Identification of novel HIV-1 capsid protein (CA) inhibitors (ACAi-010 and ACAi-024) that promotes hyper-stabilization of HIV-1 CA in vitro.

The human immunodeficiency virus type 1 (HIV-1) capsid protein (CA) is a vital viral component and an attractive antiviral target. Our prior research demonstrated that amino acid (AA)-inserts at certain positions within the HIV-1 CA result in significant CA degradation, and that such AA-insert-containing HIV-1 have significantly compromised infectivity/replicability. We posited that this degradation arises from conformational or structural-incompatibility introduced by the AA-inserts at particular sites within the CA. We also hypothesized that a compound tightly bound to certain CA domains where influence CA-stability, might alter CA-stability and inhibit viral-replication. In the current research, we initially explored compounds which can bind to such domains upon the CA by virtual docking simulations, utilizing a library comprising >six million compounds. For compounds with good docking-scores, we evaluated anti-HIV-1-capacity using HIV-1LAI. Through these methods, we identified ACAi-010 and ACAi-024, designed as CA hyper-stabilizers, exhibiting potent antiviral-activity against a broad-spectrum of HIV-1/2, containing multi-drug-resistant and CA inhibitor-resistant strains, with least cytotoxicity. We confirmed that ACAi-010/ACAi-024 induced hyper-stabilization of HIV-1 CA through ELISA using lysates from cells expressing CA-monomer or HIV-1, and through differential scanning fluorimetry. Transmission electron microscopy revealed morphological-alterations in HIV-1 virions exposed to ACAi-010/ACAi-024. HIV-1 generated with CA hyper-stabilizers exhibited disrupted replicability/infectivity, as evaluated by p24 assay/MAGI assay. We found that ACAi-010/ACAi-024 did not inhibit HIV-1 reverse-transcriptase. Finally, drug-selection experiments indicated that the emergence of ACAi-010/ACAi-024-resistant-HIV-1 was severely difficult or failed. Our findings suggest that the CA hyper-stabilizers we identified/evaluated may lead to novel and effective interventions against HIV-1, including drug-resistant-variants.