Functional and Structural Characterization of Treatment-Emergent Nirmatrelvir Resistance Mutations at Low Frequencies in the Main Protease (Mpro) Reveals a Unique Evolutionary Route for SARS-CoV-2 to Gain Resistance

Category	Detail
Condition	SARS-CoV-2 infection (COVID-19)
Key Mechanisms	Mutations in SARS-CoV-2 main protease (Mpro) confer resistance to nirmatrelvir-ritonavir by altering protein structure and inhibitor binding without compromising enzymatic activity
Target Population	Patients infected with SARS-CoV-2, including elderly and immunocompromised individuals treated with nirmatrelvir-ritonavir
Care Setting	Hospital and outpatient settings where antiviral therapy for COVID-19 is administered

Identification of novel low-frequency Mpro clinical variants (D48D/L58F/P132H and D48D/L67V/K90R/P132H) in patients treated with nirmatrelvir-ritonavir who remained SARS-CoV-2 positive.
These mutations are located outside the Mpro catalytic site but significantly reduce susceptibility to nirmatrelvir-ritonavir without impairing protease activity.
Structural and thermal analyses suggest mutations disrupt substrate binding site dynamics and protein stability, indicating a distinct evolutionary pathway for antiviral resistance.

Use RT-PCR to confirm SARS-CoV-2 infection.
Consider whole-genome sequencing in patients with persistent positivity after nirmatrelvir-ritonavir treatment to identify resistance mutations.

Administer nirmatrelvir-ritonavir as an antiviral therapy to reduce disease severity and hospitalizations, especially in elderly and immunocompromised patients.
Monitor for potential antiviral resistance in patients with incomplete or prolonged viral positivity post-treatment.

Surveillance for low-frequency resistance mutations through genomic sequencing in treated patients.
Clinical monitoring of viral clearance and symptom resolution after antiviral therapy.

Development of antiviral resistance mutations, particularly in immunocompromised individuals or with suboptimal therapy.
Potential cross-resistance to other Mpro inhibitors due to structural mutation effects.

SARS-CoV-2 infected patients treated with nirmatrelvir-ritonavir, including those with persistent infection post-treatment.

Nirmatrelvir-ritonavir effectively reduces disease severity but resistance mutations, though rare, can emerge and compromise therapeutic efficacy.

Ensure full adherence to prescribed nirmatrelvir-ritonavir treatment courses to minimize resistance development.
Incorporate genomic surveillance for resistance mutations in patients with prolonged viral positivity after treatment.
Consider alternative or adjunctive antiviral strategies if resistance mutations are detected.
Recognize that mutations outside the active site can confer resistance, underscoring the need for comprehensive molecular monitoring.

Clinical Scorecard: Analysis of Low-Frequency Nirmatrelvir Resistance Mutations in the Main Protease (Mpro) Uncovers a Distinct Evolutionary Pathway for SARS-CoV-2 Resistance Development