Identification of target sites in the VHR (DUSP3) enzyme unlocking potential avenues for future sepsis treatment

1. Introduction

Sepsis is a life-threatening condition characterized by organ dysfunction resulting from a dysregulated host response to infection [1]. It poses a significant global health burden, affecting an estimated 49 million people and contributing to 11 million deaths annually [2]. Current standard-of-care treatments are primarily supportive and do not target the underlying pathophysiological mechanisms. Despite over 100 clinical trials aimed at improving survival in sepsis, single-target therapies like endotoxin blockers and anti-TNF agents have failed [3]. This is due to the complex causes and redundant underlying mechanisms. For example, the endotoxin blocker Eritoran, a Toll-like receptor 4 (TLR4) antagonist designed to block excessive inflammation caused by bacterial endotoxins (lipopolysaccharide, or LPS), showed limited benefit because blocking TLR4 alone could not counteract the complex, multi-phase immune dysregulation of sepsis, and patients often received the drug too late for it to be effective [4]. No approved therapies exist that reverse the complex immune and metabolic dysfunctions associated with sepsis, underscoring the urgent need for novel, mechanism-based interventions. One of the major challenges in sepsis therapy is the dynamic nature of the immune response, which transitions from an early hyperinflammatory phase to a later state of immunosuppression. While modulating the early innate immune response has shown promise in preclinical models, clinical translation has been hindered by a lack of selective, druggable targets.

Dual-specificity phosphatase 3 (DUSP3), also known as Vaccinia H1-related phosphatase (VHR), is highly expressed in monocytes and macrophages and functions as a key positive regulator of innate immunity [5]. Genetic ablation of VHR provides robust protection against both endotoxin-induced and polymicrobial sepsis. VHR knockout mice remain resistant to lipopolysaccharide (LPS)- and cecal ligation and puncture (CLP)-induced sepsis, despite having normal numbers of B cells, T cells, neutrophils, monocytes, and platelets. This resistance is associated with a significant reduction in systemic proinflammatory cytokines, including TNF and IL-6.

Mechanistically, VHR deletion leads to reduced ERK1/2 activity in resident peritoneal macrophages, with no impact on other MAP kinases such as JNK1/2 or p38 [5]. Notably, this effect appears to be macrophage-specific, as ERK activity remains unchanged in B cells, T cells, and platelets. Additionally, VHR deletion does not affect NF-κB activation in response to LPS. The diminished ERK1/2 activation in VHR-deficient macrophages likely underlies their reduced cytokine production, as pharmacological inhibition of ERK1/2 similarly leads to decreased TNF secretion [6]. Importantly, VHR knockout mice are healthy, fertile, and display no spontaneous abnormalities, suggesting that pharmacological targeting of VHR may offer therapeutic benefits in sepsis without eliciting adverse effects [7].

2. Overcoming challenges in targeting VHR

VHR belongs to the superfamily of protein tyrosine phosphatases (PTPs), which are critical signaling molecules implicated in numerous human diseases but have historically been difficult to target [8,9]. A major challenge in targeting PTPs lies in their highly conserved and highly charged active sites. Inhibitors directed at these sites often lack selectivity and exhibit poor cell membrane permeability due to their polar nature [10,11]. Indeed, in highly sensitive experiments with human platelets, many reported VHR inhibitors (Table 1) exhibited off-target or nonspecific effects, with the exception of MLS-0437605, a noncompetitive inhibitor with an unknown mechanism of action [18,19]. Although allosteric inhibitors have been successfully developed for PTPs such as SHP2, these compounds typically exploit structural features outside the catalytic domain, such as additional regulatory domains [20]. However, VHR is a small PTP (185 amino acids; 20.5 kDa) composed solely of a minimal catalytic domain without any known regulatory motifs [21].

Table 1.

Selected small molecule inhibitors of VHR.

Name	Chemical Structure	VHR IC50 (μM)	Binding Site (Evidence)	Reference
RK-682		2.0	Active Site (Michaelis-Menten Kinetics)	[12,13]
4-Isoavenaciolide		1.2	Active Site Covalent (Mass Spectrometry)	[14]
GATPT		2.9	Active Site (Modeling)	[15]
Compound 1		3.7	Active Site (Modeling)	[16]
SA3		0.074	Active Site (Crystal structure; PDB ID: 3F81)	[16]
MLS-0437605		3.7	Unknown (Noncompetitive Binding in Michaelis-Menten Kinetics)	[18]

To overcome these limitations, alternative strategies are needed to target PTPs such as VHR. Proximity-inducing drugs, which recruit a target protein to a specific effector, enable selective modulation of protein function through mechanisms like degradation or post-translational modification. These approaches do not rely on direct inhibition of phosphatase activity and bypass the challenges posed by highly conserved active sites. For example, proteolysis-targeting chimeras (PROTACs) have revolutionized drug discovery over the past decade by enabling the selective degradation of previously undruggable proteins [22]. These heterobifunctional molecules bring a protein of interest (POI) into proximity with a ubiquitin ligase complex, triggering ubiquitination and subsequent degradation via the ubiquitin-proteasome system (UPS). Unlike conventional inhibitors, PROTACs eliminate all functions of the target protein – not just its catalytic activity – effectively mimicking genetic knockdown. Furthermore, their event-driven mechanism of action allows PROTACs to function catalytically and be recycled, reducing the need for high-affinity binding or sustained target engagement [23]. By exploiting newly discovered, non-conserved allosteric binding sites in VHR, proximity-inducing strategies could achieve both high specificity and efficient functional silencing.

3. Identification of novel target sites in VHR using fragment-based screening

Fragment-based drug discovery (FBDD) has emerged as a powerful complement to traditional small-molecule screening and development [24]. FBDD is based on the principle that drug-like molecules can be developed from small, structurally simple fragments that adhere to the ‘Rule of 3’ (Ro3): molecular weight < 300, cLogP ≤3, and no more than three hydrogen bond donors or acceptors. Owing to their small size and higher likelihood of fitting into diverse protein binding pockets, fragment screening is particularly well-suited for identifying novel and allosteric binding sites on protein surfaces. Furthermore, fragments typically exhibit high ligand efficiency (LE) and excellent aqueous solubility, which contributes to the high success rate of solving X-ray crystal structures of protein: fragment complexes. This enables atomic-level characterization of fragment – protein interactions and facilitates subsequent rational, structure-guided optimization. As a result, compounds evolved from fragment hits are often more likely to achieve the physicochemical properties desirable for lead-likeness.

Fragment screening relies on sensitive biophysical techniques such as NMR spectroscopy and X-ray crystallography [25,26]. Among the various NMR methods, ligand-observed fluorine NMR (¹⁹F NMR) has gained prominence for its high sensitivity and ability to identify high-quality hits. Binding of a fluorinated ligand to a protein typically results in a shift in resonance frequency or a reduction in ¹⁹F signal intensity. Our laboratory recently developed a customized ¹⁹F NMR-based fragment screening platform for VHR, incorporating automated library assembly and mixture formation using Echo acoustic compound transfer, followed by ¹⁹F NMR screening, hit confirmation, and secondary assays for characterization and validation [27]. Subsequent X-ray crystallography studies revealed two novel allosteric binding sites on VHR, distinct from its conserved active site (Figure 1(A)). At Site 1, fragment F19 engages primarily through hydrophobic and van der Waals interactions with residues T34, P35, I37, Y138, L139, Q143, M145, and I153 (Figure 1(B)). In contrast, Site 2 is more polar in nature, involving backbone carbonyls from helix H5 and the side chains of S27, Q28, N31, R155, and E159 (Figure 1 (C)). Both sites were unambiguously validated by high-resolution electron density maps and confirmed by the absence of equivalent density in a similarly packed VHR crystal structure lacking F19. Notably, F19 binding did not affect VHR enzymatic activity. However, high-affinity binders targeting these newly identified allosteric sites on VHR could be leveraged for proximity-inducing strategies such as PROTACs.

Figure 1.

Allosteric binding sites in VHR. (A) Co-crystal structure of F19 (magenta) bound to VHR at two sites distinct from the active site (PDB id 8TK3). (B-C) Close-ups of F19 bound to Site 1 (B) and Site 2 (C). In both panels, the Fo-Fc electron density omitting F19 atoms are shown as a blue mesh contoured at 3σ confidence level. Amino acids forming the binding sites are shown in stick representation. A buried, coordinated water molecule in Site 2 is indicated with an arrow. Hydrogen bonds are shown as dashed lines. (D) Chemical structure and ¹⁹F NMR binding data for F19. Reduction in peak intensity between spectra recorded in the absence (gray) and presence (blue) of VHR indicates binding. F19 selectively binds VHR over the related phosphatases MKP-6 and STEP₄₆.

Using ¹⁹F NMR, F19 was shown to selectively bind VHR over related phosphatases such as MKP-6 and STEP (Figure 1(D)). Notably, no other structures in the Protein Data Bank contain F19, and no binding or potency data for the compound are reported in ChEMBL or BindingDB. Importantly, these allosteric pockets exhibit unique residue compositions compared to other human PTPs, presenting opportunities for selective targeting. A comparison of Site 1 and Site 2 residues in VHR with those across the entire human DUSP subfamily (63 genes total) reveals that, unlike the active site, these allosteric regions are not conserved among closely related members of the PTP superfamily (Figure 2).

Figure 2.

Amino acid conservation among all 63 human dual-specificity phosphatases (DUSPs) at the active site (A), VHR allosteric site 1 (B), and VHR allosteric site 2 (C). protein sequences were aligned using clustal omega as implemented in MegAlign Pro (DNASTAR, Inc.). Amino acid conservation scores were calculated and mapped onto the crystal structure of the F19:VHR complex (PDB id: 8TK3) using the ConSurf server (https://consurf.Tau.ac.il). The protein surface, color-coded by amino acid conservation score, was visualized in PyMOL (Schrödinger, Inc.).

Notably, Site 2 appears particularly amenable to optimization, offering multiple opportunities for hydrogen bonding and featuring a well-coordinated water molecule that could be displaced to enhance binding affinity. This water molecule participates in key interactions with the protein, and its displacement may release bound water entropy, further stabilizing ligand binding. Adjacent to Site 2 lies a druggable pocket containing a non-conserved cysteine residue (C30) with an exposed side chain suitable for selective covalent modification (Figure 3). Covalent fragments targeting this second-site pocket could be linked to F19 to yield high-affinity VHR binders serving as effective ‘warheads’ for the development of VHR-directed degraders. Although irreversible covalent binding would undermine PROTAC functionality, reversible covalent binders such as cyanoacrylamides have demonstrated the ability to preserve PROTAC activity while enhancing selectivity and reducing toxicity [28]. Interestingly, according to the ICM-Pro (Molsoft, LLC) cysteine reactivity prediction model, C30 exhibited the highest reactivity score (0.76) among all VHR cysteines, including the catalytic cysteine (0.58), supporting its candidacy as a second-site target.

Figure 3.

F19 Site 2 fragment optimization strategy. Co-crystal structure of F19 (magenta) bound to VHR (PDB id: 8TK3). The VHR surface is color-coded according to binding properties: green, hydrophobic/lipophilic regions; blue, hydrogen bond donor potential; red, hydrogen bond acceptor potential; and white, neutral or aromatic lipophilic surface. The druggable pocket volume, shown as a wire mesh and identified using PocketFinder (ICM-Pro, Molsoft, LLC), reveals an adjacent pocket near the F19 Site 2 binding site, suggesting opportunities for further fragment optimization.

4. Expert opinion

Given its central role in amplifying macrophage-mediated inflammatory signaling and regulating ERK-dependent cytokine production, targeting VHR (DUSP3) represents a compelling new strategy for sepsis treatment. Unlike approaches that globally suppress pathogen recognition, VHR inhibition provides a more selective means of immune modulation, acting specifically on macrophage-driven inflammatory pathways while sparing other immune and signaling networks. Positioned at a critical regulatory node downstream of pattern-recognition receptors, VHR modulation could attenuate excessive cytokine release while maintaining essential antimicrobial defenses. Given the dynamic course of sepsis, VHR inhibition or degradation would likely be most effective during the early hyperinflammatory phase, when uncontrolled macrophage activation contributes to tissue injury and organ failure. Future small-molecule VHR modulators could be optimized for intravenous delivery to achieve rapid systemic exposure in acute settings or developed as orally bioavailable compounds for chronic or prophylactic use.

However, the development of small-molecule VHR inhibitors has been hindered by the highly conserved nature of the PTP active site and the absence of regulatory domains in VHR that would facilitate allosteric inhibition. The recent identification of non-catalytic binding sites, as described above, not only highlights the utility of fragment-based screening but also opens new avenues for selective VHR targeting. These structurally unique pockets offer opportunities for achieving the specificity that traditional active-site inhibitors lack. Optimized fragments that bind these allosteric sites may serve as warheads for the development of VHR-targeted PROTACs, enabling selective degradation of the protein rather than relying on enzymatic inhibition. To date, only a few PTP-targeted PROTACs have been reported, primarily focused on SHP2 using known allosteric inhibitors as warheads. PROTACs leveraging non-inhibitor-based binders for PTPs have not yet been described. Such an approach would represent a paradigm shift in the targeting of phosphatases and could lead to the next generation of VHR modulators, agents with the potential to transform sepsis therapy and establish a broader platform for addressing other immune-related diseases driven by aberrant dephosphorylation.