What are the new molecules for ENPP1 inhibitors?

Introduction to ENPP1
Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is an enzyme found in several biological compartments, including the extracellular matrix, cell membranes, and even in the circulatory system. It was originally characterized as a type II transmembrane glycoprotein, and its catalytic activity involves the hydrolysis of nucleotides such as ATP, as well as cyclic dinucleotides such as 2′,3′‑cGAMP. Over the past few decades, ENPP1 has evolved from being considered merely a “housekeeping” enzyme with roles in purinergic signaling and mineralization to a critical regulator of immune responses, particularly through its involvement in degrading the endogenous agonist for the stimulator of interferon genes (STING) pathway.

Role in Biological Processes
ENPP1 plays a pivotal role in maintaining homeostasis in several biochemical pathways. It is crucial in regulating extracellular nucleotide levels, thereby modulating purinergic signaling which impacts intercellular communication. More intriguingly, ENPP1’s hydrolytic action on 2′,3′‑cGAMP is a central event in the attenuation of STING-dependent innate immune responses. In this context, when ENPP1 degrades cGAMP, the resultant diminution of STING activation leads to a dampened type I interferon response. This biochemical regulation not only affects cellular metabolism and signaling cascades but also has implications for tissue mineralization and calcification, as ENPP1 generates pyrophosphate, a known inhibitor of hydroxyapatite deposition. Thus, ENPP1 is uniquely positioned at the crossroads of metabolic regulation, immune modulation, and tissue homeostasis.

Importance in Disease Context
In recent years, a rapidly growing body of evidence has unraveled the disease relevance of ENPP1. Overexpression of ENPP1 has been correlated with several pathological states ranging from metabolic disorders such as insulin resistance and osteoarthritis to various cancers. In the oncology field, rampant ENPP1 activity can lead to an immunosuppressive tumor microenvironment by reducing the levels of extracellular cGAMP, thereby blunting the activation of the STING pathway and subsequent antitumor immunity. Moreover, ENPP1 deficiency has its own set of clinical challenges such as calcification disorders; in the clinical setting, patients with ENPP1 deficiency often suffer from conditions like generalized arterial calcification of infancy (GACI) and autosomal recessive hypophosphatemic rickets type 2. The dual nature of ENPP1—in conditions of both overactivity and deficiency—highlights its central role in diverse biological contexts and underscores the need to modulate its activity therapeutically.

ENPP1 Inhibition
Given the multifaceted roles of ENPP1, targeting its enzymatic activity has emerged as a promising approach in modern drug discovery. Specifically in oncology, inhibiting ENPP1 to sustain elevated levels of 2′,3′‑cGAMP can reactivate the STING pathway, thus promoting robust type I interferon responses and ultimately enhancing antitumor immunity.

Mechanism of Action
The primary mechanism underlying ENPP1 inhibition is the interruption of its ability to catalyze the hydrolysis of substrates – most notably, the endogenous STING ligand cGAMP. In normal biological conditions, cGAMP synthesized by cyclic GMP‑AMP synthase (cGAS) activates STING, leading to an innate immune response characterized by the production of type I interferons. ENPP1, when active, degrades cGAMP, thereby attenuating the immune response. Pharmacological inhibition of ENPP1 prevents the breakdown of cGAMP, prolonging its existence in the extracellular milieu and thereby maintaining the activation of the STING pathway. Various classes of inhibitors have been developed; these molecules typically achieve inhibition by binding to the active site of ENPP1, thereby blocking substrate access. In some cases, inhibitors have been designed to be cell impermeable, ensuring that they act exclusively in the extracellular space where ENPP1 exerts its primary function. Detailed structural studies have further refined the design of inhibitors by illuminating key binding interactions within the enzyme’s active site, such as interactions with zinc ions essential for enzymatic catalysis.

Therapeutic Potential
The therapeutic potential of ENPP1 inhibitors is wide-ranging. In oncology, the principal aim is to stimulate the cGAS‑STING pathway to enhance immune-mediated tumor cell clearance. Preclinical studies have shown that ENPP1 inhibitors can delay tumor growth and improve the efficacy of conventional treatments like radiation therapy and immune checkpoint blockade when used in combination therapies. Additionally, there is emerging interest in using these inhibitors to enhance antiviral responses during viral infections, where a robust type I interferon response is crucial. The ability to modulate immune responses through a well-defined mechanism makes ENPP1 inhibition an attractive target for both cancer immunotherapy and treatment of infectious diseases.

Recent Developments in ENPP1 Inhibitors

Recent years have witnessed a surge in the discovery and development of new molecules targeting ENPP1, driven by advances in computational chemistry, structural biology, and high-throughput screening technologies. These new molecules are not only diverse in chemical structure but also bring unique features aimed at improving potency, pharmacokinetics, and safety profiles.

Newly Discovered Molecules
A number of novel molecules have recently emerged as promising ENPP1 inhibitors, each characterized by different chemical scaffolds and inhibitory profiles:

• Several patents and published articles have described new small molecules with potent ENPP1 inhibitory activity. For example, INSILICO MEDICINE IP LIMITED disclosed a series of small molecule ENPP1 inhibitors suitable for combination therapy in cancer treatment, emphasizing their application in modulating the STING pathway to enhance type I interferon levels. Although the detailed chemical structures were not disclosed in full detail in that reference, the work signifies the evolving trend of using computational design to identify drug candidates with enhanced specificity.

• Two patents by CSPC Zhongqi Pharmaceutical Technology and its collaborators report the development of crystalline forms of ENPP1 inhibitors. These patents reveal that the crystalline form of a compound represented by formula (I) has good crystallinity and chemical purity, which are important predictors of druggability and scalability in manufacturing. The distinct crystalline forms may alter the pharmacokinetic properties and hence could translate into optimized dosing regimens in the clinic.

• Another inventive work communicated in the patent JP2025502943A describes an ENPP1 inhibitor along with its pharmaceutical uses. While the exact chemical details differ slightly from the molecules reported by INSILICO MEDICINE, they share a similar strategic aim of inhibiting ENPP1 to treat diseases resulting from its aberrant activity. These molecules, developed by a Japanese entity, highlight a growing international interest in ENPP1 as a therapeutic target.

• Recent literature surveys have identified the natural compound myricetin as a moderate inhibitor of ENPP1. However, due to its poor druggability—related to issues with potency and metabolic stability—researchers have pivoted towards the design of novel analogs of myricetin. In one study, two flavonoid glycosides were identified via virtual screening on a flavonoid natural product database (FNPD); these compounds (CAS No: 1397173-50-0 and 1169835-58-8) demonstrated higher predicted inhibitory effects than myricetin itself. This work illumines the approach of using natural product scaffolds as a starting point to derive more potent and selective inhibitors through structural optimization.

• In the realm of synthetic small molecules, compound 4e has garnered attention due to its potent inhibition of ENPP1. This quinazolinone-based inhibitor exhibits an IC50 of 0.188 μM at the molecular level and is effective at downregulating ENPP1 activity in cell-based assays with an IC50 around 0.732 μM. The compound’s design capitalizes on a zinc-binding quinazolin-4(3H)-one scaffold, which was discovered after structure-based virtual screening strategies. This molecule is particularly promising given its excellent selectivity for tumor cells, which implies a reduced effect on non-cancerous tissues.

• Compound 31 is another new molecule emerging from a study utilizing a pyrido[2,3-d]pyrimidin-7-one scaffold. This small molecule inhibitor was optimized to exhibit significant activity in both enzymatic and cellular assays and has demonstrated in vivo efficacy in a syngeneic mouse model for triple-negative breast cancer. The novel scaffold not only offers high potency but also enhances the potential for specific immune-mediated antitumor effects.

• A different subset of novel inhibitors is represented by compounds based on a non-nucleotidic thioguanine core. In this strategy, researchers designed a series of compounds where the lead, designated as compound 43, showed promising in vitro potency, stability, selectivity, and favorable ADME properties. The compound successfully elicited an antitumor response in metastatic breast cancer models. This development is particularly interesting because it demonstrates how repurposing a known structure (in this case, a thioguanine analogue) can lead to compounds with entirely new mechanisms—inhibiting ENPP1 and consequently modulating immune pathways.

• Building on the structural diversity, researchers have synthesized and evaluated derivatives based on heterocyclic cores. A study reported the discovery and synthesis of 3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one and 3,4-dihydropyrido[2,3-d]pyrimidin-2(1H)-one derivatives. Through a systematic structure-activity relationship (SAR) assessment, lead compounds designated as 46 and 23 were identified as potent inhibitors with favorable microsomal stabilities across human, rat, and mouse liver microsomes. The identification of these two compounds highlights the potential of heterocyclic frameworks for achieving very low Ki values and robust potency.

• Furthermore, phosphonate-based inhibitors continue to be a major area of research. An extensive structure-aided development program led to several best-in-class ENPP1 inhibitors with Ki values under 2 nM, excellent physicochemical properties, and favorable pharmacokinetic profiles. Although the specific identifiers of these molecules are not disclosed in every report, the structure-based design approach emphasizes the importance of targeting the enzyme’s metal-binding motifs with phosphonate functionalities.

• A series of orally bioavailable phthalazinone analogs has been discovered for STING-mediated cancer immunotherapy. Among these, compound 29f emerged as a potent inhibitor with an IC50 of 68 nM in vitro and demonstrated robust enhancement of type I interferon responses while exhibiting excellent metabolic stability and bioavailability (F = 65%). The development of these analogs is significant because oral bioavailability is a highly desired attribute for chronic administration in cancer therapy.

• Another exciting new molecule is AVA-NP-695, a highly potent and selectively designed ENPP1 inhibitor. AVA-NP-695 operates by activating the STING pathway and abrogating tumor metastasis, particularly in triple-negative breast cancer models. It has been shown to inhibit epithelial–mesenchymal transition (EMT), and preclinical studies in 4T1 mouse models demonstrated superior tumor growth inhibition when compared to standard treatments such as Olaparib and PD-1 inhibitors. This molecule’s dual action in immune modulation and direct anti-metastatic effects makes it an attractive candidate for future clinical trials.

• Adding further to this repertoire, novel pyrrolopyrimidine and pyrrolopyridine derivatives have been identified through systematic SAR studies. One lead compound from this series, designated 18p, exhibited very potent ENPP1 inhibition with an IC50 of 25 nM. In preclinical models, 18p activated the STING pathway in a concentration-dependent manner, resulting in significant induction of cytokines such as IFN‑β and IP‑10, along with demonstrated antitumor effects in a 4T1 syngeneic mouse model. This discovery emphasizes the potential of designing inhibitors that target the enzyme through non-nucleotidic mechanisms.

In summary, the new molecules for ENPP1 inhibitors span a wide chemical space—from crystal forms of known scaffolds to entirely novel heterocyclic derivatives. Natural product derivatives, such as flavonoid glycosides from myricetin analogs, coexist with synthetically developed small molecules like quinazolinone-based compound 4e, pyrido[2,3-d]pyrimidin-7-one compound 31, and non-nucleotidic thioguanine derivative 43. Additionally, the emergence of phosphonate inhibitors, orally bioavailable phthalazinone analogs (e.g., compound 29f), AVA-NP-695, and pyrrolopyrimidine derivatives (18p) reflects significant progress in addressing potency, selectivity, and pharmacokinetic challenges in ENPP1 inhibition.

Preclinical and Clinical Studies
Preclinical studies have been instrumental in validating the potential of these new molecules. For instance, compound 4e’s development was accompanied by rigorous in vitro assays that measured its inhibitory activity at the molecular level, followed by cellular assays demonstrating its selectivity against metastatic breast cancer cells. Similar preclinical evidence is available for compound 31, which showed robust in vivo tumor growth inhibition in a syngeneic mouse model of triple-negative breast cancer, thereby confirming its immunomodulatory capability through the sustained activation of the STING pathway.

Phosphonate inhibitors developed through structure-aided design, demonstrating Ki values of less than 2 nM, have been evaluated for their pharmacokinetic properties, with the best candidates showing excellent absorption, distribution, metabolism, and excretion (ADME) profiles. The favorable physicochemical properties of these inhibitors have paved the way for their translational studies, as they can yield effective plasma concentrations that are sustained over time.

The orally bioavailable phthalazinone analog, specifically compound 29f, has been subjected to detailed metabolic and pharmacokinetic evaluations. Its oral bioavailability (F = 65%) and the in vitro efficacy (IC50 = 68 nM) have allowed researchers to progress into animal models that mirror the human STING pathway. These preclinical results indicate that compound 29f not only potently inhibits ENPP1 but also translates into enhanced immune activation when combined with other therapies such as immune checkpoint inhibitors.

AVA‑NP‑695 has also reached an advanced preclinical stage where its efficacy in significantly inhibiting ENPP1-mediated cGAMP degradation was demonstrated in a 4T1 breast cancer mouse model. In these studies, AVA‑NP‑695 improved tumor control by modulating the tumor microenvironment, reducing EMT, and suppressing metastasis. The molecule stands out for its dual function: immunostimulation via STING pathway activation and direct inhibition of cancer cell migration, thereby offering a multi-modal approach to cancer therapy.

The novel pyrrolopyrimidine derivative 18p has been thoroughly assessed both in vitro and in vivo. In vitro experiments confirmed its potent ENPP1 inhibitory activity (IC50 = 25 nM), and subsequent studies demonstrated that it induces the production of key cytokines associated with STING pathway activation, such as IFN‑β and IP‑10. Furthermore, the in vivo antitumor efficacy in a 4T1 syngeneic mouse model underscores the translational potential of 18p as both an immunotherapeutic agent and as a stand-alone antineoplastic agent.

Many of these new molecules are currently in different stages of preclinical development, with several advancing to early-phase clinical trials as evidenced by the number of patents that cover compositions and methods for cancer treatment using ENPP1 inhibitors. Although clinical data are still emerging, the initial safety, pharmacokinetic, and pharmacodynamic profiles reported in these studies have been promising, encouraging further development and eventual regulatory evaluation.

Challenges and Future Directions

While the recent surge in novel ENPP1 inhibitors represents significant progress, the field faces multiple challenges that must be addressed to fully harness the therapeutic potential of these molecules.

Current Challenges in Development
One of the primary challenges in developing ENPP1 inhibitors is achieving high specificity and selectivity. ENPP1 shares structural similarities with other members of the ENPP family and related phosphodiesterases. Hence, the inhibitors need to be finely tuned to interact robustly with the ENPP1 active site while minimizing off-target effects. Many of the new molecules have been designed using advanced structure-based design tools to address these specificity concerns; however, differences in isoforms and binding modes can still lead to unintended biological consequences.

Another challenge is the optimization of pharmacokinetics and pharmacodynamics. For instance, while several molecules such as the phosphonate inhibitors and phthalazinone analogs have shown excellent in vitro potency, achieving a favorable in vivo profile is complex. Molecules must be stable in the biological milieu, exhibit adequate distribution to the target tissues (especially the tumor microenvironment), and have minimal toxicity over prolonged administration. The process of transitioning from preclinical animal studies to clinical trials involves overcoming hurdles related to metabolic stability, absorption, and elimination of these compounds.

Furthermore, the tumor microenvironment itself presents challenges. In some cancers, the extracellular matrix can impede the effective delivery of ENPP1 inhibitors. This necessitates the design of inhibitors that not only exhibit potent biochemical inhibition but also have the physicochemical properties required for optimal tissue penetration. In addition, there is the risk of immunogenicity with certain types of molecules such as antibody–drug conjugates or biologics designed to target ENPP1, which necessitates rigorous preclinical safety evaluations.

The interplay between ENPP1 inhibition and the broader immune system is another area of complexity. While inhibiting ENPP1 can potentiate STING signaling and type I interferon production, the resulting immune activation needs to be carefully balanced to avoid potential inflammatory side effects or autoimmunity. This is particularly pertinent in patients with preexisting immune-related conditions. Designing molecules with the correct dose, selective tissue targeting, and controlled pharmacology remains an ongoing challenge.

Future Prospects and Research Directions
Looking ahead, the future of ENPP1 inhibitors is promising due to the robust scientific rationale, and continued improvements in drug design and development strategies. The evolution of computational modeling and structure-based drug design has already yielded several novel classes of molecules such as pyrido[2,3-d]pyrimidin-7-ones, non-nucleotidic thioguanine derivatives, heterocyclic compounds with phosphonate groups, phthalazinone analogs, and pyrrolopyrimidine derivatives. These advances not only provide potent inhibitors but also open avenues for further modifications to improve drug-like properties.

Future research directions include further refinement of these molecules through iterative medicinal chemistry efforts. Detailed SAR studies will continue to optimize potency, selectivity, and safety profiles. New screening methods, such as high-throughput cellular assays and advanced in silico modeling approaches, are critical to predict metabolic stability and bioavailability earlier in the discovery process. Moreover, integrating pharmacodynamic biomarkers, such as plasma pyrophosphate (PPi) levels and type I interferon markers, into early-phase clinical studies can help guide dosing strategies and provide early signals of efficacy.

There is also increasing interest in combination therapies. Given that ENPP1 inhibitors can potentiate STING-mediated anticancer immunity, combining these molecules with immune checkpoint inhibitors (e.g., anti-PD-1, anti-PD-L1 antibodies) or even standard chemoradiation therapy could yield synergistic effects. The patents suggest that combination strategies are being actively pursued, and preclinical studies have shown promising early results in this regard. Co-administration studies in appropriate tumor models can help determine the best regimens that maximize antitumor responses while mitigating potential adverse effects.

Additional research should focus on understanding the long-term implications of sustained ENPP1 inhibition. Chronic inhibition may alter the balance of purinergic signaling in ways that affect not only immune responses but also metabolic processes and tissue mineralization. As such, the development of reversible inhibitors or molecules with tunable pharmacokinetics may offer a safer therapeutic window, especially for chronic administration in cancer patients or those with viral infections.

Furthermore, advancements in biomaterials and drug delivery systems could aid in overcoming the challenges posed by tissue penetration and bioavailability. The use of nanocarriers, liposomal formulations, or conjugation with targeting moieties may significantly enhance delivery of ENPP1 inhibitors to the desired sites of action. These approaches can minimize systemic exposure and reduce off-target effects, thereby maximizing therapeutic efficacy.

Finally, given the emerging data from early-phase clinical studies on some of these new inhibitors—such as the promising results with AVA-NP-695 and the orally bioavailable phthalazinone analogs—the next few years are likely to see a transition of these molecules from the bench to the bedside. Continued collaboration between academia, biotech companies, and pharmaceutical industries is poised to foster innovative regulatory pathways and clinical trial designs that could accelerate the translation of these novel molecules into approved therapies.

Conclusion
In conclusion, the landscape of ENPP1 inhibitor discovery has rapidly expanded over the past few years, driven by the strong scientific rationale underlying the modulation of the cGAS‑STING pathway and its implications for cancer immunotherapy and other diseases. ENPP1 plays a central role in key physiological processes such as purinergic signaling, mineralization, and immune regulation. Its inhibition—achieved through a diverse array of new molecules—is emerging as an attractive therapeutic strategy.

Recent developments in ENPP1 inhibition highlight a remarkable diversity in chemical scaffolds. Novel molecules include small synthetic inhibitors such as the quinazolinone-based compound 4e, pyrido[2,3-d]pyrimidin-7-one compounds like compound 31, non‑nucleotidic thioguanine derivatives such as compound 43, and novel heterocyclic derivatives based on 3,4‑dihydropyrimido[4,5‑d]pyrimidin-2(1H)-one and 3,4‑dihydropyrido[2,3‑d]pyrimidin-2(1H)-one, with lead compounds 46 and 23. In addition, phosphonate inhibitors developed via structure-based design have achieved exceptional potency with Ki values less than 2 nM. Furthermore, orally bioavailable phthalazinone analogs—exemplified by compound 29f—and selective inhibitors such as AVA‑NP‑695 offer high efficacy and improved pharmacokinetic profiles, with AVA‑NP‑695 demonstrating significant antitumor activity and immune modulation in preclinical models. A recent series of pyrrolopyrimidine and pyrrolopyridine derivatives, with the lead compound 18p (IC50 of 25 nM), further expand the chemical diversity of ENPP1 inhibitors.

These novel molecules are supported by robust preclinical data demonstrating potent inhibition of cGAMP hydrolysis, effective activation of the STING pathway, and significant antitumor effects in various mouse models. Despite the encouraging results, challenges such as achieving high selectivity, acceptable pharmacokinetics, tissue penetration, and balanced immune activation still remain. Future research is geared towards optimizing these parameters, exploring combination therapies with immune checkpoint inhibitors or chemoradiation, and advancing the most promising candidates to clinical evaluation with rigorous biomarker-based trial designs.

The field holds exciting prospects as collaborative efforts in medicinal chemistry, computational modeling, and translational science continue to push the boundaries. Improvements in drug delivery systems and reversible inhibitor designs may further enhance the therapeutic index of these agents. Ultimately, the ongoing work on new ENPP1 inhibitors promises to expand the armamentarium for cancer immunotherapy and potentially for the treatment of viral infections, providing new hope for patients with challenging and refractory diseases. With continued innovation and strategic clinical development, ENPP1 inhibitors are poised to become a critical component of future personalized medicine approaches.

In summary, the novel molecules for ENPP1 inhibitors represent a multifaceted approach. They include various chemical scaffolds—from natural product derivatives and heterocyclic frameworks to phosphonate-based and phthalazinone analogs—all designed to robustly inhibit ENPP1, thereby enhancing STING-mediated immune responses in tumors. As these molecules progress through rigorous preclinical and clinical studies, the challenges associated with specificity, pharmacokinetics, and delivery are being actively addressed. The prospects for combination therapies and innovative drug-delivery strategies further support the potential for these molecules to offer meaningful improvements in disease outcomes. The evolution of ENPP1 inhibition from exploratory studies to the brink of clinical translation underscores the promise of this approach in modern drug discovery, while simultaneously highlighting the areas where further research is needed to meet the complex demands of therapeutic intervention in cancer and beyond.