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. 2024 May 23;15(7):1032–1040. doi: 10.1021/acsmedchemlett.3c00268

Design Principle of Heparanase Inhibitors: A Combined In Vitro and In Silico Study

Yuzhao Zhang 1, Meijun Xiong 1, Zixin Chen 1, Gustavo Seabra 1, Jun Liu 1, Chenglong Li 1, Lina Cui 1,*
PMCID: PMC11247634  PMID: 39015272

Abstract

graphic file with name ml3c00268_0009.jpg

Heparanase (HPSE) is an enzyme that cleaves heparan sulfate (HS) side chains from heparan sulfate proteoglycans (HSPGs). Overexpression of HPSE is associated with various types of cancer, inflammation, and immune disorders, making it a highly promising therapeutic target. Previously developed HPSE inhibitors that have advanced to clinical trials are polysaccharide-derived compounds or their mimetics; however, these molecules tend to suffer from poor bioavailability, side effects via targeting other saccharide binding proteins, and heterogeneity. Few small-molecule inhibitors have progressed to the preclinical or clinical stages, leaving a gap in HPSE drug discovery. In this study, a novel small molecule that can inhibit HPSE activity was discovered through high-throughput screening (HTS) using an ultrasensitive HPSE probe. Computational tools were employed to elucidate the mechanisms of inhibition. The essential structural features of the hit compound were summarized into a structure–activity relationship (SAR) theory, providing insights into the future design of HPSE small-molecule inhibitors.

Keywords: Heparanase inhibitor, molecular dynamics simulation, molecular modeling, drug discovery, drug design

Heparanase (HPSE), an endo-β-d-glucuronidase of the glycoside hydrolase 79 (GH79) family, plays a crucial role in modulating the extracellular matrix (ECM) functionality and stability by cleaving heparan sulfate (HS) side chains from heparan sulfate proteoglycans (HSPGs).2−4 HSPGs are fundamental components of the ECM, consisting of core proteins conjugated to HS chains, heterogeneous polysaccharides consisting of O/N-sulfated and/or N-acetylated derivatives of a variety of saccharide building blocks including d-glucosamine and uronic acids.5,6 HSPGs support the ECM’s stability and integrity, interact with the cell surface and the ECM via HS side chains, and participate in various signaling pathways.7 HPSE is the only known enzyme that cleaves HS; one identified cleavage site is at the internal glycosidic bond between a glucuronic acid (GlcUA) residue and an N-sulfoglucosamine (GlcN(NS)) residue bearing a 3-O-sulfo or a 6-O-sulfo group.8 By cleaving HS, HPSE changes the functionality of HSPGs, leading to ECM remodeling and impacting downstream pathways activated by the release of growth factors, chemokines, cytokines, and HS fragments.9 HPSE plays an essential role in various types of cancer and has been implicated in all cancer hallmarks;10 it is also involved in other diseases or pathological conditions, such as diabetes,11 bone necrosis,12 fibrosis,13 autophagy,14,15 and Alzheimer’s disease,16 making HPSE a highly promising therapeutic target.17

HPSE is first produced as a pro-enzyme, pro-HPSE, which is further activated by lysosomal or extracellular cathepsin L to its active form.4 Structurally, the active HPSE is a heterodimer consisting of an 8 kDa and a 50 kDa domain (Figure 1).18 GLU225 and GLU343 are the two catalytic residues that serve as a proton donor and a nucleophile, respectively, in the catalytic site lying in a 10 Å cleft.19 This catalytic cleft is one of the most important sites for HPSE inhibition. In addition, heparin binding domain-2 (HBD-2), consisting of the sequence of GLN270-LYS280, and the glycine loop (GLY349-GLY351) that lies close to HPSE substrate binding pocket are also suggested to be druggable regions (Figure 1).17 Generally, any residues that participate in substrate binding are potential targets for inhibition.

Figure 1.

Figure 1

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Structure and functional domains of human heparanase (HPSE) (PDB ID:5E98).

Currently, the only HPSE inhibitors that have advanced to clinical trials are heparin/HS derivatives/mimetics (PI-88,20PG545,21SST0001,22M-402,23 and sulodexide(24)); however, to date, none has been approved as a drug. Due to their heterogeneous nature, polysaccharide-based HPSE inhibitors suffer from challenges in product standardization and characterization. Additionally, the large molecular weight and high hydrophilicity of these molecules limit their bioavailability, complicating their clinical applications. Besides oligo- and polysaccharides, small-molecule, nucleic acid, and antibody-based therapeutics targeting HPSE have also been reported. Small-molecule HPSE inhibitor discovery has been attempted for decades, with various scaffolds reported.25 To date, few of the small-molecule inhibitor candidates have been evaluated in animal models, and none has entered clinical trials, leaving a gap in the discovery of HPSE small-molecule inhibitors. Traditional medicinal chemistry methods have been widely applied in the development of HPSE inhibitors, and only a few computational methods incorporating HPSE inhibitor design studies have been reported.25 By employing high-throughput screening (HTS),26,1 pharmacophore modeling,27,28 and molecular dynamics (MD) simulation,29,30 some promising small-molecule HPSE inhibitors were discovered or inhibition mechanisms were revealed. Herein, we report the discovery of a novel HPSE inhibitor hit and provide design insights into novel small-molecule HPSE inhibitors.

In our previous study, we discovered a novel HPSE inhibitor hit via an HTS study using the ultrasensitive heparanase activatable disaccharide probe (HADP).1 The hit, TC LPA5 4 (Figure 2A), is a known LPA5-selective receptor antagonist (IC50 = 0.8 μM),31,32 exhibiting promising HPSE inhibitory activity (IC50 = 10 μM, Figure 2B). In the initial molecular docking study, we discovered a potential binding mode of TC LPA5 4 with HPSE (Figure 2C). Specifically, carboxypyrazole ring 1 formed polar interactions with LYS159 and ARG303, while the methoxy group at ring 2 formed a hydrogen bond with THR97. Although we did not observe any interaction of HPSE with chlorobenzene ring 3 or cyclohexane ring 4, we hypothesized that the overall topology of the small molecule and the planar angle formed by the two phenyl rings with ring 1 were crucial for the HPSE inhibitory activity. In our initial attempt to optimize the structure of TC LPA5 4 for HPSE inhibition, we retained the overall structure feature and used ring 1 as a key binding moiety while adding polar groups on ring 2 to yield a 2-aminophenol ring (Figure 3A). We also changed ring 4 to a pyrazole ring with the intention of forming interactions with the polar residues ARG272 and GLN270, which were within close distance of TC LPA5 4 according to the preliminary docking study. The resulting small molecule, MX4-62, was then synthesized (Figure 3A and SI). Unfortunately, MX4-62 did not exhibit any HPSE inhibitory effect (Figure 3B), suggesting that further optimization using more sophisticated computational tools is necessary. It is noteworthy that molecular docking, despite its utility, may not yield reliable results due to its dependence on rigid protein structures. Therefore, caution is needed, as a high-scoring conformation in docking studies may not correlate with inhibitory action.

Figure 2.

Figure 2

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(A) Structure of the hit compound, TC LPA5 4, and the numbering of each ring of the compound. (B) Heparanase (HPSE) inhibitory activity of TC LPA5 4 (figure adapted from ref (1)). HPSE inhibitory IC50 = 10 μM. (C) Binding conformation of TC LPA5 4 with HPSE identified from a molecular docking study.

Figure 3.

Figure 3

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(A) Synthetic route to MX4-62. Conditions: (a) Pyrazole; 10 mol% Cu2O; 2.0 equiv of Cs2CO3; DMF; 100 °C; 24 h. Yield: 73%. (b) Dimethyl oxalate; 1.3 equiv tBuOK; THF; 0 °C to rt under argon; overnight. The reaction mixture was directly used in the next step. (c) 5-Hydrazinyl-2-nitrophenol; 0.1 M HCl; MeOH; 50 °C; 24 h. Two-step yield: 74%. (d) 20% w/w Pd/C; H2 balloon. Yield: 53%. (e) 1 M NaOH; THF/H2O (1:1); rt; 12 h. Yield: 67%. (B) Heparanase inhibition assay of MX4-62 against recombinant human HPSE.

Before we further designed any small molecules, we made efforts to understand the potential key interactions that may form between good binders and the enzyme from both mechanistic and structural insights. For HS cleavage, the enzymatic reaction, GLU225 serves as a proton donor that protonates the glycosidic oxygen, and then GLU343 acts as a nucleophile to attack the anomeric carbon and accomplish the cleavage. These two catalytic residues are crucial HPSE activity inhibition sites. To identify other critical interactions involved in HPSE–substrate or HPSE–inhibitor binding, we conducted a structural analysis on HPSE. The endogenous saccharide substrate interacts with ASP62, ASN64, THR97, ASN224, GLN270, ARG272, HIS296, ARG303, GLU343, GLY349, GLY350, GLY389, and TYR391, as observed in the structure of HPSE cocrystallized with a heparin-derived tetrasaccharide (dp4) (PDB ID: 5E9C, Figure 4A).33 To compare the substrate binding and inhibitor binding modes with HPSE, we analyzed the interactions established in other HPSE crystal structures, including PDB IDs 5E98, 7PR7, 7PR8, and 7PRT(34) (Figure 4B–E). The 3D interaction diagrams of the small molecules with the HPSE structure are shown in Figure S1. Since 7PR7, 7PR8, and 7PRT are HPSEs cocrystallized with a small-molecule inhibitor, the ligand–protein interactions provide a fundamental understanding for the subsequent design of inhibitors. In summary, THR97, ASN224, GLU343, GLY349, GLY350, and TYR391 are essential residues in both substrate binding and inhibitor binding. Meanwhile, it is important to note that interactions with ASP62, ASN64, GLN270, GLN272, and GLY389 are also observed in substrate binding, which provides another effective approach to inhibiting HPSE by competing with the binding sites of the endogenous substrate.

Figure 4.

Figure 4

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Interaction maps of (A) heparanase (HPSE) with dp4 (PDB ID:5E9C), (B) HPSE with M04S02a (PDB ID:5E98), (C) HPSE with VL166 (PDB ID:7PR7), (D) HPSE with GR109 (PDB ID:7PR8), and (E) HPSE with CB678 (PDB ID:7PRT).

To study the binding mode of the hit in a more versatile manner, we conducted a 500 ns MD simulation of the HPSE-TC LPA5 4 complex and discovered three stable binding conformations of TC LPA5 4 (Figure S2), two of which provided insights into the inhibition mechanism. The third conformation, with fewer interactions, was excluded from the discussion (Figure 5A). Different from HS mimetic HPSE inhibitors, small-molecule HPSE inhibitors inhibit HPSE mainly through interactions with the catalytic residues GLU225 and GLU343.35 Moreover, TC LPA5 4 interacted with GLN270 and ARG272, two residues residing in another potential inhibitory domain, HBD-2 (GLN270-LYS280). Additionally, TYR298, TYR348, and LYS231 were identified as characteristic binding residues that stabilized TC LPA5 4 in the binding pocket. The 500 ns MD simulation revealed diverse interactions, including hydrogen bonding with GLU225, GLU343, and GLN270, ionic interaction with LYS231, water bridges with GLU343, TYR298 and GLY349, and hydrophobic interactions with TYR298 and TYR348. The overall interaction heatmap is shown as Figure 5A, while the categorized interaction heatmaps are provided in Figure S3.

Figure 5.

Figure 5

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Binding conformations of TC LPA5 4 with heparanase (HPSE) identified from the 2500 frames sampled during the 500 ns molecular dynamics (MD) simulation. (A) Frequency heatmap of HPSE residues interacting with TC LPA5 4. (B) The first binding conformation of TC LPA5 4 with HPSE and the detailed interactions. (C) The second binding conformation of TC LPA5 4 with HPSE and the detailed interactions.

The first binding conformation sustained for the initial 100 ns of MD (Figure 5B). In this conformation, carboxypyrazole ring 1 established hydrogen bonds with ASN224, GLU343, and THR97 and an ionic interaction with LYS232, while the π–π-stacking with TYR348 stabilized the conformation. In the second conformation, the carboxylic acid of TC LPA5 4 formed frequent hydrogen bonds with GLU225, water bridges with GLU343, and occasional ionic interactions with LYS231 (Figure 5C). The interactions with TYR298 and TYR348, including water bridges and π–π-stacking, stabilized this conformation. Additionally, the molecule’s methoxy phenyl ring 2 interacted with GLN270 by forming hydrogen bonds and water bridges. This conformation was maintained for 300 ns. Apart from the frequent interactions with GLU225, GLU343, TYR298, and TYR348, the interactions with THR97, ASN224, GLN270, the residues participating in substrate binding, were also highlighted.

Using a similar HPSE–hit binding conformation, we performed a 500 ns MD simulation of the HPSE-MX4-62 complex (Figure 6). The carboxypyrazole ring formed water bridges with GLU225 and hydrogen bonds with GLN270 and LYS231. Additionally, MX4-62 was in proximity to TYR348, which stabilized the binding conformation through hydrophobic interactions (Figure 6A). However, we observed that the flexible polar side chain of ARG303 interacted with MX4-62 at multiple sites, specifically with the amino and alcohol functional groups and the ring system of the small molecule. These interactions caused MX4-62 to move away from the catalytic cleft, forcing the aminophenol ring to interact with ASN390 (Figure 6B). ASN390 acted as an anchor by forming hydrogen bonds with the amino and hydroxyl groups, while the carboxypyrazole ring continued to interact with LYS231. As MX4-62 no longer fit inside the binding pocket, it started forming interactions with residues located at the periphery of the pocket. These interactions resulted in a change in the binding conformation of MX4-62, causing it to move toward the edge of the binding pocket and form an ionic interaction with LYS98 and a hydrogen bond with TYR391. Furthermore, ASP62 formed water bridges with the carboxylic acid group, while the aminophenol ring continued to anchor MX4-62 to other residues such as ARG303 and ALA388. Despite these interactions, the carboxypyrazole ring was further extracted from the catalytic cleft (Figure 6C). During the remaining MD simulation, MX4-62 moved away from the HPSE binding pocket and did not form any interaction with the key residues identified in HPSE-hit binding. We concluded that the aminophenol group played a crucial role in forming interactions with ARG303 and ARG390, causing the small molecule to move away from the binding site and resulting in an unfavorable binding profile (Figure S4). Due to this weak binding profile, MX4-62 failed to form a stable binding conformation in the binding pocket, explaining its lack of potency.

Figure 6.

Figure 6

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Binding pattern of MX4-62 in the HPSE binding pocket. (A) Initial conformation of MX4-62 in complex with HPSE. (B) Interaction summary of MX4-62 with HPSE from the 5–10 ns time frame of the MD simulation. (C) Interaction summary of MX4-62 with HPSE from the 10–20 ns time frame of the MD simulation.

We further conducted a computational structure–activity relationship (SAR) study using MD simulations to investigate TC LPA5 4 and its structural features concerning HPSE inhibition. To assess the importance of the four-ring structure and substitutions in binding, we designed nine derivatives of TC LPA5 4 (Figure 7) and subjected each system to a 200 ns MD simulation (Figure S5). We categorized the nine derivatives into three classes based on their structural features and modifications. Class 1 retained the four rings while making modifications to ring 2 and ring 3 substitutions. Class 2, with ring 4 removed to investigate its role in receptor binding, also had modifications on rings 2 and 3 to further investigate the substitutions. Class 3 had rings 2 and 3 removed to investigate the role of the overall shape of the small molecule and its impact on binding. To achieve better comparison, all the systems were built based on the TC LPA5 4 MD starting point (Figure S5). Throughout the simulations, we recorded ligand–protein interactions frequencies and categorized them into four types: hydrogen bonds, water bridges, ionic interactions, and hydrophobic interactions. Additionally, we calculated the binding free energy using the molecular mechanics generalized Born surface area (MM-GBSA) method (Figure 7). The energy calculation data used to plot the figure are provided in Table S1. Our study revealed the crucial role of the four-ring system of the TC LPA5 4 in inhibitory activity. Removing ring 4 or both rings 3 and 4 led to the loss of the GLU225 and GLU343 interactions and impaired energy profiles (Figure 7B,C). In Class 1 molecules, both methoxy and chloride substitutions increased the contact frequency with HPSE residues. Removing both resulted in fewer π–π interactions with TYR298 and impaired binding free energy (−40.1 kcal/mol). Although removing chlorine substitution improved the binding free energy (−63.0 kcal/mol) compared to the hit (−55.7 kcal/mol), it resulted in fewer interactions (Figure 7A). Class 2 molecules showed binding profiles comparable to Class 1 but with a lower interaction frequency and weaker binding free energy of around −40 kcal/mol (Figure 7B), suggesting the importance of ring 4 in binding through space-occupying. In contrast, the study on Class 3 molecules revealed the critical role of the ring 1–ring 2 core of TC LPA5 4 for the basic binding, emphasizing designs not smaller than 3a (Figure 7C). Specifically, 3a managed to stay in the pocket during the simulation due to the methoxy group’s ability to occupy space and provide useful interactions, while 3b flew out of the pocket due to poor fitting, resulting in poor binding free energy (−28.1 kcal/mol). In summary, the first part of the computational SAR study provides insights into the importance of the four-ring system and the substitution groups in the TC LPA5 4 structure for HPSE inhibition. The root-mean-square deviation (RMSD) of the protein backbone and ligand and root-mean-square fluctuation (RMSF) of protein residues are provided in Figure S6 and Figure S7.

Figure 7.

Figure 7

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Binding profiles of (A) Class 1, (B) Class 2, and (C) Class 3 molecules with heparanase (HPSE), highlighting interactions categorized into hydrogen bond, water bridge, hydrophobic, and ionic interactions. The x-axis represents the HPSE residue, while the left y-axis denotes the interaction frequency of a small molecule with the corresponding residue. The figures also depict the absolute value of binding free energy (dG) of the molecule with HPSE on the right y-axis, measured in kcal/mol.

We conducted an additional series of 200 ns MD simulations to further study the substitution groups and their positions on TC LPA5 4. Each ligand–protein system was built based on the hit MD starting point (Figure S8). We designed Class 4 molecules (Figure 8A) to investigate the effects of ring 2 substitutions on the binding profile. First, we changed the methoxy substitution position on ring 2 from meta to ortho and para (4a and 4b). The ortho-substitution was excluded from further optimization study due to impaired interaction and binding free energy (−27.8 kcal/mol). However, para-OMe-substituted 4b showed a similar binding profile to the hit compound with more frequent interactions with GLU225 and TYR298, even though with impaired binding free energy (−38.0 kcal/mol), indicating para-substitution on ring 2 could be an optimization direction. Next, we investigated hydrogen bond donor (HBD) substitution groups by designing 4c, with the methoxy group changed to a hydroxyl group. However, 4c showed much fewer interactions with GLU225 and lost almost all interactions with GLU343. The m-F-substituted 4d showed a weaker binding profile, suggesting that the size of the substitution group was essential in the SAR. Overall, we concluded that a meta- or para-substituted hydrogen bond acceptor (HBA) no smaller than fluorine might be suitable to optimize ring 2.

Figure 8.

Figure 8

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Binding profiles of (A) Class 4, (B) Class 5, and (C) Class 6 molecules with heparanase (HPSE), highlighting interactions categorized into hydrogen bond, water bridge, hydrophobic, and ionic interactions. The x-axis represents the HPSE residue, while the left y-axis denotes the interaction frequency of small molecules with the corresponding residue. The figures also depict the absolute value of binding free energy (dG) of the molecule with HPSE on the right y-axis, measured in kcal/mol.

Next, we investigated ring 3 using Class 5 molecules (Figure 8B). We started with 5a with the meta-substituted chlorine replaced with the ortho-position. 5a exhibited a much weaker interaction profile, likely due to the constrained rotation of ring 3, altering the molecule’s conformation. Interestingly, the binding free energy of 5a with HPSE significantly improved (−67.2 kcal/mol), suggesting the importance of the planarity of the three-ring structure for binding. We then replaced the m-Cl with m-Br to yield 5b but found that the larger halide group did not improve interactions with desired HPSE residues nor the binding free energy. However, we observed multiple interaction improvements with m-F (5c) or m-OMe (5d), suggesting that HBA substitutions on ring 3 were beneficial. Moreover, 5c and 5d shared similar binding profiles, with 5c displaying a comparable binding free energy to the hit (−55.0 kcal/mol) and 5d of −48.3 kcal/mol. Additionally, a methyl substitution (5e) yielded a weaker binding profile but comparable binding free energy to the hit. We therefore concluded that meta-substitution benefited the overall interaction profile, with fluorine and methoxy substitution options worth exploring.

Lastly, we examined ring 4 and the potential optimization strategies using Class 6 molecules (Figure 8C). We first replaced ring 4 with a phenyl ring to yield 6a and found a very different binding profile from TC LPA5 4. This finding confirmed that the nonaromatic substitutions at the ring 4 position were important to the overall binding profile. We then added para-substitutions on ring 4 to yield molecules 6b, 6c, and 6d. We observed a preference for bulkier substitutions, as they improved both the interaction frequency profile and binding free energy. This observation prompted us to design 6e, which exhibited a favorable binding profile with increased interactions with GLU225 and LYS231 and comparable binding free energy to the hit (−53.9 kcal/mol). The RMSD of the protein backbone and ligand and RMSF of protein residues are provided in Figure S9 and Figure S10, respectively.

In this study, we have employed a multidisciplinary approach combining HTS, medicinal chemistry optimization, molecular docking, and MD simulations to investigate TC LPA5 4, a promising inhibitor hit of HPSE. Our analysis of cocrystallized complexes of HPSE with small molecules allowed us to identify a list of essential residues in HPSE inhibitor design. Based on the MD simulation, we proposed two binding conformations for TC LPA5 4 and summarized the binding profile. Further MD simulations on its derivatives allowed us to identify the minimal core structure and key functional groups (Figure 9). Based on these results, we have developed an SAR theory and proposed potential modifications to guide model generation for further lead optimization. Overall, our study has yielded a promising candidate for our ongoing optimization efforts and provides insights into the design of small-molecule HPSE inhibitors.

Figure 9.

Figure 9

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Summary of the TC LPA5 4 structure–activity relationship (SAR) with heparanase (HPSE).

Acknowledgments

This work is supported by research grants to Prof. L. Cui from the University of Florida (UF Startup Fund) and the National Institute of General Medical Sciences of National Institutes of Health (Maximizing Investigators’ Research Award for Early-Stage Investigators, R35GM124963).

Glossary

Abbreviations

HPSE

heparanase

HS

heparan sulfate

HSPG

heparan sulfate proteoglycan

HTS

high-throughput screening

HADP

ultrasensitive heparanase activity detecting probe

GH79

glycoside hydrolase

ECM

extracellular matrix

GlcUA

glucuronic acid

GlcN(NS)

N-sulfaglucosamine

HBD-1

heparin binding domain 1

HBD-2

heparin binding domain 2

MD

molecular dynamics

HBD

hydrogen bond donor

HBA

hydrogen bond acceptor

MM-GBSA

molecular mechanics generalized Born surface area

RMSD

root-mean-square deviation

RMSF

root-mean-square fluctuation

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.3c00268.

  • Experimental procedure for computational study, analog synthesis, characterization, and biological activity evaluation; 3D style ligand–protein interaction diagrams; RMSD and RMSF plots, interaction frequency heatmaps, and bar plots of MD simulations; binding free energy calculation results (PDF)

Author Contributions

Y.Z. and L.C. designed the project. M.X. and Z.C. synthesized and characterized the compounds. Z.C. performed the enzyme assays. J.L. synthesized the probe for the enzymatic assay. G.S. and C.L. performed the initial docking study, and Y.Z. performed all other computational studies. Y.Z. and L.C. wrote the paper. All authors reviewed the manuscript and provided feedback.

The authors declare no competing financial interest.

Supplementary Material

ml3c00268_si_001.pdf (3.1MB, pdf)

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