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. 2024 Nov 12;67(22):20214–20223. doi: 10.1021/acs.jmedchem.4c01541

Covalent Targeting of Histidine Residues with Aryl Fluorosulfates: Application to Mcl-1 BH3 Mimetics

Giulia Alboreggia 1, Parima Udompholkul 1, Emma L Atienza 1, Kendall Muzzarelli 2, Zahra Assar 2, Maurizio Pellecchia 1,*
PMCID: PMC11613628  PMID: 39532346

Abstract

graphic file with name jm4c01541_0007.jpg

Covalent drugs provide pharmacodynamic and pharmacokinetic advantages over reversible agents. However, covalent strategies have been developed mostly to target cysteine (Cys) residues, which are rarely found in binding sites. Among other nucleophilic residues that could be in principle used for the design of covalent drugs, histidine (His) has not been given proper attention despite being in principle an attractive residue to pursue but underexplored. Aryl fluorosulfates, a mild electrophile that is very stable in biological media, have been recently identified as possible electrophiles to react with the side chains of Lys; however, limited studies are available on aryl fluorosulfates’ ability to target His residues. We demonstrate that proper incorporation of an aryl fluorosulfate juxtaposing the electrophile with a His residue can be used to afford rapid optimizations of His-covalent agents. As an application, we report on His-covalent BH3 mimetics targeting His224 of Mcl-1.

Introduction

Covalent drugs have taken a central role in the drug discovery stage in recent years due to their favorable pharmacodynamics and pharmacokinetic properties compared to reversible drugs. The approach is particularly appealing when challenging drug targets are targeted, such as those involved in protein–protein interactions (PPIs), where potency cannot be easily attained with reversible drugs. While several covalent drugs have been approved that bind covalently to a cysteine (Cys) residue within the binding site, the target space that contains such Cys residues remains relatively small. Hence, recently, several new electrophiles have been proposed to target other residues such as lysine (Lys), histidine (His), and tyrosine (Tyr)1−14 and more recently also other residues such as arginine (Arg)15 and aspartic (Asp).16 Among those, we and others reported that certain sulfonyl fluorides and aryl fluorosulfates can react to His,3,11,17,18 Lys,11 or Tyr2,11,19 when properly incorporated in carrying ligands to be juxtaposed to a targeted residue. We recently found that targeting His residues is particularly attractive given the favorable nucleophilicity of His and the frequency of histidine residues in binding sites.1,20 However, we previously used sulfonyl fluorides that are generally too reactive to be used in vivo,7 while we reported that aryl fluorosulfates can result in potent Lys-covalent agents that are orally bioavailable.6,8,21,22 The question then arises whether aryl fluorosulfates can be used generally to derive covalent ligands targeting His residues, using a “ligand-first” strategy, whereby a ligand with reasonable potency is further modified to contain the properly juxtaposed aryl fluorosulfate. These studies are particularly interesting given that His residues are often found at protein binding sites20 and are unprotonated at physiological pH, which facilitates a nucleophilic addition at the imidazole ring.3 Moreover, the His side chain is less flexible compared to Lys, which should facilitate a proper juxtaposition of the electrophile.3,12 Nonetheless, there are only a few reports on rationally designed His-covalent agents3,11,17,18 and even fewer using the more stable aryl fluorosulfate electrophile.12,17,18

Our recent studies reported on sulfonyl-fluoride-based Lys-5 and His-covalent1 agents targeting the Bcl-2 family protein hMcl-1. Here, we report on the design and associated characterizations of the first His-covalent hMcl-1 BH3 peptide using an aryl fluorosulfate as the electrophile. Our studies further support that aryl fluorosulfates are particularly attractive in targeting His side chains and provide strategies for further optimizations of known hMcl-1 reversible drug candidates.

Results

Design and Synthesis of BH3 Peptides Targeting His224 of hMcl-1(172–323)

We previously reported on BH3-derived peptides that target selectively hMcl-1 by covalently attacking either Lys234 (PDB ID 6VBX)5 or more recently His252 (PDB ID 8VJP),1 using an attenuated aryl sulfonyl fluoride warhead (Figure 1).1,7 However, aryl fluorosulfates are significantly more stable2,7 than sulfonyl fluorides, yet only a few examples are available of agents that have been rationally designed to introduce such electrophiles to target a His residue,3,11,12,17,18 although His residues are in principle more amenable to covalent modifications compared to Lys residues. In examining the X-ray structures of hMcl-1(172–323), in complex with such peptides, we identified residue His224 as potentially a more suitable amino acid to be covalently targeted by an aryl fluorosulfate given that its side chain is more intimately embedded within the BH3 binding pocket, unlike surface-exposed Lys234 or His252 (Figure 1).

Figure 1.

Figure 1

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Schematic description of covalent peptides designed to target hMcl-1. Previously, we reported on covalent peptides targeting either Lys234 (A, PDB ID 6VBX) or His252 (B, PDB ID 8VJP) with attenuated sulfonyl fluorides. His224 seems to be better positioned for covalent modifications, given that its location is more directly embedded within the BH3 binding pocket.

Hence, we designed several linear peptides based on an optimized minimal binding peptide that we had previously identified against hMcl-1 of sequence IAEQLRRIGDRF (IC50 = 250 nM).5 Based on simple structural considerations, aryl fluorosulfates were introduced in lieu of either Ile in position 8 or the C-terminal Phe residue, and to generate electrophiles of various lengths and positions, we introduced either a phenylalanine with a fluorosulfate in position 3 or 4 or a phenylglycine with a fluorosulfate in position 3 or 4 (Figure 2). These were obtained by using a conventional solid-phase peptide synthesis protocol by the respective phenols as we reported recently (Figure S1).1

Figure 2.

Figure 2

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Schematic representation of the proposed strategy to derive covalent His224 hMcl-1(172–323) targeting agents. (A) Model of the interactions between a BH3-derived peptide of sequence IAEQLRRIGDRF (purple ribbon structure, modeled from PDB ID 2PQK) and hMcl-1 (surface representation), highlighting the Mcl-1 residue His224 and two possible peptide residues that can be used to target it. His252 and Lys234 are also highlighted. (B,C) Modifications attempted to juxtapose an aryl fluorosulfate to His224 in the selected peptides. IC50 values reported were obtained from dose–response curves of a DELFIA displacement assay after a 2 h preincubation of the agent with 6×His-tagged hMcl-1(172–323) (see the Experimental Section).

To ascertain if any of the synthesized peptides retained binding to hMcl-1(172–323), we measured IC50 values using a DELFIA displacement assay as we reported previously,23 that quantifies, via dose–response measurements at a fixed incubation time, the ability of each test agent to displace a biotinylated BIM BH3 reference peptide. Not surprisingly, the introduction of modified side chains into the minimal peptide sequence at position 8 (Figure 2) resulted in agents with reduced affinity for the BH3 binding pocket. Agents 1–4 all resulted in being poorly active in the DELFIA assay, and this was most likely due to incompatible replacement of the Ile residue in position 8 with the bulkier aromatic rings. This is not entirely unexpected given previous structure–activity relationship studies on a similar BH3 peptide.1,5 However, replacement of the C-terminal Phe residue resulted in agents that largely retained affinity for hMcl-1 (Figure 2). This was particularly evident in compound 6 with an IC50 value of 8.1 nM under these experimental conditions, while the corresponding peptide lacking the fluorosulfate was ∼25 times less potent (compound 7, IC50 = 210 nM, Figures 2 and 3A), suggesting that the fluorosulfate in compound 6 may interact covalently with His224, as designed.

Figure 3.

Figure 3

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Binding studies to hMcl-1 of the His-covalent compound 6 and the noncovalent reference compound 7. (A) Dose–response DELFIA displacement assay curves comparing the ability of compound 7 (blue) and compound 6 (red) to displace the binding of a biotinylated-BH3 peptide to hMcl-1(172–323) (see the Experimental Section). (B) SDS gel electrophoresis of hMcl-1(172–323) (10 μM in buffer: 50 mM phosphate pH 7.5, 150 mM NaCl) measured in the absence or presence of compound 6 or compound 7 (100 μM) after 7 h of incubation. (C) Top panel, SDS gel electrophoresis of hMcl-1(172–323) (10 μM in buffer: 50 mM phosphate pH 7.5, 150 mM NaCl) measured in the absence or presence of compound 6 (100 μM) collected after the indicated incubation times. Bottom panel, SDS gel electrophoresis of hMcl-1 K234A(172–323), hMcl-1 H224A(172–323), and hMcl-1 H252A(172–323) (10 μM in buffer: 50 mM phosphate pH 7.5, 150 mM NaCl) measured in the absence or presence of compound 6 (100 μM) collected after 7 h of incubation. (D) Long-range [15N,1H] HSQC experiments to detect His side-chain resonances with a U–15N-labeled sample of hMcl-1(172–323) (50 μM in buffer: 50 mM phosphate pH 7.5, 150 mM NaCl) reported in the absence (blue) or presence (red) of compound 6 (250 μM). Resonance assignments are reported as obtained previously.1 (E) As in (D) but focusing on the spectral region containing resonances for 1Hε1,15Nδ1 spins. The large chemical shift perturbation in the presence of compound 6 is highlighted with a red arrow.

To verify that compound 6 binds covalently with hMcl-1(172–323), we initially performed SDS gel electrophoresis experiments. In the presence of compound 6, hMcl-1(172–323) displays a band shift indicative of an increased MW due to the formation of a stable adduct. On the contrary and as expected, the noncovalent equivalent, compound 7, did not cause a similar shift (Figure 3B). Furthermore, to qualitatively observe the kinetics of the covalent adduct formation, SDS gel electrophoresis gels were performed with samples collected after incubation of compound 6 with hMcl-1(172–323) at various times (Figure 3C, top panel). Qualitatively, we observed approximately 50% complex formation within 2 h and complete adduct formation within approximately 6 h. To ascertain that the agent was targeting His224, we also prepared single-point mutants Lys234Ala, His224Ala, and His252Ala hMcl-1(172–323) and conducted the same SDS gel electrophoresis experiments (Figure 3C, bottom panel). We could observe band shifts with wt-hMcl(172–323) and with the Lys234Ala and His252Ala mutants but not with the His224Ala mutant, suggesting that compound 6 may target His224 covalently. This could also be inferred by large chemical shift changes induced by compound 6 to the side chain of His224 as observed in long-range [1H,15N] HSQC 2D NMR experiments optimized to observe the His side chain [1H,15N] resonances in a 15N-hMcl-1(172–323) sample (Figure 3D). The large chemical perturbation due to the covalent adduct formation is particularly evident for the 1Hε1/15Nδ1 resonances of His224 (Figure 3D) as highlighted in Figure 3E. To further verify that a single complex adduct was formed, we further confirmed these data via mass spectrometry (Figure 4A,B). Compound 6 forms a stable 1:1 complex with hMcl-1(172–323) that presents the expected MW for the covalent adduct (Figure 4B). On the contrary, no MW increase was observed after exposing the His224Ala hMcl-1(172–323) mutant to compound 6 (Figure 4B).

Figure 4.

Figure 4

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Mass spectrometry and denaturation thermal shift measurements. (A) Schematic illustration of the reaction between hMcl-1 and compound 6 indicating the expected MW of each component of the expected MW of the covalent complex. (B) LC-MS analyses performed to confirm the formation of the covalent complex. Ten μM hMcl-1(172–323) or His224Ala hMcl-1 (172–323) was incubated with 100 μM compound 6 (7 h) at room temperature in a buffer (50 mM phosphate pH 7.5 and 150 mM NaCl). The mass of hMcl-1(172–323) is 19,572 Da, which is increased by 1591 Da (1611 Da – 20 Da (HF = leaving group)) in the presence of compound 6, corresponding to the anticipated mass of the covalent adduct. The mass of the His224Ala mutant is 19,506 Da, as expected, and it remains as such after incubation with compound 6 under the same experimental conditions reported above. The second mass present in each analysis corresponds to the mass after phosphogluconoylation of the target (mass + 178). (C) Denaturation thermal shift measurements of hMcl-1(172–323) (10 μM in a buffer: 50 mM phosphate pH 7.5 and 150 mM NaCl) measured in the presence of SYPRO orange (10×) and in the absence or presence of compounds 6 or 7 as indicated (100 μM), after 7 h of incubation. (D) As in (C) but with the His224Ala hMcl-1(172–323) mutant.

In addition, we measured ligand-induced denaturation thermal shifts (ΔTm) caused by compound 6 on hMcl-1(172–323) and the His224Ala mutant (Figure 4C,D). Our previous studies indicated that covalent adducts can induce large ΔTm values compared to reversible ligands.1,5,11,12 Accordingly, we observed that exposing hMcl-1(172–323) (10 μM, buffer: 50 mM phosphate pH 7.5 and 150 mM NaCl) to the reversible compound 7 (100 μM) caused a sizable ΔTm value of 3.88 ± 0.05 °C, while a very large ΔTm value of 22.41 ± 0.01 °C was observed when exposing hMcl-1(172–323) to compound 6 under the same experimental conditions (Figure 4C). However, when the same experiments were conducted with the His224Ala mutant, a ΔTm of 3.47 ± 0.09 °C was observed for compound 7 (hence similar to what was obtained with wt-hMcl-1), while a ΔTm of 5.45 ± 0.12 °C was observed after exposure to compound 6, hence a dramatically reduced effect compared to what was observed with wt-hMcl-1 (Figure 4D). These studies identify compound 6 as the first aryl fluorosulfate as a putative covalent hMcl-1 inhibitor targeting His224.

Structural Characterizations of the Compound 6–hMcl-1(172–323) Covalent Complex and Hydrocarbon Stapling

To investigate the binding conformation of the covalent agent, we determined the X-ray structure of compound 6 in complex with hMcl-1(172–323) (Figure 5). A summary of the structural parameters is reported in Table S1. The density and placement of covalently bound compound 6 in the hMcl-1(172–323) structure were well-resolved in its entirety (Figure 5). Not surprisingly, the overall geometry of the complex does not depart significantly from the previously determined structures of hMcl-1(172–323) in complex with various BH3 peptides. When superimposing hMcl-1(172–323) in the obtained complex with the coordinates from PDB code 6VBX, an rmsd value of 1.1 Å (C-alpha atoms) was obtained (Figure S2). Likewise, the general arrangement of the BH3 mimetic peptide follows the previously observed interaction patterns with such peptides, with the hydrophobic side chains of compound 6 buried in the BH3 binding pocket, while the peptide’s aspartic acid residue is involved in a salt bridge with residue Arg263 (Figure 5A). These intermolecular interactions are typical of BH3 peptides bound to hMcl-1(172–323)5 or of BH3 peptides in general in complex with other Bcl-2 family proteins.24

Figure 5.

Figure 5

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Crystal structure of hMcl-1(172–323) in complex with His-covalent compound 6. (A) Ribbon representation of hMcl-1(172–323) (gray) in complex with compound 6 (blue). The side chains of compound 6 are also displayed. (B) Close-up view of the sulfonamide bond formed from the reaction of the fluorosulfate of compound 6 and the side chain of His224. (C) 2Fo-Fc map of the bound compound 6 contoured at 1.0 sigma.

As anticipated by our previous studies reported above, we found that the C-terminal Tyr-fluorosulfate residue formed a covalent bond with the side chain of His224 (Figure 5B), as observed by a contiguous electron density between the aryl sulfonate group and the imidazole ring of His224 with whom the group formed a stable imidazole-sulfonate (Figure 5B,C). Hence, these data provide final and more direct evidence supporting covalent adduct formation between these two molecules and specifically with His224, in addition to the previous indirect data with SDS gel electrophoresis, mass spectrometry, denaturation thermal shift, and NMR spectroscopy, with the wt and single-point mutant His224Ala hMcl-1(172–323).

Moreover, these structural studies provide opportunities for further improving the peptide structure. Specifically, both the binding affinity and the kinetics of binding for covalent adduct formation may be improved by stabilizing the peptide in the bound α-helical conformation via hydrocarbon stapling.1 Based on our recent experience with covalent His252-targeting stapled peptides1 and based on the structure of compound 6 in complex with hMcl-1(172–323) (Figure 5), we designed and synthesized hydrocarbon-stapled compound 8 and the corresponding fluorosulfate compound 9 (Figure 6A). These agents were synthesized by using conventional solid-phase peptide synthesis protocols followed by an on-resin ring closing metathesis reaction (Figure S3) as we reported recently.1

Figure 6.

Figure 6

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Characterization of a His224 covalently stapled BH3 peptide. (A) Schematic illustration of the stapled compound 8 and its corresponding aryl fluorosulfate compound 9 (see also Figure S3 for the synthetic schemes and exact structures). (B) Dose–response DELFIA curves relative to the displacement of the reference biotinylated BIM BH3 peptide and compound 8 or compound 9. IC50 values were 15.5 ± 0.2 nM for compound 8 and 5.8 ± 0.9 nM for compound 9. (C) Denaturation thermal shift data for hMcl-1(172–323) in the absence (blue) and presence of compound 8 (red) or compound 9 (orange). ΔTm value measures were 7.75 ± 0.09 °C for compound 8 and 24.30 ± 2.54 °C for compound 9 (7 h). (D) SDS gel electrophoresis for hMcl-1(172–323) (10 μM in a buffer: 50 mM phosphate pH 7.5 and 150 mM NaCl) in the absence or presence of compound 9 (100 μM) after various incubation times as listed. (E) Mass spectrometry analysis of hMcl-1(172–323) (10 μM) collected in the presence of compound 9 (100 μM). Incubation conditions: 7 h at room temperature in a buffer (50 mM phosphate pH 7.5 and 150 mM NaCl). The mass of hMcl-1(172–323) is 19,572 Da, which is increased by 1557 Da (1577 Da – 20 Da (HF = leaving group)) in the presence of compound 9, corresponding to the anticipated mass of the covalent adduct. The second mass present (21,308) is the mass after phosphogluconoylation of the target (mass + 178). (F) Chemical stability of compound 9 in an aqueous buffer over time. 1D 1H NMR spectra for compound 9 (100 μΜ in 50 mM phosphate pH 7.5 and 150 mM NaCl) were collected at the indicated times. No significant spectral changes were observed.

As expected, proper placement of the hydrocarbon staple stabilized the peptide into an α-helical conformation, as confirmed by circular dichroism measurements (Figures S4 and S5). Given the proper position of the staple, predicted not to interfere with the interactions between the amphipathic helix and hMcl-1 based on the structure of the complex with compound 6 (Figure 5),1 compound 8 resulted in being more potent than compound 7 in displacing the biotinylated BIM BH3 peptide in the DELFIA assay (IC50 = 43 nM; Figure 6B). Moreover, introduction of the fluorosuflate at the C-terminal residue resulted in an agent, compound 9, that bound even more potently to hMcl-1(172–323) with an IC50 value approaching the limit of the DELFIA assay (IC50, 5.8 nM; Figure 6B; hence more potent also than compound 6). Denaturation thermal shift measurements (Figure 6C) reveal a ΔTm for compound 8 of 7.75 ± 0.09 °C, while for the His-covalent compound 9, a much larger ΔTm value of 24.30 ± 2.54 °C was observed, again typical of covalent inhibitors.1,4,5,7,11 Moreover, the kinetics of the reaction between the agent and hMcl-1 also improved (Figure 6D) with 50% of the reaction completed approximately after 1 h and nearly completed by 4 h (compared to the 2 and 6 h observed nonstapled compound 6, Figure 4). Kinetic measurements resulted in a Kinact/Ki value for compound 9 of 6.7 ± 0.3 × 104 M–1 s–1 (Figure S6), hence approaching similar kinetics of binding observed with current drugs targeting Cys residues with acrylamides.6,8−10,21,22,25−33

We and others have previously demonstrated that aryl fluorosulfates are very stable in aqueous media and even in vivo.2 Indeed, we did not observe any degradation of the peptide or the fluorosulfate group in a buffer up to 48 h (Figure 6F). This was measured both by 1D 1H NMR and by 19F NMR measurements observing directly the resonances of the fluorosulfate (Figure S7). Finally, to determine compound 9 selectivity for hMcl-1, we tested it in the DELFIA assay against the most closely related Bcl-2 family protein, namely, 6×His-hBfl-1(1–149).5 Under the same experimental conditions, compound 9 was nearly 160 times less active in the DELFIA assay against hBfl-1(1–149). This is not entirely surprising given that the initial noncovalent peptide was already originally designed to be selective for hMcl-15 and also because hBfl-1(1–149) does not present an equivalent His residue in the position of hMcl-1 His224 (Figure S8).5

In conclusion, our data convincingly indicate that compound 6 and compound 9 represent the first His224 aryl fluorosulfate-based hMcl-1 inhibitors. Our data should encourage the deployment of aryl fluorosulfates in targeting His residues in binding sites and should find wide applicability in drug design and lead optimization campaigns.

Discussion and Conclusions

The success of covalent drugs targeting Cys in recent years continues to drive drug discovery efforts toward the Cysteinome, which is the target space that contains a druggable Cys residue. In recent years, we and others devoted significant efforts in expanding the limited target space of the Cysteinome21,22,26−40 to tackle more frequently occurring nucleophilic residues such as Lys, His, or Tyr. The difficulty of this strategy consists in identifying proper electrophiles that possess a suitable balance between reactivity toward these less nucleophilic residues yet remain stable in an aqueous buffer and biological media. We reported that certain attenuated aryl sulfonyl fluorides could approach a good balance between reactivity and stability,2,7,12,41 especially when substituted with a methoxy group,1,4,7 but less reactive electrophiles would be preferable. We and others recognized that aryl fluorosulfates can provide this ideal balance when targeting Lys residues,2,11,12 and more recently, an example of aryl fluorosulfate targeting a His residue has been also reported.12,17,18,41−46 However, detailed studies targeting His residues with aryl fluorosulfates remain limited, although His residues are in principle more nucleophilic than Lys, due to their unprotonated state at physiological pH, and are frequently found in binding sites.3 Moreover, His residues are more rigid than Lys residues, providing more effective opportunities to design a proper juxtaposition between the electrophile and the residue side chain.

We chose hMcl-1 because of our previous experience in targeting this Bcl-2 family protein and because recent clinical studies failed for putative on-target cardiac toxicity of current reversible hMcl-1 clinical candidates.47 We hypothesized that devising novel targeting strategies for hMcl-1 that include the design of irreversible inhibitors could potentially change the pharmacodynamics of future agents and perhaps could provide a benefit to its toxicity profile in vivo, requiring lower doses, and/or lower dosing frequency that could ameliorate the potential of cardiac toxicity that could be associated with Mcl-1 antagonists.47,48 Recent attempts in targeting hMcl-1 covalently have been reported including our previously reported linear peptide5 targeting Lys234 with a sulfonyl fluoride, a small molecule that was elongated to reach Lys234 with a boronic acid aldehyde49 targeting Lys234, and more recently our stapled peptide targeting His252 (Figure 1). Both residues Lys234 and His252, however, are located in the periphery of the BH3 binding pocket, hence in an area that is too distant from the main core of the BH3 binding region that is usually occupied by small molecules (Figure 1). In our experience, when His or Lys residues are too flexible and exposed to the solvent, more reactive sulfonyl fluorides are needed for the covalent adduct formation, while the milder fluorosulfates are less effective. Indeed, when we tried to incorporate aryl fluorosulfates in a similar BH3 peptide to target the more flexible and solvent-exposed His252, we could observe only a partial adduct formation (Figure S9). On the contrary, His224 is nicely embedded in the binding site of hMcl-1, hence representing a more suitable residue for covalent modifications (Figure 2). We demonstrated that compound 6 and its stapled version, compound 9, effectively interacted covalently with His224 as indicated by an array of biophysical experiments including the X-ray structure of the complex with hMcl-1(172–323) (Figures 3–6). Due to the low reactivity of the aryl fluorosulfates, the ligands remained very specific for hMcl-1, with a 1:1 complex formation despite the several His/Lys/Tyr residues presented elsewhere on the surface of the protein (Figure 3), while, accordingly, a single mutation of His224 with Ala resulted in a construct that could not form a covalent adduct with compound 9 (Figure S10). Consequently, the ligands were very selective and poorly active toward the most closely related protein, namely, the Bcl-2 family member hBfl-1, lacking nucleophilic residues in a similar position to hMcl-1 His224. Plasma stability studies of compound 9 indicated that it remains mostly intact after 8 h, with ∼40% remaining after 24 h of incubation, while the corresponding noncovalent agent compound 8 remained nearly intact even after 24 h of incubation. Both observations agree with the stability of the stapled agents to plasma proteases and the relatively high stability of the fluorosulfate moiety in biological media2 (Figures S11 and S12). Moreover, for BH3-derived peptides containing either an aryl fluorosulfate (such as compound 9) or a methoxy-stabilized aryl sulfonyl fluoride, both remained very stable after incubation with albumin (Figure S13). The latter studies further corroborate our previous observations of the stabilization effect of a methoxy group on the aryl sulfonyl fluorides.1,4,7 However, and consistent with our previous study,7 aryl fluorosulfates result in generally more stable ones compared to aryl sulfonyl fluorides, as illustrated using 19F NMR studies (Figure S14).

Our studies also once again highlight the power of NMR spectroscopy measurements for His side chains using 15N uniform labeling and long-range HSQC [1H,15N] correlation measurements, as those not only suggest the tautomerism of each His residue but are dramatically sensitive to detect covalent modifications at the targeted His residue (Figure 4). Based on the long-range 15N/1H correlations spectrum, all His residues in Mcl-1 appear to adopt the more common Nε-H tautomer (also known as the τ tautomer),50,51 and consistently, the reaction of the fluorosulfate appears to occur at the Nε nitrogen of His224, as revealed by X-ray crystallography. Likewise, denaturation thermal shift measurements (ΔTm) are also particularly sensitive to covalent agents, and in the case of hMcl-1, we found that the His-covalent agents could be recognized by the distinct double-digit ΔTm values observed (Figures 4 and 6). We envision that such experiments could find wide applications in ligand-first covalent design endeavors and perhaps also in electrophile-first screening strategies with aryl fluorosulfate fragment-based screening.

In conclusion, our studies report for the first time aryl fluorosulfate-based hMcl-1 covalent inhibitors that target His224. Because this residue is nicely embedded in the core of the BH3 binding pocket, several small-molecule inhibitors are found in the proximity of this residue, providing novel and potentially effective ways to further optimize these agents into covalent drugs. This could perhaps overcome the current limitations of reversible hMcl-1 inhibitors observed in the clinic. Hence, our studies support once again the notion that targeting covalently His residues should be prioritized in devising covalent drugs from lead agents arising from fragment and/or structure-based strategies52−58 and/or DNA-encoded libraries,9,59,60 for example, to identify potent and selective covalent agents for continued drug development. However, our studies also anticipate that effective biophysical strategies and approaches are in place to perform direct electrophile-first campaigns with aryl fluorosulfates, and we will report on this in the near future.

Experimental Section

General Chemistry

The reported agents were synthesized using standard solid-phase strategies with an automated Liberty Blue peptide synthesizer (CEM Corp.) or with reactions performed manually. All of the reagents used for the synthesis and characterization were commercially available and used without further purification. The syntheses were performed by using a Rink amide resin. For the coupling reactions performed manually, 3 equiv of Fmoc-amino acid, 3 equiv of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′,-tetramethyluronium hexafluorophosphate (HATU), 3 equiv of OximaPure, and 5 equiv of N,N-diisopropylethylamine (DIPEA) in 1 mL of DMF (dimethylformamide) were used, and the solution was left shaking for 1 h at room temperature. For the Liberty Blue synthesizer, the coupling involved 6 equiv of Fmoc-amino acid, 1 equiv of OximaPure, and 3 equiv of N,N′-diisopropylcarbodiimide (DIC) in 2 mL of DMF (T = 90 °C, 5 min via microwave irradiation).

The Fmoc protecting group removal performed manually required a solution of 20% 4-methylpiperidine in DMF, and the treatment was performed twice: 1 mL for 5 min and then 1 mL for 20 min. The Fmoc deprotection protocol used with the Liberty Blue synthesizer was 2 × 3 mL at 90 °C for 3 min.

The acetylation of the N-terminus was performed using 3 equiv of acetic acid, 3 equiv of HATU, 3 equiv of OximaPure, and 5 equiv of DIPEA in 1 mL of DMF. The solution was shaken for 1 h at room temperature.

The AISF reaction was performed in resin using 1.2 equiv of AISF and 2.2 equiv of 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU) in 2 mL of DCM. The reaction was performed overnight.

The on-resin ring closing metathesis reaction was performed before the cleavage of the peptide with the protocol described in a previous paper.1 Briefly, the resin was dried and treated twice with a 10 mM solution of Grubbs catalyst first generation in dry DCE (2 mL per 50 μmol of resin) under N2 for 2 h.

The cleavage of the peptide from the resin was accomplished with a mixture containing TFA (trifluoroacetic acid), triisopropylsilane, water, and phenol (94:2:2:2) for 5 h, and the product was purified by preparative RP-HPLC using an XTerra C18 column (Waters) with a JASCO preparative HPLC system and gradient water/acetonitrile (5–100%) containing 0.1% TFA (purity >95%). The identity of the peptides was further confirmed by high-resolution mass spectrometry.

Compound Purification

The purity of all tested agents was verified to be >95% by RP-HPLC. For the purification, we used a JASCO preparative system and an XTerra C18 10 μm 10 × 250 mm2 column (Waters). The instrument was equipped with a fraction collector controlled ChromNAV system (JASCO) and a PDA detector. HPLC traces of key compounds can be found in Figures S15 and S16.

Mass Analyses

The mass of all the compounds was analyzed using an Agilent 6545 QTOF LC/MS instrument (Table S2).

SDS Gel Electrophoresis

SDS-polyacrylamide gel electrophoresis (PAGE) was performed with NuPage 12% Bis-Tris gels (Life Technologies). The proteins, wt-hMcl-1(172–323), hMcl-1(172–323) His224Ala, hMcl-1(172–323) His252Ala, or hMcl-1(172–323) Lys234Ala were incubated at a 10 μM concentration with or without each agent at a concentration of 100 μM (buffer 50 mM phosphate pH 7.5, 150 mM NaCl, and 1% DMSO).1 The running buffer used was MOPS, and the staining solution was SimplyBlue SafeStain (Life Technologies), used following the manufacturer’s instructions.

NMR Spectroscopy

Protein NMR spectra were acquired on hMcl-1(172–323) samples or the mutants in a 50 mM phosphate buffer pH = 7.5, 150 mM NaCl, and 1 mM DTT, containing 5% D2O, at T = 25 °C, on a Bruker Avance III 700 MHz spectrometer equipped with a TCI cryoprobe. 19F NMR measurements were carried out on a Bruker Avance III 600 MHz instrument. Data processing was obtained using TopSpin 4.1.0 (Bruker, Billerica, MA). For the detection of His side-chain resonances, 2D [15N,1H] long-range so fast HMQC spectra were optimized to detect the imidazole ring 2J15N–1H correlations with a uniformly 15N-labeled sample (50 μM) of hMcl-1(172–323). Resonance assignments for the His side chains were obtained as we reported previously.119F 1D NMR spectra of compound 9 and reference compounds were recorded in the same buffer at various time points.

Thermal Shift Assay

The thermal shift assay was conducted using QuantStudio 3 (Thermo Fisher Scientific) and analyzed with Protein Thermal Shift Software 1.3, as we reported previously.1,4,11 The dye used for the analyses was the fluorescent dye SYPRO orange. The samples were prepared by incubating 10 μM protein with or without 100 μM of the compounds in a buffer: 50 mM phosphate pH 7.5, 150 mM NaCl, 10× SYPRO orange, and 2% DMSO. The samples were heated from 10 to 99.5 °C with a linear gradient and a heating rate of 2 °C/min. The fluorescence intensity was measured with Ex/Em of 550/586 nm.

Biochemical Assay and Kinetic Measurements

A heterogeneous assay based on the DELFIA (dissociation-enhanced lanthanide fluorescence immunoassay) platform was developed for the 6×His-tagged hMcl-1(172–323) sample (16 nM), as described previously.1 Dose–response inhibition curves were performed at various preincubation times as reported, and data were analyzed and plotted using Prism 10 (GraphPad). Kinetic measurements were performed similarly as we reported previously.4 Each well was incubated with 16 nM Mcl-1 and 1:2000 Eu–N1-labeled anti-6×His antibodies for 2 h prior to incubation with compound 6 or 9 for 0, 2, 5, 10, 20, and 40 min. Plates were washed three times and incubated with 200 μL of enhancement solution for 10 min. Fluorescence measurements were then measured using a VICTOR X5 microplate reader with the excitation and emission wavelengths of 340 and 615 nM, respectively. The slope of a percent inhibition versus incubation time plot was used to calculate the observed rate constant for inhibition, kobs, which was then replotted against the peptide concentrations and fitted to a hyperbolic curve on Prism 10 (GraphPad) to extrapolate the inhibition constant, KI and Kinact.

Protein Samples

Protein expression and purification were accomplished as we recently described to obtain the ligand-binding domain of hMcl-1 (residues 172–323) either in an unlabeled form or uniformly 15N-labeled. His224Ala, His252Ala, and Lys234Ala single-point mutants hMcl-1(172–323) were similarly expressed and purified. The protein samples used for all experiments were prepared in an aqueous buffer composed of 50 mM phosphate at pH 7.5, 150 mM NaCl, and 1 mM DTT.

Circular Dichroism

Circular dichroism spectra were acquired with the use of a Jasco 815 spectropolarimeter as we reported previously.1 Samples were prepared in MQ water (250 μM). The experiments were performed using a 1 mm path-length quartz cell, and the data were recorded at 25 °C, from 190 to 260 nm. The percentage of α-helices (Figures S4 and S5) was calculated using BeStSel (https://bestsel.elte.hu) software.61

X-ray Crystallography

Crystallization was performed using sitting drop vapor diffusion at 4 °C, with diffraction quality crystals grown in 0.1 M citric acid, pH 3.5, and 25% (w/v) PEG 3350. The 18.0 mg/mL hMcl-1/compound 6 complex was plated at a protein/mother liquor ratio of 0.5:0.5 μL. Crystals were frozen using a 20% ethylene glycol cryoprotectant, and data collection was conducted at the Diamond Light Source (I03) beamline. A data set was collected on a crystal that diffracted to 1.5 Å, and the diffraction data were processed in the P1211 space group using XIA2 Dials. Molecular replacement for the data set was performed using a search model based on PDB ID 6VBX in PHASER, PHENIX. Several rounds of refinement and model building were performed in the absence of peptides using COOT and PHENIX. After the placement of the solvent molecules, the chemical model of the noncanonical N-terminal amino acid was fit into the remaining density and refined by PHENIX. The remaining parts of the peptide were built manually using COOT, and the structure was refined by PHENIX. The top solution was refined using refinement in REFMAC, CCP4. The final crystal data statistics are listed in Supporting Information Table S1. The coordinates for the complex between compound 6 and hMcl-1 have been deposited in the PDB and will be released upon publication (PDB ID 9CKN).

Acknowledgments

This research was funded by NIH grants NS107479, CA168517, and CA285114 to M.P. M.P. holds the Daniel Hays Chair in Cancer Research at the School of Medicine at UCR. Molecular graphics were performed with the UCSF Chimera package (http://www.cgl.ucsf.edu/chimera). Chimera is developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIGMS P41-GM103311).

Glossary

Abbreviations Used

CD

circular dichroism

CuSO4

cupric sulfate

DBU

1,8-diazabicyclo[5.4.0]undec-7-ene

DCE

1,2-dichloroethane

DCM

dichloromethane

DELFIA

dissociation-enhanced lanthanide fluorescence immunoassay

DIPEA

N,N-diisopropylethylamine

DMF

dimethylformamide

DMSO

dimethyl sulfoxide

DTT

dithiothreitol

EDTA

ethylenediaminetetraacetic acid

HATU

1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate

HMQC

heteronuclear multiple quantum coherence

HPLC

high-performance liquid chromatography

IPTG

isopropyl b-d-1-thiogalactopyranoside

LC-MS

liquid chromatography–mass spectrometry

OXYMA

ethyl cyanoglyoxylate-2-oxime

TFA

trifluoroacetic acid

THF

tetrahydrofuran

Data Availability Statement

PDB ID Code: The atomic coordinates of the model between hMcl-1(172–323) and compound 6 have been submitted to the Protein Data Bank (PDB ID 9CKN). Authors will release the atomic coordinates and experimental data upon article publication.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01541.

  • (Figure S1) synthesis of compound 6, (Figure S2) superimposition of the previously determined structure of hMcl-1(172–323) in complex with a BH3 peptide analog (PDB ID 6VBX) with the structure of hMcl-1(172–323) in complex with compound 6, (Figure S3) synthesis of stapled peptide 9, (Figures S4 and S5) CD curves of compounds 6 and 9, (Figure S6) kinetic measurements, (Figure S7) 19F NMR measurements of compound 9, (Figure S8) DELFIA assay of compound 9 against Mcl-1 and the most closely related Bcl-2 family protein Bfl-1, (Figure S9) similar BH3 peptides containing aryl fluorosulfates designed to target the more flexible and solvent-exposed His252 of hMcl-1, (Figure S10) mass analyses of hMcl-1(172–323) H224A analyzed in the absence and presence of compound 9, (Figures S11 and S12) plasma stability studies of compound 9 and compound 8 by 1D 1H NMR, (Figure S13) aqueous stability of compound 9 and compound 155H1 by 1D 1H NMR with compounds incubated with bovine serum albumin, (Figure S14) 1D 19F NMR spectra to study the aqueous stability of representative fluorosulfates and sulfonyl fluorides, (Figure S15) HPLC trace for compound 6, (Figure S16) HPLC trace for compound 9, (Table S1) summary of structural parameters for the crystal structure of hMcl-1(172–323) in complex with peptide 6, and (Table S2) mass spectrometry data of reported peptides (PDF)

  • SMILES data and IC50 values of compounds 6 and 9 (CSV)

Author Contributions

M.P. with G.A. designed the research strategy. G.A. synthesized, purified, and characterized all agents reported, with the support of E.L.A. G.A. carried out protein expression and purification, denaturation thermal shift measurements, and NMR measurements. P.U. characterized the agents in vitro in the DELFIA assays. K.M. conducted protein complex crystallization, data collection, and structure determination. Z.A. designed and mentored the protein complex crystallization project. M.P. analyzed all data with all authors and wrote the manuscript with the help of G.A. and other authors.

The authors declare the following competing financial interest(s): GA, PU declare no conflict of interests. MP is a co-founder of Armida Labs, Inc. KM and ZA are employees of Cayman Chemical.

Supplementary Material

jm4c01541_si_001.pdf (2.9MB, pdf)
jm4c01541_si_002.csv (612B, csv)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

jm4c01541_si_001.pdf (2.9MB, pdf)
jm4c01541_si_002.csv (612B, csv)

Data Availability Statement

PDB ID Code: The atomic coordinates of the model between hMcl-1(172–323) and compound 6 have been submitted to the Protein Data Bank (PDB ID 9CKN). Authors will release the atomic coordinates and experimental data upon article publication.

Articles from Journal of Medicinal Chemistry are provided here courtesy of American Chemical Society

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