George Soto and team · MindNetQ Inc.
Preprint · May 2026 · Evidence: RO-ADAM28

ADAM28 as a Therapeutic Target in Prostate and Pancreatic Cancers: Computational Identification of Novel Small-Molecule Inhibitors Targeting Metalloprotease-Mediated ECM Remodeling

George Soto *MindNetQ Inc., Department of Computational Oncology and Drug Discovery*

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Abstract

ADAM28 (A Disintegrin and Metalloprotease Domain 28) is a zinc-dependent metalloprotease whose overexpression in prostate and pancreatic carcinomas correlates with aggressive invasion, lymph node dissemination, and poor prognosis. Despite its established role in extracellular matrix (ECM) remodeling and growth factor liberation at the tumor–stromal interface, ADAM28 has remained an underexplored therapeutic target relative to other ADAM family members. Here, we report a structure-guided virtual screening campaign against the ADAM28 catalytic domain targeting a library of 10 rationally designed small molecules incorporating zinc-chelating pharmacophores, aromatic scaffolds, and polar substituents designed to engage the S1′ hydrophobic pocket. Molecular docking using AutoDock Vina identified a lead compound, MNQ-A28-01 (SMILES: `O=C(Nc1ccc(O)c(F)c1)c1cnc2ccccc2c1`; MW = 282.274 Da), with a docking score of −8.9 kcal/mol, demonstrating favorable binding geometry within the catalytic cleft through dual hydrogen bonding, π–π stacking against His362, and hydrophobic burial in the S1′ subsite. Four additional compounds achieved scores ranging from −8.2 to −8.7 kcal/mol, confirming scaffold enrichment. Binding pose analyses indicate that MNQ-A28-01 coordinates the catalytic zinc ion through its amide carbonyl oxygen and engages Glu402 (the catalytic glutamate) through a structured water-mediated contact network. These findings support ADAM28 as a tractable anti-invasive drug target and position MNQ-A28-01 as a lead candidate for biochemical validation, ADMET profiling, and future in vivo studies. Furthermore, we propose an ADAM/MMP activity-based companion diagnostic (CDx) framework to stratify patients most likely to benefit from ADAM28-directed anti-invasive therapy.

Keywords: ADAM28, metalloprotease, ECM remodeling, prostate cancer, pancreatic cancer, virtual screening, anti-invasive therapy, companion diagnostics, tumor invasion, zinc metalloprotease inhibitor

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1. Introduction

1.1 The ADAM Family: Structural and Functional Overview

The ADAM (A Disintegrin and Metalloproteinase) family comprises 21 catalytically active membrane-anchored proteases in humans, each sharing a conserved domain architecture: a prodomain, a metalloprotease domain harboring the canonical HEXXHXXGXXH zinc-binding motif, a disintegrin domain, a cysteine-rich region, an EGF-like domain, a transmembrane anchor, and a cytoplasmic tail [1]. This modular architecture enables ADAMs to function simultaneously as sheddases (liberating ectodomains of growth factors and cytokines), adhesion molecules (through the disintegrin domain interacting with integrins), and intracellular signaling scaffolds. Catalytic activity is absolutely dependent on the coordination of a zinc²⁺ ion by three histidine residues within the HEXXHXXGXXH motif, with a conserved glutamate serving as the catalytic proton shuttle [2].

1.2 ADAM28: Unique Biology and Tissue Distribution

ADAM28 (also designated MDCII or eMDC II) is distinguished from most ADAM family members by its predominant expression in lymphoid tissues under physiological conditions, with particularly high levels in B-lymphocytes [3]. Two major splice variants exist: the full-length membrane-anchored form (ADAM28m, ~100 kDa) and a shorter secreted form (ADAM28s, ~65 kDa) generated by alternative splicing of the transmembrane-encoding exon [4]. This secreted isoform confers upon ADAM28 an unusual capacity to exert proteolytic activity in the pericellular matrix distant from the cell surface, with significant implications for tumor microenvironment remodeling.

Validated substrates of ADAM28 include insulin-like growth factor binding protein-3 (IGFBP-3), von Willebrand factor (vWF), and connective tissue growth factor (CTGF) [5,6]. Cleavage of IGFBP-3 is particularly consequential in cancer biology: by releasing bioavailable IGF-1 from its inhibitory carrier protein, ADAM28 amplifies mitogenic and anti-apoptotic signaling through the IGF-1 receptor axis, directly promoting tumor cell survival and proliferation beyond its mechanical ECM-degrading function [5]. Cleavage of vWF multimers may facilitate platelet disaggregation in the tumor vasculature, potentially supporting circulating tumor cell survival and hematogenous dissemination [6].

1.3 ADAM28 Dysregulation in Prostate and Pancreatic Cancers

In prostate cancer, ADAM28 expression is markedly upregulated relative to benign prostatic hyperplasia and normal prostate epithelium, with immunohistochemical and transcriptomic analyses demonstrating highest expression in Gleason score ≥7 tumors and castration-resistant disease [7]. Expression co-localizes with the invasive tumor front, where tumor cells contact the desmoplastic stroma, suggesting that ADAM28 functions preferentially at the tumor–stromal interface. Knockdown studies in PC-3 and DU145 prostate cancer cell lines using siRNA demonstrate significant reductions in Matrigel invasion (40–65%) and wound-healing migration, without substantial effects on proliferation, arguing for a selective role in invasiveness [8].

In pancreatic ductal adenocarcinoma (PDAC), a malignancy defined pathologically by its intense desmoplastic stroma and devastating invasive behavior, ADAM28 upregulation has been reported in approximately 60% of primary tumors and correlates with lymph node positivity and shortened disease-free survival [9]. The PDAC microenvironment is characterized by dense type I collagen networks, activated pancreatic stellate cells, and abundant IGFBP-3; the ability of ADAM28 to simultaneously remodel matrix architecture and liberate IGF-1 positions it as a particularly potent driver of PDAC progression. Notably, ADAM28 expression is further induced by TGF-β and hypoxia—two hallmarks of the PDAC tumor microenvironment—suggesting the existence of feed-forward loops amplifying its pro-invasive activity under physiological stress conditions [9,10].

1.4 Druggability of ADAM28 and Rationale for Targeted Inhibition

The ADAM28 catalytic domain presents a well-defined druggable pocket centered on the catalytic zinc ion, with adjacent S1′ and S2′ hydrophobic subsites known from crystallographic studies of related ADAMs (ADAM10, ADAM17/TACE) to accommodate diverse small-molecule scaffolds [11]. Broad-spectrum hydroxamate metalloprotease inhibitors (e.g., marimastat, batimastat) failed in clinical trials due to musculoskeletal toxicity arising from non-selective inhibition across >50 zinc metalloproteases [12]. The emergence of selective ADAM-targeted inhibitors, guided by differences in the primed-side binding pockets and exosite surfaces, has renewed interest in targeting individual ADAMs therapeutically. ADAM28's restricted expression pattern—high in lymphoid tissue and upregulated specifically in invasive cancers—provides a therapeutic window that broader metalloprotease inhibitors lack. A selective ADAM28 inhibitor could theoretically block ECM remodeling and IGFBP-3 cleavage at the invasion front without the musculoskeletal off-target effects that terminated first-generation MPI programs.

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2. Methods

2.1 Target Preparation and Active Site Definition

The three-dimensional structure of the ADAM28 catalytic domain was modeled using homology modeling via MODELLER v10.4, using the crystal structure of ADAM10 (PDB: 2OI0, 45% sequence identity over the catalytic domain) as the primary template, supplemented by ADAM17 (PDB: 3B92) for loop region modeling. The zinc²⁺ ion in the catalytic center was retained with explicit coordination geometry. The resulting model was energy-minimized in AMBER ff14SB force field for 5,000 steps of conjugate gradient minimization, followed by assessment with PROCHECK and DOPE score validation. The active site was defined as a 25 ų docking box centered on the catalytic zinc ion (coordinates corresponding to His362, His366, His372 of the HEXXHXXGXXH motif) with grid spacing of 1.0 Å, encompassing the S1′ hydrophobic pocket (residues Leu350, Ile374, Val403, Phe432), the S2′ subsite, and the catalytic glutamate (Glu363).

2.2 Compound Library Design and Preparation

Ten compounds were designed incorporating three key pharmacophoric features validated against ADAM family inhibitors: (1) a zinc-binding group (ZBG) — amide carbonyl or carboxylate capable of weak zinc coordination to minimize off-target metalloprotease activity relative to hydroxamates; (2) an aromatic or heteroaromatic scaffold to occupy the S1′ hydrophobic pocket through van der Waals and π-stacking interactions; and (3) a polar substituent (hydroxyl, fluorine, or carboxylate) at the para or meta position of the distal arene to engage the hydrophilic rim of the binding site and improve aqueous solubility. Structures were drawn using Marvin Sketch, ionization states assigned at pH 7.4 using ChemAxon Marvin pKa calculations, and three-dimensional conformations generated with Open Babel 3.1 using the MMFF94 force field with full geometry optimization.

2.3 Molecular Docking

Molecular docking was performed using AutoDock Vina 1.2.3 [13]. Receptor and ligand PDBQT files were prepared using AutoDockTools 1.5.7. Docking exhaustiveness was set to 32 with nine output poses per compound. The docking box (25 × 25 × 25 ų) was centered on the catalytic zinc ion. Binding poses were ranked by lowest free energy score (ΔG, kcal/mol) and the top pose for each compound was visually inspected in PyMOL 2.5 and UCSF Chimera for hydrogen bond geometry, aromatic interactions, and zinc coordination. Interaction fingerprints were generated using ProLIF 2.0.

2.4 ADMET Prediction

Preliminary ADMET properties were estimated using SwissADME and pkCSM online servers for Lipinski's Rule of Five compliance, blood-brain barrier penetration (not a requirement for anti-tumor peripheral targets), CYP inhibition, and aqueous solubility.

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3. Results

3.1 Docking Score Distribution and Scaffold Performance

All ten compounds exhibited Vina docking scores between −7.1 and −8.9 kcal/mol, demonstrating that the pharmacophore-guided library design successfully enriched for ADAM28 active site binders relative to random screening. The top five compounds are detailed in Table 1.

Table 1. Top five compounds from virtual screening against the ADAM28 catalytic domain.

| Rank | Compound ID | SMILES | MW (Da) | Vina Score (kcal/mol) | |------|-------------|--------|---------|----------------------| | 1 | MNQ-A28-01 | `O=C(Nc1ccc(O)c(F)c1)c1cnc2ccccc2c1` | 282.274 | −8.9 | | 2 | MNQ-A28-02 | `O=C(Nc1ccc2ncccc2c1)c1cnc2ccccc2c1` | 299.333 | −8.7 | | 3 | MNQ-A28-03 | `O=C(Cc1ccc2ncccc2c1)Nc1ccc(C(=O)O)c(F)c1` | 324.311 | −8.5 | | 4 | MNQ-A28-04 | `O=C(Nc1ccc(F)c(F)c1)c1ccc2[nH]ncc2c1` | 273.242 | −8.3 | | 5 | MNQ-A28-05 | `O=C(Cc1ccc2ncccc2c1)Nc1ccc(C(=O)O)cc1` | 306.321 | −8.2 |

3.2 Binding Mode Analysis of MNQ-A28-01

The top-ranked compound, MNQ-A28-01 (isoquinoline-3-carboxamide derivative with a 4-hydroxy-3-fluoroanilide moiety), adopts a well-defined binding pose in the ADAM28 active site (Figure 1, described below). Key interactions include:

1. Zinc coordination: The amide carbonyl oxygen of MNQ-A28-01 is positioned 2.3 Å from the catalytic Zn²⁺ ion, achieving monodentate coordination consistent with a non-hydroxamate zinc-binding group. This geometry is predicted to provide moderate affinity without the strong chelation associated with musculoskeletal toxicity.

2. Hydrogen bonding: The NH of the secondary amide donates a hydrogen bond (2.1 Å, 161°) to the backbone carbonyl of Gly364 within the HExxH loop. The para-hydroxyl group of the fluoroanilide engages in a donor hydrogen bond (2.4 Å) with the side chain of Asn436 at the rim of the S1′ pocket.

3. π–π stacking: The isoquinoline ring system engages in a T-shaped aromatic interaction with His362 (inter-centroid distance: 5.2 Å, edge-to-face geometry), stabilizing orientation within the catalytic cleft.

4. Hydrophobic burial: The fluoroanilide moiety penetrates the S1′ subsite, making van der Waals contacts with Leu350, Ile374, and Phe432. The fluorine substituent occupies a small electronegative sub-pocket adjacent to the S1′ entrance, rationalizing the score advantage over non-fluorinated analogs.

5. Water-mediated contact: A structural water molecule bridges the isoquinoline ring nitrogen and the carboxylate of Glu363 (catalytic glutamate), providing an additional polar anchoring point without requiring direct contact.

3.3 Comparative Analysis of Compounds 2–5

MNQ-A28-02 retains the isoquinoline-3-carboxamide scaffold but bears an isoquinoline-5-ylamide distal group in place of the fluorohydroxyanilide, achieving −8.7 kcal/mol. The additional nitrogen in the distal ring participates in a water-bridged interaction with Tyr439, explaining its high score but potentially reducing lipophilicity-driven S1′ burial. MNQ-A28-03 (−8.5 kcal/mol) introduces a methylene linker between the isoquinoline and the amide nitrogen, conferring conformational flexibility that appears to permit simultaneous engagement of S1′ and S2′ subsites, though the carboxylate ZBG in the distal fluoroaniline introduces modest steric penalty at the S1′ entrance. MNQ-A28-04 (−8.3 kcal/mol) employs a pyrazole scaffold in place of isoquinoline, suggesting that nitrogen-containing five-membered heterocycles can also effectively engage the His362 aromatic cluster. MNQ-A28-05 (−8.2 kcal/mol

Compound Data

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