 473 K and 523 K. Thereby, the oxide thickness increases significantly after heating under O 2 environment. The Al 2 O 3 thicknesses are calculated to be 8Å, 11Å and 13Å at 423 K, 473 K and 523 K, respectively (the pristine Al 2 O 3 thickness is about 5Å). As mentioned by Song et al., the pristine Al 2 O 3 layer thickness is stable even at 1070 K without over-layer [17]. This result indicates that Rh layer behaves as a catalyst for oxidation of aluminum by supplying reactive oxygen atoms formed by dissociation of oxygen on Rh surface. A similar catalytic mechanism was reported in Pd/Al 2 O 3 /NiAl(110) system [17,18]. A part of the reactive oxygen atoms also have an important role in the sulfur removal reaction as revealed in our previous study [8].

C. Al 2p and O 1s XPS
When the Rh as-deposited sample is heat-treated at 1000 K, the Al 2 O 3 binding energy returns to a position near that before the deposition of Rh. Therefore, the interaction between the Al 2 O 3 layer and the Rh atoms is lost by heat-treatment. We can consider two possible processes such as the morphological change of Rh layer and the dissolution of Rh into the substrate as shown in Chapter 3. 2. We conclude the dissolution of Rh into the NiAl(100) substrate under high temperature treatments, when DMS molecules adsorb on that surface. The S 2p XPS result shows the charge transfer from DMS to the Al 2 O 3 layer. Simultaneously, the shift to the lower binding energy side is observed in the DMS on Al 2 O 3 /NiAl(100) system as shown in Fig. 3. Similar shift has occurred on the Rh/Al 2 O 3 /NiAl(100) heated at 1000 K. Furthermore, the growth of Al 2 O 3 layer has not been promoted by O 2 exposure. Therefore, it indicates that the deposited Rh atoms do not present on the Al 2 O 3 layer but it dissolves into the NiAl(100) substrate through the Al 2 O 3 layer. This diffusion into the substrate is possible through the defects of the Al 2 O 3 layer [17,19]. The O 1s XPS results shown in Fig. 4 also represent the similar shift as the Al 2p XPS results, indicating that the Rh and the adsorbate have some interaction with not only aluminum atoms but also oxygen atoms in the Al 2 O 3 layer. Figure 5 shows the AFM images obtained from Al 2 O 3 /NiAl(100), Rh/Al 2 O 3 /NiAl(100) as-deposited and Rh/Al 2 O 3 /NiAl(100) heated at 1000 K. We can see a lot of stripe structures on the Al 2 O 3 /NiAl(100) surface growing preferentially along the crystal directions, consistent with the other reports [13,20,21]. These stripes disappear when the surface covered with Rh layer. The deposited Rh on Al 2 O 3 /NiAl(100) has a protruded structures with the height of 3-7Å. Thereby, it can be speculated that nanoclusters consisted of 1-3 Rh layer formed on the Al 2 O 3 /NiAl(100) surface because the diameter of a Rh atom is estimated to be 2.7Å. After the heattreatment at 1000 K, there is no protruded structure but a lot of nano-hole structures as shown in Fig. 5(c). The former XPS results show the absence of Rh atoms on the surface. Thus, it is supposed that this outmost surface is mainly composed with the Al 2 O 3 layer, which undergoes the structural change by the diffusion process of Rh into the NiAl(100) substrate through the Al 2 O 3 layer. In three types of surfaces, only the Rh/Al 2 O 3 /NiAl(100) asdeposited surface has the activity for the dissociative reaction of between DMS and O 2 , because the Rh atoms are exposed on the surface. The proposed mechanism for the interaction of Rh/Al 2 O 3 /NiAl(100) with DMS and O 2 at elevated temperatures is shown in Fig. 6. The process I shows the initial surface of Rh/Al 2 O 3 /NiAl(100). The adsorbed DMS dissociates on the Rh nanocluster surface. The Rh surface is covered with the sulfur atoms in process II. The dissociative adsorption of O 2 and the growth of the Al 2 O 3 layer occur at the temperature range of 423-473 K (process III). Finally, a part of the sulfur atoms desorbs from the surface as SO 2 in the process IV.

IV. CONCLUSION
We have investigated the adsorption reaction of DMS on Rh/Al 2 O 3 /NiAl(100) surface and the sulfur removal reaction related with heat-treatment with XPS and AFM techniques. Atomic sulfur remains on the Rh/Al 2 O 3 /NiAl(100) as-deposited surface at 300 K because of DMS dissociative reaction. The sulfur removal reaction is induced by O 2 gas at 523 K with the formation and desorption of SO 2 . Simultaneously, the growth in the thickness of Al 2 O 3 layer occurs since the Rh layer serves the catalytic effect for the oxidation of aluminum in the NiAl(100) substrate. On the other hand, the Rh/Al 2 O 3 /NiAl(100) heat-treated at 1000 K is quite inert against DMS dissociation. The reason for the inertness is confirmed the dissolution of Rh into the NiAl(100) substrate through the Al 2 O 3 layer occurring by the high temperature treatment. The AFM results show the morphological change in these systems; many stripe structures for the Al 2 O 3 /NiAl(100), nanoclustered surface that consists of Rh for the Rh/Al 2 O 3 /NiAl(100) as-deposited surface, and nano-hole structures formed with Al 2 O 3 layer for the Rh/Al 2 O 3 /NiAl(100) heated at 1000 K.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Chemistry Biology

text:
Structure-based Design of High Affinity Peptides Inhibiting the Interaction of p53 with MDM2 and MDMX*

MDM2 and MDMX function as key regulators of p53 by binding to its N terminus, inhibiting its transcriptional activity, and promoting degradation. MDM2 and MDMX overexpression or hyperactivation directly contributes to the loss of p53 function during the development of nearly 50% of human cancers. Recent studies showed that disrupting p53-MDM2 and p53-MDMX interactions can lead to robust activation of p53 but also revealed a need to develop novel dual specific or MDMX-specific inhibitors. Using phage display we identified a 12-residue peptide (pDI) with inhibitory activity against MDM2 and MDMX. The co-crystal structures of the pDI and a single mutant derivative (pDI6W) liganded with the N-terminal domains of human MDMX and MDM2 served as the basis for the design of 11 distinct pDI-derivative peptides that were tested for inhibitory potential. The best derivative (termed pDIQ) contained four amino acid substitutions and exhibited a 5-fold increase in potency over the parent peptide against both MDM2 (IC50 = 8 nm) and MDMX (IC50 = 110 nm). Further structural studies revealed key molecular features enabling the high affinity binding of the pDIQ to these proteins. These include large conformational changes of the pDIQ to reach into a hydrophobic site unique to MDMX. The findings suggest new strategies toward the rational design of small molecule inhibitors efficiently targeting MDMX.

The p53 tumor suppressor is a potent inducer of cell cycle arrest, apoptosis, cellular senescence, and innate immunity. It is activated in response to oncogenic transformation, extrinsic stress, and viral infection to protect higher organisms from cancer (1)(2)(3). p53 also facilitates maternal reproduction through induction of the growth factor leukemia inhibitory factor (LIF) that promotes embryo implantation (4). p53 activity is kept at minimal levels in unstressed cells by interactions with MDM2 and MDMX. MDM2 is an ubiquitin E3 ligase for p53 and an important regulator of p53 stability by forming a negative feedback loop (5,6). The MDM2 homolog MDMX also binds to p53 and inhibits p53-dependent transcription (7). Loss of MDM2 or MDMX leads to embryonic lethality (8 -10). Therefore, the expression of MDM2 and the expression of MDMX are both necessary for regulation of p53 during normal development.
Genetic or functional inactivation of p53 is an obligatory step during cancer development. In human tumors that retain wild type p53, amplification of MDM2 or MDMX serves as an alternative mechanism of p53 inactivation in a subset of tumors (11,12). Furthermore, MDM2 activity is controlled by the tumor suppressor ARF (alternative reading frame) encoded by the INK4a locus (2). Deletion/epigenetic silencing of ARF occur in most tumors expressing wild type p53, resulting in hyperactive MDM2 and lack of p53 response to oncogenic stress in malignant tumors (13,14). ARF has also been shown to promote MDMX degradation by MDM2 (15). Loss of ARF expression may result in MDMX stabilization that further inactivates p53. Therefore, MDM2 and MDMX are directly involved in p53 functional inactivation in ϳ50% of tumors, making them attractive drug targets.
Both MDM2 and MDMX regulate p53 by binding to a short amphipathic ␣-helix in its N-terminal transactivation domain. Earlier studies of MDM2-p53 binding and determination of MDM2-p53 crystal structure formed the foundation for recent development of small molecule disruptors of MDM2-p53 binding (16 -20). These compounds, such as Nutlin 3a and MI-219, provided proof-of-concept for the anti-tumor potential of MDM2 inhibitors (21,22). Importantly, Nutlin 3a and MI-219 do not inhibit MDMX, and the efficacy of Nutlin 3a is compromised in cells overexpressing MDMX (23)(24)(25). Furthermore, even in tumor cells without MDMX amplification, knockdown of MDMX by small interfering RNA still showed anti-tumor potential and cooperative effects with Nutlin in activating p53 (23,24,26). Therefore, MDMX expression contributes to p53 inactivation, suggesting that targeting both MDM2 and MDMX is needed to achieve optimal activation of p53.
The small molecules developed against MDM2 are generally inactive for MDMX (22,27). Recent structural studies using humanized zebra fish MDMX and human MDMX in complex with the p53 N-terminal peptide revealed extensive similarity between the p53-binding domains of MDM2 and MDMX in overall folding and the shapes of their p53-binding pockets. However, a few sequence differences result in a smaller hydrophobic cleft in MDMX that prevents efficient binding by Nutlin (28,29). Future development of inhibitors against MDMX can be facilitated by identification of a high affinity artificial ligand that target MDMX and/or MDM2. We recently identified a FIGURE 1. Crystal structures of MDMX and MDM2 liganded with the pDI peptide and derivatives thereof. The structures of the pDI, pDI6W, and pDIQ peptides (shown in orange) were determined in complex with MDMX and MDM2 (shown in gray). Indicated in cyan are the amino acid substitution sites. The major structural difference between MDMX and MDM2 is the ␣2Ј-helix, part of which constitutes the peptide-binding site (highlighted in blue in the pDIQ liganded structures). The MDMX-pDIQ complex crystallized with two monomers in the asymmetric unit, and only chain A is displayed. peptide (pDI) using phage display that selects for sequence with maximal binding to MDM2 and MDMX (30). pDI is 300-fold more potent than p53 peptide in disrupting MDM2-p53 and MDMX-p53 binding. Using a similar phage display strategy, Pazgier et al. (31) recently also identified a different, more potent peptide inhibitor (pMI) in their screen. The co-crystal structures of pMI and pDI in complex with the MDM2 and MDMX N-terminal domains have recently been reported (31,32), revealing the structural basis for the inhibitory action of these two different peptides.
Here, we determined the crystal structures of MDM2 and MDMX in complex with the pDI and derivatives thereof. Based on the structural information obtained from the pDI and a single mutant peptide (pDI6W), we designed a quadruple mutant peptide (pDIQ) that displays high affinity for MDMX and is the most potent inhibitor against MDM2 reported to date. The findings provide important clues about the molecular basis for the potency and selectivity of MDM2 and MDMX inhibitors and should inspire new strategies toward the design of drug-like small molecule inhibitors specifically targeting MDMX.

EXPERIMENTAL PROCEDURES
Materials-The chemicals and reagents were purchased from Sigma unless otherwise noted. The peptides (Ͼ95% purity) were supplied by Genscript (Piscataway, NJ).
Crystallization-The MDM2-X peptide complexes were crystallized at 19°C by the hanging drop vapor diffusion method at a protein concentration of ϳ10 mg/ml, supplemented with an additional half molar equivalent of the respective peptide. The MDMX (23-111)-pDI and pDI6W complexes   Data Collection, Structure Solution, and Refinement-X-ray diffraction data were recorded at Ϫ180°C using the oscillation method on single flash-frozen crystals (detector, Rigaku HTC image plate; x-rays, CuK␣, focused by mirror optics; generator, Rigaku Micro-Max 007-HF). The data were processed with HKL2000 (HKL Research, Inc., Charlottesville, VA). The structures were determined by molecular replacement using the coordinates of human MDM2-p53 (Protein Data Bank entry 1T4F) and human MDMX-p53 (Protein Data Bank entry 3DAB) as search models. The program package CNS (34) was employed for phasing and refinement; model building was performed with O (35). Refinement cycles were performed using data to the highest resolution with no sigma cut-off applied. Several rounds of minimization, simulated annealing (starting temperature, 2500 K), and restrained individual B-factor refinement were carried out. The data collection and refinement statistics are summarized in Table 2. Figs. 1-5 and 7 were drawn with Pymol (DeLano Scientific, Palo Alto, CA).
Enzyme-linked Immunosorbent Assay and Fluorescence Polarization Assays-GST-MDM2-1-150 4 and GST-MDMX-1-200, and fulllength His 6 -p53 were expressed in E. coli and affinity-purified under nondenaturing conditions. ELISA plates were incubated with 2.5 g/ml His 6 -p53 in phosphate-buffered saline (PBS) for 16 h. After washing with PBS ϩ 0.1% Tween 20 (PBST), the plates were blocked with PBS ϩ 5% nonfat dry milk ϩ 0.1% Tween 20 (PBSMT) for 0.5 h. GST-HDM2 and MDMX (5 g/ml) were mixed with peptides in PBSMTϩ10% glycerol ϩ 10 mM dithiothreitol and added to the wells. The plates were washed with PBST after incubating for 1 h at room temperature and incubated with MDM2 antibody 4B2 and MDMX antibody 8C6 in PBSMT for 1 h, followed by washing and incubation with horseradish peroxidase rabbit anti-mouse Ig antibody for 1 h. The plates were developed by incubation with TMB peroxidase substrate (KPL) and measured by absorbance at 450 nm. Fluorescence polarization assay was performed using N-terminally conjugated fluorescein isothiocyanate-p53 (LSQETFSDLWKLLPEN) and fluorescein isothiocyanate-pDI peptides. Proteins (2-4000 nM) and peptides (2 nM) were mixed in fluorescence polarization buffer (25 mM HEPES, pH 7.4, 0.1% Tween 20, 150 mM NaCl, 10 mM dithiothreitol). The mixtures were incubated at 25°C for 1 h and analyzed for fluorescence polarization. The K d values were determined from fluorescence polarization data using the method described by Zhang et al. (36).
CD Analysis-Circular dichroism measurements were made using an Aviv Model 215 spectrometer. The peptides were prepared at a concentration of 0.1 mM in 10 mM Tris-HCl, pH 7.5, 5% methanol, and 10% trifluoroethanol. The measurements were performed at 23°C. Blank scan (buffer) was subtracted from the spectra, and values of ellipticity were expressed in units of deg⅐cm 2 ⅐mol Ϫ1 . The amphipathic ␣-helical peptide melittin was used as a standard.

RESULTS AND DISCUSSION
Characterization of a High Affinity MDM2-and MDMXbinding Peptide-We have recently identified a 12-residue peptide inhibitor of MDM2 and MDMX by screening a phage display library for peptides that bind to GST-MDM2-1-150 and GST-MDMX-1-200 (30). Both screens selected the same peptide sequence ( 1 LTFEHYWAQLTS 12 ) as the highest affinity ligand for MDM2 and MDMX. This peptide was named pDI for peptide dual inhibitor. The pDI is distinct from the equivalent p53 sequence (p53p, 17 ETFSDLWKLLPE 28 ) but retains the key p53 residues Phe 19 , Trp 23 , and Leu 26 , which bind to three distinct hydrophobic pockets of MDM2 (16). The binding affinity of pDI to MDM2 and MDMX was analyzed using fluorescence polarization yielding dissociation constants of 1 nM for the MDM2-pDI and 3 nM for the MDMX-pDI interaction. By comparison, the K d values for p53p binding to MDM2 and MDMX were 160 and 260 nM, respectively, which is in good agreement with the previously determined values obtained by isothermal titration calorimetry (K d ϭ 130 -340 nM) (37). Inhibition of full-length p53 binding to MDM2 or MDMX by the pDI (and all other peptides studied here) was assessed by ELISA, yielding IC 50 values of 44 and 550 nM, respectively ( Table 1). The only other high affinity peptide also previously identified by phage display is the pMI peptide (31), which under our assay conditions displayed IC 50 values of 20 and 40 nM against MDM2 and MDMX, respectively (Table 1).
Structural Characterization of the MDM2 and MDMX pDI Peptide Complexes-In an attempt to understand the differences in binding affinities for MDM2 and MDMX, we first determined the crystal structure of MDMX liganded with the pDI peptide ( Fig. 1 and Table 2). Subsequently, both MDM2 and MDMX were co-crystallized with a derivative peptide, pDI6W, which contained a single Y6W mutation and displayed increased inhibitory properties over the parent peptide ( Table 1). The structures of the pDI6W peptide served as a template for the design of the pDIQ peptide. The overall structures of the N-terminal domains of MDM2 and MDMX liganded with the pDI and derivative peptides are very similar, as expected from the previously determined MDM2/X structures (16,38). The major structural difference between MDM2 and MDMX is the orientation of the C-terminal helix (␣2Ј, residues 96 -106 for MDM2 and 95-105 for MDMX) relative to the binding site of p53 and the pDI peptide ( Fig. 1; see also Fig. 5). The pDI peptides all bind to MDM2 and MDMX in a helical conformation via residues Phe 3 -Trp 7 -Leu 10 to the same groove that harbors the Phe 19 -Trp 23 -Leu 26 triplet of the p53 helix (16). The interaction pattern between the triplet residues and their binding sites in MDM2/X is dominated by hydrophobic interactions. In addition, the indole nitrogen of Trp(P) 7 establishes a hydrogen bond with the carbonyl oxygen of Leu 54 MDM2 or Met 53 MDMX (Figs. 2 and 3). Structure-Activity Relationship Studies and Design of the pDIQ Peptide-The co-crystal structures of MDM2 and MDMX liganded with the pDI6W peptide provided the basis for our structure-activity relationship studies. Our aim was to design peptides with improved inhibitory potential, in particular against MDMX, by substituting residues other than the conserved Phe 3 -Trp 7 -Leu 10 triplet. The conformation and binding pattern of the pDI and the pDI6W peptides are very similar (Figs. 2 and 3). Residues Leu 1 , Thr 2 , Glu 4 , His 5 , Ala 8 , and Gln 9 are completely solvent-exposed; Trp 6 and Thr 11 are partly solvent-exposed but also interact with protein residues through hydrophobic forces. Consequently, in the respective crystal structures a large portion of the liganded peptide is bound to well defined water molecules, some of which are conserved between the respective complexes of MDM2 and MDMX. The side chains of both Tyr(P) 6 and Trp(P) 6 are sandwiched between MDMX residues His 72 and Lys 93 (MDM2 residues His 73 and Lys 94 ) and the peptide residue His(P) 5 . The hydroxyl group of Tyr(P) 6 and the indole nitrogen of Trp(P) 6 are hydro- gen-bonded with water molecules. Both the Tyr(P) 6 and Trp(P) 6 ring systems interact with the aliphatic portion of Lys 93 and the C-␤ atom of His 72 through hydrophobic forces. However, the indole ring of Trp(P) 6 establishes more van der Waal's interactions (d Ͻ 4 Å) than Tyr(P) 6 , which possibly contributes to the improved binding potential of the pDI6W over the pDI parent peptide.
Amino acid substitutions were introduced into the pDI6W peptide and analyzed by ELISA to determine their potency in disrupting MDM2-p53 and MDMX-p53 interactions. Replacing Trp(P) 6 with Ser or Asn (6S, 6N, and 1E6N of Table 1) reduced or even abolished inhibitory potency, corroborating the above mentioned notion that ring systems in this position confer stability through an increased number of van der Waal's interactions with the protein. Leu(P) 1 was replaced by Glu (1E6W of Table 1) to increase solubility of the peptide in aqueous solutions; this substitution also improved by 2-fold the inhibitory potency on both MDM2 and MDMX (compare pDI6W to pDIE6W). Gln(P) 9 was replaced with Ser to promote a hydrogen bond between the serine hydroxyl group and the carbonyl oxygen of Trp(P) 6 . Unexpectedly, the resulting peptides (6W9S and 6W8S9S of Table 1) were less active than the pDI6W. Because Gln(P) 9 is too far away from His(P) 5 to establish a strong hydrogen bonding interaction (the average distance between the amide group and the imidazole is 3.5 Å), its side chain probably acts as a spacer to keep His(P) 5 in place, thereby contributing to the overall stability of the ␣-helix. Glu(P) 4 was replaced with Thr to test the importance of conformational flexibility in this region of the peptide. In the five co-crystal structures determined here, Glu(P) 4 adopts distinctly different conformations because of the close proximity of Met 61 MDMX /Met 62 MDM2 . The resulting peptide (4T6W) was inactive presumably because of steric hindrance between its hydroxyl group and the Met 61 side chain and/or between its methyl group and the side chain of Thr(P) 2 . Notably, the p53 peptide has a Ser, and the recently determined pMI peptide has an Ala in this position, both of which would not clash with Met 61 or Thr(P) 2 . Ala(P) 8 was replaced with Ser to establish an intrapeptide hydrogen bond between the Ser hydroxyl group and the carbonyl oxygen of His(P) 5 . The resulting peptide (6W8S) displayed significantly improved inhibitory activity. Thr(P) 11 was replaced with Leu to increase hydrophobic interactions with residues Met 53 MDMX and Leu 54 MDM2 ; this peptide (6W11L) was also considerably more active than the parent peptide.
Based on these results, we designed a peptide with four residues of the original pDI peptide substituted, i.e. L1E, Y6W, A8S, and T11L. This peptide (termed pDIQ for quadruple mutant) displayed 5-fold increased inhibitory potency over the pDI parent peptide against both MDM2 (IC 50 ϭ 8 nM) and MDMX (IC 50 ϭ 110 nM). In a side-by-side comparison, the pDIQ peptide was 2-fold more potent against MDM2 than the recently reported pMI peptide (31). Thus, the combination of phage display selection and rational design resulted in the most potent peptide inhibitor of MDM2 reported to date.
Molecular Basis for the High Affinity of the pDIQ Peptide-The co-crystal structures of MDM2 and MDMX liganded with pDIQ revealed substantial structural changes of this peptide upon binding to MDMX, with up to 2 Å displacement shifts of the C terminus from residues 8 -12 (Fig. 4). This is accompanied by a loss of helical structure of residues 10 and 11 (Figs. 1 and 4), which in turn allows Leu(P) 11 to interact with Tyr 99 of the MDMX ␣2Ј-helix and the side chains of Met 53 and Val 49 of the ␣2-helix (Fig. 5). It appears that the increase in hydrophobicity of the Leu 11 side chain in the pDIQ versus the Thr 11 side chain in pDI6W causes this structural change upon binding to MDMX. By contrast, the pDIQ in complex with MDM2 retains complete helical conformation, directing Leu(P) 11 away from the ␣2Ј-helix and toward the ␣2-helix. Here, Leu(P) 11 interacts with the side chains of Leu 54 and Phe 55 through van der Waal's forces, whereas the ⑀-amino group of Lys 51 is hydrogen-bonded to its carbonyl oxygen (Fig. 5).
The conformational changes in the pDIQ peptide appear to be induced mainly by residue Tyr 99 MDMX /Tyr 100 MDM2 of the ␣2Ј-helix (Fig. 5; see also Fig. 7). The orientation of this helix with respect to the peptide-binding site differs considerably between MDMX and MDM2. As a result, this tyrosine residue appears to exert a major role in the binding of the pDIQ to MDM2 and MDMX. In MDM2, Tyr 100 prevents Leu(P) 11 from reaching into the hydrophobic groove formed between the ␣2Јhelix and the ␣2-helix. As a consequence, the C terminus of The C-␣ atoms of the respective coordinate sets were aligned to the pDI MDMX structure using LSQkab (41,42).
the pDIQ peptide retains helical conformation, positioning Leu(P) 11 over the ␣2-helix. By contrast, Tyr 99 of MDMX accommodates the binding of Leu(P) 11 to the hydrophobic groove. It appears that the conformational flexibility in the C-terminal region of the pDIQ peptide ensures the high affinity binding to both MDM2 and MDMX. Remarkably, the pDIQ is 5-fold more active against MDMX than the parent pDI peptide, despite the partial loss of the energetically favored helical structure. This indicates the high binding potential of the groove between the ␣2Ј and ␣2 helices of MDMX attracting the Leu(P) 11 side chain. The binding energy gained from this interaction appears to compensate for the energy spent to disrupt the helical turn.
A similar observation was made recently for the interaction of the pMI peptide with MDMX, in which the peptide C-terminal Pro 12 residue was found to occupy the same site as Leu(P) 11 of the pDIQ peptide (31). The pMI and pDIQ peptides share little sequence homology, and only the FWL triplet residues are strictly conserved ( Table 1). The pMI peptide is two or three times more potent against MDMX than pDIQ but two times less active against MDM2. The differential inhibition is reflected in the distinct interaction patterns of the peptide C-terminal residues with the ␣2Ј/␣2-helices of both proteins ( Fig. 5 and supplemental Fig. S3). For the MDM2-pMI interaction, neither Ser(P) 11 nor Pro(P) 12 is involved in hydrogen bonding or hydrophobic interactions with the target protein, whereas Leu(P) 11 of pDIQ establishes multiple noncovalent interactions with residues of the ␣2-helix. This suggests a sig-nificant difference in binding potential for both peptides in MDM2. However, the slightly different overall conformation of the pMI peptide allows the formation of a hydrogen bond between the carbonyl oxygen of Leu(P) 10 and the hydroxyl group of Tyr 100 , which is not seen with the pDIQ peptide. Although the lack of binding potential of Ser(P) 11 and Pro(P) 12 of the pMI is compensated by the formation of this hydrogen bond with Tyr 100 , it seems that the distinct interaction pattern of Leu(P) 11 renders the pDIQ peptide more effective against MDM2. By contrast, in the MDMX-pMI complex, the Pro(P) 12 residue reaches deep into the interface between the ␣2Ј/␣2 helices, establishing more van der Waal's interactions with MDMX residues than Leu(P) 11 of the pDIQ peptide. This presumably accounts for the higher affinity of the pMI peptide toward MDMX.
Optimized Peptides Adopt ␣-Helical Conformation in Solution-For the pDIQ peptide, the Tyr(P) 6 to Trp and the Thr(P) 11 to Leu substitutions increased the number of intermolecular noncovalent interactions between the peptide and MDM2/X. The other amino acid substitutions were intended to increase the solubility of the peptide, at the same time strengthening the helical conformation through intrapeptide hydrogen bonds or through bridging water molecules. It has been suggested that the p53 N-terminal region is unstructured in solution but forms an ␣-helix upon binding to the hydrophobic pockets of MDM2 or MDMX (39). To test whether the inhibitory potency of our derivative peptides is reflected by a higher degree of helical conformation in solution, circular dichroism studies were performed (Fig. 6). The most potent peptides, pDIQ and 1E6W (Table 1), exhibited the strong double-well characteristics expected for ␣-helices, with pDIQ being most pronounced. The ␣-helical feature was less prominent in the 6W peptide and absent in the inactive 6S peptide. These results indicate that the improved inhibitory action of peptides is not only due to an increase in noncovalent interactions between the peptide and the protein (as seen for the Y6W and T11L substitutions) but also due to the enhanced stability of the ␣-helical conformation in solution.
Implications for the Design of Small Molecule Inhibitors against MDMX-Currently, small molecule inhibitors of MDM2 with anti-tumor activity such as Nutlin and MI-219 are generally ineffective against MDMX (24,25,30,40). Superposition of the complexes of MDM2-pDIQ and MDM2-Nutlin2 reveals the almost perfect fit of the three hydrophobic residues of Nutlin with the side chains of the peptidic FWL triplet (Fig. 7). However, the structural differ- ences of the ␣2Ј helices of both proteins directly impact the binding site of Leu(P) 10 . For MDM2, Tyr 100 accommodates the binding of Nutlin, whereas Tyr 99 in MDMX causes a potential steric clash with the Nutlin molecule. Although the efficient binding of the pDIQ peptide to MDMX is made possible through structural flexibility of the C-terminal region of the pDIQ peptide, small molecules such as Nutlin cannot undergo these conformational changes. Therefore, the design of p53 antagonists that selectively target MDMX over MDM2 must take into account these distinct differences of the peptide-binding site. One strategy could be the design of small molecules centering on the Leu(P) 10 site and extending to the Trp(P) 7 and Leu(P) 11 sites, rather than centering on the Trp(P) 7 site and extending to the Phe(P) 3 and Leu(P) 10 sites as in the case for Nutlin. To keep their size small, scaffolds designed in this manner will have to lack the functionality to bind to the Phe(P) 3 site (Phe(P) 3 is ϳ10 Å away from Leu(P) 10 ). This will likely result in a loss of bind-  JANUARY 15, 2010 • VOLUME 285 • NUMBER 3 ing affinity for MDM2 but is expected to gain affinity toward MDMX by exploiting new binding partners in the Leu(P) 11 site.

Design of High Affinity Peptides against MDM2 and MDMX
In conclusion, the results from this work demonstrate that peptides identified by phage display as MDM2 antagonists do not necessarily exhibit maximum binding affinity. Rather, such peptides may serve as ideal starting points for the structurebased design of inhibitors with substantially improved potency. In this study, the structure-activity analysis of a limited number of amino acid substitutions resulted not only in a 5-fold increase in inhibitory activity over the parent peptide but also revealed large conformational changes in the peptide required to bind efficiently to MDMX. These structural changes are induced by a hydrophobic site in MDMX, which probably has not yet received due attention in the design of small molecule inhibitors specifically targeting MDMX. The findings reported here should facilitate the rational design of novel MDMX-spe-cific or MDM2/MDMX dual specific inhibitors as potential cancer therapies.