Comprehensive Report on the Investigational Dual ATM/DNA-PK Inhibitor XRD-0394

1. Introduction to XRD-0394

XRD-0394 is a novel, orally bioavailable, small-molecule investigational drug candidate currently under development for the treatment of various cancers.[1] It represents a first-in-class therapeutic approach targeting the DNA Damage Response (DDR) pathway through the dual inhibition of two critical serine/threonine protein kinases: Ataxia Telangiectasia Mutated kinase (ATM) and DNA-dependent Protein Kinase catalytic subunit (DNA-PKcs).[1] This dual inhibitory action confers potential radiosensitizing and antineoplastic activities.[1]

The compound is being developed by XRad Therapeutics, Inc., a clinical-stage biopharmaceutical company specializing in the discovery and development of dual kinase inhibitors targeting DNA damage repair pathways for cancer therapy.[3] The initial clinical development strategy for XRD-0394 focuses on its potential to enhance the efficacy of radiation therapy (RT) when used in combination.[3] Concurrently, XRad Therapeutics is exploring the therapeutic potential of XRD-0394 in combination with other modalities, including targeted radionuclides, immune checkpoint inhibitors, polyadenosine diphosphate-ribose polymerase (PARP) inhibitors, and antibody-drug conjugates (ADCs).[3]

Chemically, XRD-0394 is identified by the CAS Number 2595308-10-2.[2] Its molecular formula is C26​H30​FN5​O4​S, corresponding to a molecular weight of 527.615 g/mol.[2] The IUPAC name is N-(5-(7'-fluoro-3'-methyl-2'-oxo-2',3'-dihydrospiro[cyclobutane-1,1'-pyrrolo[2,3-c]quinolin]-8'-yl)-2-(2-(isopropylamino)ethoxy)pyridin-3-yl)methanesulfonamide.[6] Synonyms include ataxia telangiectasia mutated kinase/DNA-dependent protein Kinase inhibitor XRD-0394, XRD 0394, and XRD0394.[1]

Table 1: Summary of XRD-0394 Key Information

Feature	Detail	Source(s)
Drug Name	XRD-0394	1
Developer	XRad Therapeutics, Inc.	3
Mechanism of Action	Dual inhibitor of ATM and DNA-PKcs; DDR inhibitor; Radiosensitizer	1
Biological Targets	ATM, DNA-PK (PRKDC)	1
Modality	Small Molecule	1
Route	Oral	1
Highest Phase	Phase 1a Completed (NCT05002140); Phase 0/I Planned (NCT06829173)	9
Initial Focus	Combination with Radiotherapy	1

2. Mechanism of Action

XRD-0394 exerts its potential therapeutic effects by targeting key components of the DNA Damage Response (DDR) pathway, a complex cellular network essential for maintaining genomic integrity.[1] The DDR pathway identifies DNA lesions, signals their presence, and orchestrates repair processes. DNA double-strand breaks (DSBs), such as those induced by ionizing radiation used in cancer therapy, are particularly cytotoxic lesions requiring efficient repair for cell survival.[1] Cancer cells often exhibit an enhanced ability to repair DNA damage, contributing significantly to their resistance to radiotherapy and certain chemotherapies.[1]

XRD-0394 is designed to counteract this resistance by simultaneously inhibiting two pivotal kinases within the DDR: ATM and DNA-PKcs.[1]

• Ataxia Telangiectasia Mutated (ATM): ATM is a primary sensor and transducer of DSBs. Upon activation, it phosphorylates numerous downstream targets, initiating cell cycle checkpoints to allow time for repair and promoting DNA repair pathways, most notably Homologous Recombination (HR).[1] ATM activity is frequently upregulated in various cancer types.[1] Inhibition of ATM by XRD-0394 disrupts this signaling cascade, potentially preventing checkpoint activation and hindering HR-mediated repair.[1]
• DNA-dependent Protein Kinase (DNA-PK): DNA-PK is the catalytic subunit (DNA-PKcs, encoded by PRKDC) of a complex crucial for the Non-Homologous End Joining (NHEJ) pathway.[1] NHEJ is the predominant mechanism for repairing DSBs caused by ionizing radiation throughout the cell cycle.[1] By inhibiting DNA-PK activity, XRD-0394 interferes with NHEJ, preventing the ligation of broken DNA ends.[1]

The rationale for developing a dual inhibitor stems from the intricate and sometimes compensatory nature of DNA repair pathways. While ATM primarily governs HR and DNA-PK drives NHEJ, these pathways can partially compensate for each other, especially in cancer cells which may harbor defects in one pathway (e.g., BRCA mutations impairing HR) and become hyper-reliant on the other.[5] Inhibiting only ATM might allow cells to survive through NHEJ, and vice-versa. By simultaneously blocking both major DSB repair routes, XRD-0394 aims to create a more comprehensive and durable blockade of DNA repair.[5] This dual inhibition strategy is expected to be more effective than single-agent inhibition in sensitizing tumor cells to DNA-damaging therapies like radiation, leading to increased accumulation of lethal DNA damage, subsequent tumor cell apoptosis, and potentially overcoming resistance mechanisms.[1]

3. Preclinical Evidence

Preclinical studies have provided substantial evidence supporting the proposed mechanism of action and therapeutic potential of XRD-0394.

Potency and Selectivity:

XRD-0394 demonstrates high potency against its intended targets, with reported IC50 values of 0.39 nM for ATM and 0.89 nM for DNA-PKcs.2 Importantly, it exhibits selectivity over other related kinases in the PI3K/PIKK family, suggesting a specific mode of action with potentially fewer off-target effects.2

In Vitro Findings:

Laboratory studies using cancer cell lines have confirmed the biological activity of XRD-0394:

• Target Engagement: In MCF7 human breast cancer cells, XRD-0394 pretreatment followed by ionizing radiation (IR) resulted in potent, dose-dependent inhibition of the autophosphorylation of DNA-PKcs (at Ser2056) and ATM (at Ser1981), as well as phosphorylation of the downstream ATM substrate KAP1 (at Ser824). This inhibition occurred without affecting the total protein levels of ATM or DNA-PKcs, confirming specific kinase inhibition.[2]
• Radiosensitization: Consistent with its mechanism, XRD-0394 significantly enhanced IR-induced tumor cell killing across various in vitro models.[2]
• Combination Potential with Other Agents:
• PARP Inhibitors (PARPi): XRD-0394 displayed synergistic effects when combined with PARP inhibitors, particularly in cancer cells deficient in BRCA1 or BRCA2.[2] This synergy is based on the principle of synthetic lethality; BRCA-deficient cells are reliant on PARP and potentially NHEJ for DNA repair, and inhibiting these pathways alongside ATM/DNA-PK leads to catastrophic DNA damage.
• Topoisomerase I Inhibitors: The compound also potentiated the cytotoxic effects of topoisomerase I inhibitors.[2] These inhibitors induce DNA breaks that require DDR pathways for repair, making their inhibition by XRD-0394 an effective combination strategy.
• Single-Agent Activity: Notably, XRD-0394 demonstrated single-agent cytotoxic activity in BRCA1/2-deficient cells, further supporting the synthetic lethality concept.[2]
• Genetic Context: A CRISPR screen identified that alterations in the Fanconi Anemia (FA)/BRCA pathway sensitize tumor cells to XRD-0394, providing further genetic evidence for the synthetic lethal interaction and validating this approach in multiple cell types.[12]

In Vivo Findings:

Animal model studies corroborated the in vitro observations:

• Radiosensitization: XRD-0394 significantly enhanced the tumor-killing effects of IR in in vivo models.[2]
• Target Inhibition: Oral administration of XRD-0394 prior to focal radiotherapy in mice bearing MDA-MB-321 xenografts resulted in the inhibition of both ATM activity (measured by reduced KAP1 phosphorylation) and DNA-PK activity (measured by reduced autophosphorylation) within the tumors.[2]

The dual inhibitory action of XRD-0394 underpins its potential utility beyond just enhancing conventional radiotherapy. The preclinical synergy observed with PARP inhibitors suggests a targeted approach for HR-deficient tumors. Similarly, the potentiation of topoisomerase I inhibitors opens avenues for combination chemotherapy regimens. These findings collectively indicate that XRD-0394 is not merely a radiosensitizer but a versatile DDR inhibitor with potential applications across various therapeutic combinations and cancer types, particularly those with specific DNA repair deficiencies.

4. Pharmacokinetics and Pharmacodynamics (PK/PD)

Understanding the absorption, distribution, metabolism, and excretion (pharmacokinetics, PK) of XRD-0394, along with its effect on the body (pharmacodynamics, PD), is crucial for its clinical development.

Preclinical PK:

Studies in mice demonstrated oral bioavailability. Following a single oral gavage dose of 10 mg/kg, plasma concentrations reached 840-1,125 ng/mL (equivalent to 1.6-2.1 µM) and were sustained for approximately 4 hours before declining to undetectable levels by 24 hours.2 This profile suggested that oral administration could achieve potentially therapeutic concentrations.

Clinical PK/PD (Phase 1a - NCT05002140):

The first-in-human Phase 1a trial provided initial PK and PD data in patients with advanced cancer receiving palliative radiotherapy.8

• Pharmacokinetics: Following a single oral dose of 160 mg, plasma concentrations of XRD-0394 were sustained above the preclinical target level of 530 ng/mL (1 µM) for up to 15 hours. The median time to reach maximum plasma concentration (tmax) was approximately 2.3 hours, and the mean terminal elimination half-life (t½) was approximately 11.1 hours.[12]
• Pharmacodynamics: Target engagement was confirmed at the 160 mg dose level. Analysis of tumor biopsy samples taken after irradiation showed inhibition of ATM kinase activity, as evidenced by a reduction in the phosphorylation of its substrate, KAP1 (pKAP1).[8]

These initial human PK/PD findings are encouraging. They demonstrate that oral administration of XRD-0394 can achieve plasma concentrations exceeding the level associated with preclinical efficacy (1 µM) for a duration relevant to radiotherapy schedules.[12] The observed half-life of ~11 hours supports manageable dosing regimens, such as administration shortly before daily radiation fractions, ensuring the drug is present at active concentrations during the period of maximal radiation-induced DNA damage.[12] Furthermore, the direct evidence of target inhibition (reduced pKAP1) in human tumor tissue at these concentrations provides crucial validation of the drug's mechanism of action in patients.[8] This convergence of adequate PK exposure and pharmacodynamic effect at a well-tolerated dose (see Section 6) provides a solid foundation for advancing XRD-0394 into further clinical studies.

5. Clinical Development Program

The clinical development of XRD-0394 is currently in the early stages, focusing initially on establishing safety, tolerability, PK, PD, and its potential as a radiosensitizer.

Table 2: Overview of Clinical Trials for XRD-0394

NCT ID	Phase	Status	Condition(s)	Intervention(s)	Sponsor / Lead	Purpose / Key Details	Source(s)
NCT05002140	1a	Completed (Jul 2023)	Metastatic, Locally Advanced, or Recurrent Cancer	Single Dose XRD-0394 + Palliative RT (4Gy x 5)	XRad Therapeutics Inc.	Dose-finding (40, 80, 160mg), Safety, PK, PD (pKAP1 inhibition in biopsies). N=12 enrolled.	8
NCT06829173	0/I	Not Yet Recruiting	High Grade Gliomas	XRD-0394 + RT + Temozolomide	NYU Langone Health	Assess safety and tolerability of combination therapy in glioma patients. N=39 planned.	10

Phase 1a Trial (NCT05002140):

This first-in-human, multicenter, open-label study evaluated single ascending doses (40 mg, 80 mg, 160 mg) of XRD-0394 administered orally in combination with a standard palliative radiation therapy regimen (4 Gy x 5 daily fractions).8 The trial enrolled 12 subjects with metastatic, locally advanced, or recurrent solid tumors requiring palliative RT.10 Key objectives included assessing safety and tolerability (primary), determining the PK profile, and demonstrating pharmacodynamic target engagement (ATM inhibition via pKAP1 reduction in tumor biopsies).8 The study successfully identified the 160 mg dose as achieving target plasma concentrations and demonstrating target inhibition, without dose-limiting toxicities.12 The trial completed in July 2023.10 Sponsors and collaborators included XRad Therapeutics, Memorial Sloan Kettering Cancer Center, Stanford Cancer Institute, and potentially researchers affiliated with Duke University, UNC Chapel Hill, Pharmaron, Allucent, University of Washington/Fred Hutch, MD Anderson, and University of Toronto based on publication authorships.12

Phase 0/I Trial (NCT06829173):

A Phase 0/I study sponsored by NYU Langone Health is planned but not yet recruiting.10 This trial aims to assess the safety and tolerability of XRD-0394 in combination with standard radiation therapy and temozolomide specifically in patients with high-grade gliomas.10 This marks a transition towards evaluating XRD-0394 in a specific tumor type where its radiosensitizing properties could be particularly beneficial.

The progression from the initial Phase 1a study in a broad advanced cancer population to a planned trial focused on high-grade glioma indicates a strategic clinical development direction. High-grade gliomas are notoriously difficult to treat, often exhibiting resistance to standard therapies including radiation. Given that ATM and DNA-PK are critical for repairing radiation-induced DNA damage, inhibiting these kinases with XRD-0394 presents a strong mechanistic rationale for improving treatment outcomes in this patient population. This focused approach aligns the drug's mechanism with a significant unmet clinical need in a specific oncology setting.

6. Safety and Tolerability Profile

Early clinical data suggest a favorable safety profile for XRD-0394.

Preclinical Safety:

Detailed preclinical toxicology studies specific to XRD-0394 were not fully described in the provided materials. Standard safety pharmacology assessments in preclinical models reportedly showed no adverse effects on major organ systems like respiratory, cardiovascular, or central nervous systems, though this information comes from a source primarily discussing an ASO and should be interpreted cautiously for the small molecule XRD-0394.

Clinical Safety (Phase 1a - NCT05002140):

The Phase 1a dose-escalation study, evaluating single oral doses of XRD-0394 (up to 160 mg) combined with palliative radiotherapy, reported that the drug was well-tolerated.12 Crucially, no Dose-Limiting Toxicities (DLTs) were observed within the dose range tested.12 The ability to reach a dose (160 mg) that demonstrated both target plasma exposure and pharmacodynamic evidence of target engagement (inhibition of pKAP1) without encountering DLTs is a positive indicator.12 This suggests a potentially acceptable therapeutic window, which is particularly important for agents intended for use in combination regimens where overlapping toxicities can be a concern. While specific adverse event types and frequencies were not detailed beyond the drug being "well-tolerated," the absence of DLTs at biologically active doses supports further clinical investigation.

7. Therapeutic Potential and Future Directions

XRD-0394 holds considerable therapeutic potential, primarily as a radiosensitizer but also in combination with other anticancer agents, based on its dual inhibition of ATM and DNA-PK.

Radiosensitization:

The primary development focus is leveraging XRD-0394's ability to inhibit DNA repair, thereby sensitizing cancer cells to the cytotoxic effects of ionizing radiation.1 This applies broadly to various radiotherapy modalities, including Intensity-Modulated Radiation Therapy (IMRT), Stereotactic Body Radiation Therapy (SBRT), proton beam therapy, and brachytherapy.3 Furthermore, it holds potential for use with targeted radionuclides delivered via conjugates or other means.3 An important potential benefit is the possibility of achieving adequate tumor control with lower doses of radiation when combined with XRD-0394, which could reduce radiation-related toxicities for patients.3

Combination Therapies:

The dual mechanism of inhibiting both HR- and NHEJ-related kinases opens multiple avenues for combination therapy beyond radiation:

• PARP Inhibitors: Preclinical data strongly support combining XRD-0394 with PARP inhibitors, especially in tumors with pre-existing defects in HR, such as those with BRCA1/2 mutations.[2] By blocking compensatory repair pathways (NHEJ via DNA-PK inhibition) and potentially further impairing any residual HR (via ATM inhibition), XRD-0394 can induce synthetic lethality with PARPi. XRD-0394 also showed single-agent activity in BRCA-deficient models.[2]
• Topoisomerase I Inhibitors: Potentiation of topoisomerase I inhibitors was observed preclinically.[2] These agents cause DNA breaks that rely on DDR pathways for repair; inhibiting ATM and DNA-PK simultaneously is expected to enhance their efficacy.
• Immunotherapy: While less explored in the provided snippets, DDR inhibition can sometimes increase tumor immunogenicity. XRad Therapeutics lists combination with immune checkpoint inhibitors as an area of exploration.[3]
• Antibody-Drug Conjugates (ADCs): Combination with ADCs, particularly those carrying DNA-damaging payloads, is another potential strategy being considered.[3]

Future Directions:

The positive preclinical data and the favorable safety, PK, and PD results from the Phase 1a trial provide a strong rationale for continued clinical development.5 Future trials are warranted to explore XRD-0394 in combination with radiotherapy (beyond palliative settings), PARP inhibitors (in biomarker-selected populations like HR-deficient tumors), and targeted topoisomerase I inhibitors.5 The planned Phase 0/I trial in high-grade glioma (NCT06829173) represents the next concrete step, evaluating the combination with standard-of-care chemoradiation in this challenging disease.10

The versatility suggested by the preclinical combination data indicates that XRD-0394 is positioned not just as a classical radiosensitizer but as a broader DDR inhibitor. Its potential applicability spans enhancing standard radiotherapy, exploiting synthetic lethality in genetically defined tumors, and potentially augmenting chemotherapy and targeted therapies, marking it as a compound with multiple plausible avenues for clinical exploitation.

8. Conclusion

XRD-0394 is a novel, first-in-class, orally bioavailable small molecule inhibitor targeting the critical DNA Damage Response kinases ATM and DNA-PKcs. Developed by XRad Therapeutics, Inc., its primary mechanism involves disrupting DNA double-strand break repair pathways, thereby sensitizing cancer cells to DNA-damaging agents, particularly ionizing radiation.

Preclinical studies have robustly demonstrated XRD-0394's potency and selectivity, its ability to enhance radiation-induced tumor cell killing in vitro and in vivo, and its potential to synergize with PARP inhibitors (especially in BRCA-deficient contexts) and potentiate topoisomerase I inhibitors.[2]

The initial Phase 1a clinical trial (NCT05002140) successfully established the safety and tolerability of single oral doses up to 160 mg when combined with palliative radiotherapy in patients with advanced cancers.[12] Importantly, this dose achieved plasma concentrations exceeding preclinical targets and demonstrated pharmacodynamic evidence of target engagement (ATM inhibition) in patient tumor samples, validating the drug's mechanism and dosing strategy in humans.[8]

Currently, XRD-0394 has completed Phase 1a development, and a Phase 0/I trial (NCT06829173) is planned to evaluate its combination with standard chemoradiation in patients with high-grade glioma.[10] Based on the available data, XRD-0394 shows significant promise as a DDR inhibitor with potential applications as a radiosensitizer and in combination with other targeted therapies (e.g., PARPi) and chemotherapies (e.g., Topo I inhibitors). Further clinical investigation is warranted to fully define its efficacy and safety profile across various cancer types and therapeutic combinations.

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