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. 2026 Jul 17;11(29):44506–44514. doi: 10.1021/acsomega.6c06074

Evaluating Alkaline Phosphatase-Instructed Self-Assembly of d‑Peptide Diesters for Selectively Inhibiting Immunosuppressive Cancer Cells

Meihui Yi †, Gabriel Ashton-Rickardt †, Yuchen Qiao †, Yali Huang †, Hongjian He †,‡, Bing Xu †,*
PMCID: PMC13425513  PMID: 42540203

Abstract

Hypoxia-driven adenosinergic signaling suppresses antitumor immunity and enables many tumors to evade cancer immunotherapy. Alkaline phosphatase (ALP) contributes to this immunosuppression by hydrolyzing extracellular adenosine triphosphate (ATP) to adenosine. Rather than inhibiting this broadly essential enzyme, we exploit elevated ALP activity in tumors as a tumor-specific trigger for enzyme-instructed self-assembly (EISA). Using a potent naphthalene-capped phosphopeptide diester precursor (1P), which exhibits in vivo efficacy as a starting point, we designed and synthesized a series of analogs (2P–15P) that vary the N-terminal capping group and/or the position of the phosphotyrosine trigger to explore structure–activity relationships. We evaluated these analogs for anticancer activity in ALP-overexpressing cancer cells (e.g., Saos-2), using ALP-low cells (e.g., HEK-293) as controls. One analog (6P) exhibited potent, selective cytotoxicity, with submicromolar GI50 values (∼0.3 μM) in ALP-high cancer cells. Notably, 6P demonstrated submicromolar GI90 values and surpassed the efficacy of cisplatin and paclitaxel against Saos-2 cells. These findings highlight the potential of rationally designed d-phosphopeptide diesters to enable ALP-responsive self-assembly and selectively inhibit the growth of ALP-overexpressing tumors. This strategy offers a promising platform for the development of supramolecular therapeutics targeting immunosuppressive tumor microenvironments.

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Introduction

Cancer remains a formidable challenge to human health, often evading therapy through complex survival mechanisms. − Despite the transformative progress of immunotherapy, − hypoxia-driven adenosinergic signaling frequently suppresses antitumor immunity, allowing many tumors to escape immune surveillance. − Alkaline phosphatase (ALP), an ectoenzyme overexpressed in various malignant tissues, significantly contributes to this immunosuppression by hydrolyzing extracellular adenosine triphosphate (ATP) into adenosine. − While conventional strategies focus on inhibiting such enzymes, − their effectiveness diminishes in the presence of ALP overexpression. On the other hand, the elevated levels of ALP within the tumor microenvironment (TME) present a compelling opportunity to exploit this enzyme as a selective trigger for therapeutic intervention.

Thus, instead of blocking its essential physiological functions, we and others harness ALP to trigger enzyme-instructed self-assembly (EISA). − EISA uses ALP-mediated dephosphorylation as the initiating biochemical event to convert small, soluble peptide precursors into larger, nondiffusive supramolecular nanostructures in situ. The local formation of supramolecular assemblies on or near cancer cells perturbs cellular functions and compromises cell viability. Unlike traditional inhibitors, which lose efficacy as enzyme levels rise (amplification), EISA analogs become more potent as enzyme expression increases, turning a common drug-resistance mechanism into a therapeutic advantage. We have found that the naphthalene-capped phosphopeptide diester precursor Nap-ffpye­(OMe)2 (1P) inhibits an ALP-overexpressing osteosarcoma (Saos-2 and Saos-2-lung) in cell assay and strongly suppress tumor growth in an orthotopic mouse model without harming normal organs. Importantly, the mechanism underlying enzyme-instructed peptide self-assembly is increasingly understood. Recent cryo-structural work has shown that alkaline-phosphatase-triggered peptide self-assembly can drive extrinsic lytic cell death, providing direct structural and mechanistic support for EISA-enabled pyroptosis for enhancing immunotherapy. In addition, earlier work on prion-like nanofibrils of small molecules demonstrated selective inhibition of cancer cells through disruption of cytoskeletal dynamics, establishing that supramolecular assemblies can exert anticancer activity through defined cellular mechanisms rather than nonspecific aggregation.

In contrast to most studies that use ALP-based EISA for targeting various cancers through incorporation of additional therapeutic agents, the study of 1P 55 represents the first case of EISA being used to target immunosuppressive tumors in vivo without other therapeutic agents.

Building upon the previous identification of a naphthalene-capped d-phosphopeptide diester (1P) with promising ALP-responsive self-assembly and anticancer properties arising from its response to both surface and intracellular ALP (Scheme ), we sought to systematically investigate how subtle structural modifications influence assembly propensity, enzyme responsiveness, and the resulting bioactivity. To this end, we developed a series of analogs (2P–15P) by altering the N-terminal capping group and/or repositioning the phosphotyrosine trigger. These modifications aimed to fine-tune the EISA process and modulate interactions with ALP-overexpressing tumor cells for developing potent anticancer EISA agents.

1. General Structures of the d-Peptide Diesters and EISA at the Plasma Membrane and ER for Inhibiting Immunosuppressive Cancer Cells.

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To evaluate the biological relevance of these structural variations, we conducted cytotoxicity assays across different cancer cell lines with varying ALP expressions, including osteosarcoma and ovarian carcinoma, as well as ALP-low control cell lines (HEK-293). This approach enabled us to assess both potency and selectivity linked to ALP activity. Among the analogs tested, one analog (6P) emerged as particularly effective, exhibiting strong antiproliferative effects with GI50 values in the submicromolar range (∼310 nM) in both ALP-high cell lines. Notably, this analog demonstrated GI50 values (0.3 μM) exceeding the efficacy of clinical chemotherapeutics such as cisplatin (41 μM) and paclitaxel (1.0 μM) against Saos-2 cells. In addition, for 2P, cytotoxicity exhibited a strong correlation with ALPL expression across different cell lines. These results underscore the utility of molecular design in modulating ALP responsive supramolecular building blocks and support the development of d-phosphopeptide diesters as an effective class of tumor-selective agents. Collectively, our findings establish a structure–activity framework for engineering EISA-based therapeutics against ALP-overexpressing tumor cells. Although this study does not directly evaluate immune-related outcomes, the focus on ALP-high Saos-2 cells is biologically relevant because ALP participates in adenosinergic immunosuppression and our previous study showed that ALP-triggered EISA suppresses Saos-2 orthotopic bone tumor growth in vivo. Thus, the immunosuppression-related context provides motivation for this work, while direct immune modulation remains an important direction for future study.

Results and Discussion

Molecular Design and Synthesis

As shown in Scheme , the analogs were designed from the parent precursor 1P 55 to systematically vary two key structural parametersthe N-terminal capping group and the position of the phosphotyrosine triggerthereby enabling us to correlate aromaticity, hydrophobicity, and rigidity with self-assembly propensity, ALP-mediated responsiveness, and the resulting cell-selective bioactivity. The parent precursor, 1P, has a basic structure consisting of an N-terminal capping group (R), a central d-triaromatic amino-acid motif, and a C-terminal d-glutamic acid dimethyl ester. Within the triaromatic motif, X1, X2, and X3 denote positions that can be either hydrogen or phosphate. To investigate the influence of N-terminal capping groups on biological activity, we varied R to generate precursors 2P and 5P–15P. In addition, while maintaining the biphenyl group as the N-terminal cap, we systematically relocated the phosphate from X3 to X1 or X2, yielding 3P and 4P, respectively. This design allows us to elucidate the role of phosphate position in regulating enzyme-instructed self-assembly and biological activity. In addition, to verify the role of the phosphate group, we removed the phosphate from 2P to obtain compound 2.

2. Molecular Structures of 1P, and Analogs 2P−15P .

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All peptides without methylation were synthesized by solid-phase peptide synthesis (SPPS) (Scheme S2). Briefly, Fmoc-protected amino acids were loaded onto 2-chlorotrityl chloride resin. After removal of the Fmoc protecting group with piperidine in N, N-dimethylformamide (DMF), subsequent amino acids or the N-terminal capping group were coupled sequentially to elongate or cap the peptide chain. The peptides were then cleaved from the resin using trifluoroacetic acid (TFA). To obtain methylated analogs,

the resulting peptides (1 equiv) were dissolved in dichloromethane under stirring, followed by the addition of bromotrimethylsilane (30 equiv). The reaction mixture was stirred at room temperature overnight. After removal of solvents by air drying, methanol was added, and the mixture was stirred at room temperature for an additional 24 h. Following purification by HPLC, we obtained analogs 2P–15P in good yields with purity over 95% as shown in Figure S8–S23.

Self-Assembly

First, we determined the critical micelle concentration (CMC) of these analogs using Rhodamine 6G fluorescence. As shown in Table and Figure S1, 14P, which contains a benzoic acid capping group, 1P with a naphthylene group, and 2P–4P bearing biphenyl motifs exhibited a progressive decrease in CMC from approximately 343.1 μM to 50 μM and further to 10–20 μM.

1. Critical Micelle Concentration (CMC) and Log Po/w of Analogs 1P−15P .

Analogues CMC (μM) Consensus Log Po/w
1P 50.0 3.60
2P 14.6 4.51
2 6.00 5.22
3P 17.8 4.60
4P 19.7 4.57
5P 21.9 4.35
6P 11.4 4.38
7P 12.4 4.29
8P 7.70 5.75
9P 7.74 4.83
10P 9.24 3.71
11P 9.43 4.46
12P 39.8 2.05
13P 22.1 3.90
14P 343.1 2.99
15P 182.6 3.11
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a

The Consensus Log P was Calculated Using SwissADME.

These results indicate a positive correlation between the number of aromatic units and self-assembly capability. To further tune hydrophobicity, a hydrophobic C4H6 chain or a hydrophilic boronic acid moiety was introduced onto the biphenyl capping group, yielding 8P and 12P, respectively. Consistent with increased hydrophobicity, 8P displayed a markedly lower CMC (7.7 μM) than 12P (39.8 μM), demonstrating that the self-assembly behavior can be modulated by incorporating hydrophobic or hydrophilic elements into the capping motif. Among 2P, 3P, and 4P, which differ only in the position of the phosphate group, only minor variations in CMC were observed, suggesting that phosphate positioning has a limited effect on self-assembly. In contrast, comparison of 2P with its dephosphorylated counterpart 2 revealed a substantial decrease in CMC upon phosphate removal, highlighting the enhanced self-assembly propensity induced by dephosphorylation.

Containing a biphenyl capping group but with an additional methylene spacer between the capping motif and the peptide backbone, 5P exhibited a higher CMC, indicating increased conformational flexibility that disfavors assembly. Conversely, 13P, bearing a naphthyl group with one fewer carbon linker compared to 1P, showed a lower CMC, consistent with increased molecular rigidity. Notably, 9P, which features a rigid olefinic linkage between the two phenyl rings, also exhibited a low CMC. Collectively, these results suggest an inverse relationship between molecular rigidity and CMC, where increased rigidity and aromaticity promote stronger self-assembly.

In addition, we explored the possibility of other heteroaromatic rings as capping groups to the peptides. 6P contains a thiophene–phenyl conjugated capping group with 2-substituted thiophene, whereas 7P is the corresponding regioisomer featuring 3-substituted thiophene. 10P incorporates a furan aromatic capping group. 11P bears a bithiophene group. These capping groups share a rigid, π-conjugated aromatic framework, composed of a phenyl ring conjugated with a five-membered heteroaromatic ring, and all shown very similar low CMC values. 15P features an imidazole–phenyl capping group, introducing both aromaticity and hydrogen-bonding capability. The relatively large CMC value could be attributed to the deprotonation of imidazole, which makes it more hydrophilic compared to other heteroaromatic capping groups. To better visualize the relationship between CMC and hydrophobicity, the LogP was calculated using SwissADME and plotted with CMC and molecular weight. According to Figure , there is a clear negative correlation between CMC and LogP (Pearson r = −0.70), a common feature of amphiphilic molecules.

1.

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Relationship between CMC and logP of the analogs.

Cell Inhibition

After determining the CMC values of these analogs, we next evaluated their cytotoxicity against different cell lines to better understand how molecular structure influences their biological activity. First, the importance of the phosphate group was confirmed by comparing analog 2P with its dephosphorylated counterpart 2 (Figure a,b and S2a). In ALP overexpressing Saos-2 osteosarcoma cells, 2P exhibited a 24 h GI50 of approximately 0.60 μM, whereas compound 2 showed a 24 h GI50 greater than 10 μM. Over 3 days, 2P exhibited a dose-dependent response, whereas compound 2 showed no dose–response behavior. The substantial loss of activity upon removal of the phosphate group highlights its critical role as an enzymatic trigger for enzyme-instructed self-assembly (EISA). As shown in Figure S2b, 2P exhibited only mild effects on HEK-293 cells, which express ALP at normal levels; moreover, the decrease in HEK-293 viability observed after 72 h was not dose-responsive, suggesting that it is unlikely to result from a specific ALP-dependent EISA mechanism and may instead reflect nonspecific long-term culture or compound-exposure effects.

2.

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Cellular inhibition of 1P−15P. a. The cell viability of Saos-2 cells treated with 1P−15P for 24 h. b. The GI50 of 1P−15P against Saos-2 cells for 24 h. c. The correlation between 24 h GI50 of analogs and their CMC.

Additionally, treatment with the tissue nonspecific alkaline phosphatase (ALPL) inhibitor 2,5-dimethoxy-N-(quinolin-3-yl) benzenesulfonamide (DQB) rescued Saos-2 cells from 2P induced cytotoxicity in a dose dependent manner (Figure S2c). Together, these results support the essential role of the phosphate group and ALP triggered EISA in mediating the observed biological activity. The markedly lower activity of the dephosphorylated compound 2 compared with its phosphorylated precursor 2P further confirms that 2P does not act as a conventional prodrug releasing 2, but instead requires the phosphate group as an ALP-responsive trigger for productive EISA and cytotoxicity.

While retaining the phosphate group near the C-terminus, we investigated the influence of N-terminal capping groups on cellular activity using Saos-2 cells. Among the analogs, 6P exhibited the highest cytotoxicity toward Saos-2 cells, with a 24 h GI50 of approximately 0.31 μM. Notably, 2P, 6P, 7P, 8P, and 9P displayed similar GI50 values in the submicromolar range, consistent with their comparable CMC values. In contrast, 1P, 14P, and 15P, which possess higher CMC values, showed reduced cytotoxicity toward Saos-2 cells. The trends observed over the 3 days were similar, as shown in Figure S3. To further examine this relationship, the correlation between cytotoxicity (GI50) and CMC was analyzed (Figure c), revealing a strong positive linear correlation between CMC and cellular activity (Pearson r = 0.92). Given that lower CMC values indicate stronger self-assembly propensity, these results further confirm that increased cytotoxicity of EISA arises from the enhanced self-assembly ability of the peptide building blocks.

Among the low-CMC analogues, 6P exhibited the strongest cytotoxicity toward Saos-2 cells, suggesting that CMC alone does not fully account for cellular activity. The enhanced activity of 6P may arise from the thiophene-containing N-terminal capping group, which provides a rigid, π-conjugated, and moderately hydrophobic motif that favors productive supramolecular assembly after ALP-mediated dephosphorylation. Compared with other low-CMC analogues, such as 7P, 8P, and 9P, the 2-substituted thiophene–phenyl cap in 6P may better balance hydrophobicity, aromatic stacking, and molecular geometry, thereby promoting the formation of more effective assemblies in the cellular environment. Consistent with this possibility, TEM (Figure ) showed that 6P formed fiber-like structures even before ALP treatment and generated more extensive, well-defined fibrous networks after ALP treatment. Although these TEM results do not establish a direct structure–activity relationship, they suggest that the thiophene-containing cap of 6P may enhance ALP-responsive assembly and/or interactions of the resulting assemblies with Saos-2 cells. Thus, the superior potency of 6P likely reflects the combined effects of low CMC, favorable heteroaromatic capping, efficient enzyme responsiveness, and productive cellular interactions.

4.

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Enzyme-instructed self-assembly of peptide derivatives. a. The TEM images of 1P, 6P, and 8P at 10 μM with or without the treatment with 1 U/mL ALP for 24 h. b. The TEM images of 2P, 3P, and 4P at 20 μM with or without the treatment of 1 U/mL ALP for 24 h.

In addition, the position of the phosphate group within the peptide sequence appears to be an important determinant of activity. Notably, the cellular responses to 2P, 3P, and 4P varied across different cell lines (Figures a and S5–S7). For 2P, in which the phosphate group is located distal to the aromatic capping group, two distinct response patterns were observed, with some cell lines exhibiting clear dose–response behavior while others showed minimal or no response. In contrast, 3P, where the phosphate group is positioned proximal to the aromatic capping group, consistently displayed dose–response behavior across all tested cell lines, albeit with varying sensitivity. Notably, 4P, in which the phosphate group is located between these two cases, exhibited intermediate behavior, with cellular responses falling between those observed for 2P and 3P.

3.

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Relationship between gene expression and cell inhibition. a. The cell viability of different cell lines being treated with 2P, 3P, and 4P for 24 h. b. The cell viability of different cell lines at 2 μM for 24 h versus ALPL gene expression levels according to Depmap CCLE.

To better understand this relationship, we examined DepMap CCLE Cell Line Gene Expression Profiles for ALPL, ALPP, ALPP2, and ALPI. As shown in Figure b, 2P showed the strongest relationship between gene expression of ALPL and cell viability (Pearson r = – 0.87). For 3P and 4P, the correlation between ALPL expression and cytotoxicity was moderate (Pearson r = – 0.65 and −0.74, respectively), suggesting that additional factors, including other alkaline phosphatases, alternative enzymes, and distinct cellular pathways, may be involved. To further understand the contribution of other alkaline phosphatases, we analyzed the expression of ALPP, ALPP2, and ALPI. However, as shown in Figure S4, the correlations between their gene expression levels and cell viability were weak, with Pearson r values below 0.5. These results suggest that the spatial positioning of the phosphate group relative to the aromatic capping moiety plays a critical role in modulating peptide activity, potentially by influencing enzymatic accessibility and subsequent self-assembly.

Enzyme-Instructed Self-Assembly

Based on the correlations observed above, peptide self-assembly is likely involved in modulating cellular activity. To gain preliminary insight into the morphology of the assemblies, transmission electron microscopy (TEM) was performed on several key precursors. As shown in Figure a, three derivatives (1P, 6P, and 8P) that differ only in capping groups exhibit distinct self-assembly behaviors, both before and after enzymatic treatment. At 10 μM, 1P, which has a relatively high CMC, primarily forms aggregates and shows little morphological change upon ALP treatment. In contrast, 6P and 8P, with CMC values below 10 μM, are capable of forming fiber-like structures even in the absence of enzyme. Upon ALP treatment, more extensive and well-defined fibrous networks are observed for these compounds. Three additional derivatives (2P, 3P, and 4P), which differ in the position of the phosphate group, were further examined. As their CMC values fall within the range of 10–20 μM, a concentration of 20 μM was selected for TEM analysis. As shown in Figure b, despite their similar CMC values, these compounds exhibit markedly different assembly behaviors. At this concentration, 2P undergoes a morphological transition from aggregates to fibrous structures upon enzymatic treatment. In contrast, 3P maintains a similar morphology before and after enzyme treatment, indicating limited structural change. Notably, 4P is capable of forming fibrous structures even in the absence of enzyme, and its morphology shows minimal change upon enzymatic treatment. These results demonstrate that peptide self-assembly is strongly influenced by both the capping group and the position of the phosphate group, which together determine assembly behavior and responsiveness to enzymatic treatment.

While TEM provides preliminary insight into the morphology of the assemblies, the structural information remains limited and precludes direct correlation of TEM with cellular activity. Therefore, more advanced techniques, such as cryo-electron microscopy (cryo-EM) or cryo-electron tomography (cryo-ET), which enable direct visualization of peptide assemblies in cellular environments, are required to elucidate the relationship between supramolecular structure and biological function.

Conclusion

In summary, this study establishes that the anticancer activity of ALP-responsive d-peptide diesters is governed by two coupled design parameters: the propensity of the precursors to assemble and the accessibility of the phosphotyrosine motif to enzymatic dephosphorylation. By systematically varying the N-terminal capping groups and the position of the phosphate trigger, we found that changes in aromaticity, hydrophobicity, and molecular rigidity alter CMC and thereby tune assembly propensity, which strongly correlates with cellular activity. At the same time, shifting the phosphotyrosine closer to the aromatic cap diminishes the relationship between activity and ALPL expression, indicating that enzyme accessibility is an equally important determinant of productive EISA. Together, these results show that potent ALP-triggered bioactivity arises from a balance between efficient enzymatic conversion and a strong tendency of the resulting molecules to assemble in situ.

More broadly, these findings provide a practical framework for designing EISA analogs for ALP-overexpressing tumors: effective precursors should combine low-CMC scaffolds with phosphate placements that remain readily processed by ALP in cellular environments. Within this framework, the exceptional potency of 6P is especially informative. Because 6P, which bears a heteroaromatic thiophene-containing N-terminal cap, emerged as the most active and selective analog, heteroaromatic N-terminal groups appear to be a particularly promising design space for further optimization. Moreover, the trend exhibited by the N-terminal capping groups may provide a reference point for other EISA based anticancer therapeutics. − Future studies should therefore expand this motif class systematically, for example by varying heteroaromatic identity, substitution pattern, electronic properties, and ring connectivity, to determine whether the strong activity of 6P reflects a broader advantage of heteroaromatic capping groups in promoting favorable enzyme responsiveness and supramolecular assembly. Nevertheless, further in vivo validation, pharmacokinetic analysis, and comprehensive toxicity evaluation will be required to establish 6P as a viable lead analog for therapeutic development.

Although the present study focuses on the cellular structure–activity relationship of these ALP-responsive d-peptide diesters, our previous in vivo work with the related precursor 1P demonstrated effective inhibition of bone tumor growth without obvious systemic toxicity, supporting the feasibility of this EISA strategy in vivo while highlighting the need for future studies to directly quantify hydrolysis kinetics, CMC-dependent pharmacokinetic effects, tissue retention, and biological clearance.

Supplementary Material

ao6c06074_si_001.pdf (1.7MB, pdf)

Acknowledgments

The work in the B.X. laboratory was supported by NIH grants CA142746 and EY036512. This work made use of equipment purchased under National Science Foundation MRI Program Award # 2319804. TEM samples were prepared and imaged at the Brandeis Electron Microscopy Facility. CLSM was performed at the Brandeis Light Microscopy Core Facility. LC/MS analyses were conducted at the Brandeis University Mass Spectrometry Facility.

Glossary

Abbreviations

ALP

alkaline phosphatase

EISA

enzyme-instructed peptide self-assembly

ff

di-d-phenylalanine

CMC

critical micelle concentration

TEM

transmission electron microscopy

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c06074.

  • Detailed experimental procedures and materials; LC/MS results of individual compounds; additional cell viability data (PDF)

#.

(M.Y.) Laboratory for Biomaterials and Drug Delivery, Department of Anesthesiology, Division of Critical Care Medicine, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, United States

∇.

(G.A.-B.) Division of Chemistry and Chemical Engineering, California Institute of Technology

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

NIH grants CA142746 and EY036512.

The authors declare no competing financial interest.

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