Development of Small-Molecule Anti-HIV-1 Agents Targeting HIV-1 Capsid Proteins.

The capsid of human immunodeficiency virus type 1 (HIV-1) forms a conical structure by assembling oligomers of capsid (CA) proteins and is a virion shell that encapsulates viral RNA. The inhibition of the CA function could be an appropriate target for suppression of HIV-1 replication because the CA proteins are highly conserved among many strains of HIV-1, and the drug targeting CA, lenacapavir, has been clinically developed by Gilead Sciences, Inc. Interface hydrophobic interactions between two CA molecules via the Trp184 and Met185 residues in the CA sequence are indispensable for conformational stabilization of the CA multimer. Our continuous studies found two types of small molecules with different scaffolds, MKN-1 and MKN-3, designed by in silico screening as a dipeptide mimic of Trp184 and Met185 have significant anti-HIV-1 activity. In the present study, MKN-1 derivatives have been designed and synthesized. Their structure-activity relationship studies found some compounds having potent anti-HIV activity. The present results should be useful in the design of novel CA-targeting molecules with anti-HIV activity.

Exploratory Studies of Effective Inhibitors against the SARS-CoV-2 Main Protease by Halogen Incorporation and Amide Bond Replacement.

In the development of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) drugs, its main protease (Mpro), which is an essential enzyme for viral replication, is a promising target. To date, the Mpro inhibitors, nirmatrelvir and ensitrelvir, have been clinically developed by Pfizer Inc. and Shionogi & Co., Ltd., respectively, as orally administrable drugs to treat coronavirus disease of 2019 (COVID-19). We have also developed several potent inhibitors of SARS-CoV-2 Mpro that include compounds 4, 5, TKB245 (6), and TKB248 (7), which possesses a 4-fluorobenzothiazole ketone moiety as a reactive warhead. In compounds 5 and TKB248 (7) we have also found that replacement of the P1-P2 amide of compounds 4 and TKB245 (6) with the corresponding thioamide improved their pharmacokinetics (PK) profile in mice. Here, we report the design, synthesis and evaluation of SARS-CoV-2 Mpro inhibitors with replacement of a digestible amide bond by surrogates (9-11, 33, and 34) and introduction of fluorine atoms in a metabolically reactive methyl group on the indole moiety (8). As the results, these compounds showed comparable or less potency compared to the corresponding parent compounds, YH-53/5h (2) and 4. These results should provide useful information for further development of Mpro inhibitors.

Development of anti-HBV agents targeting HBV capsid proteins.

Hepatitis B is a viral hepatitis, which is caused by infection of hepatitis B virus (HBV). This disease progresses to chronic hepatitis, cirrhosis and liver cancer. To treat hepatitis B, exclusion of virus and covalently closed circular DNA (cccDNA) that is formed in hepatocyte nucleus is necessary. A hepatitis B capsid protein (HBc) is an indispensable protein, which forms the capsid that encapsulates viral DNA. Since HBc is correlated to the transcriptional regulation of cccDNA, this protein would be an attractive target for complete cure of hepatitis B. By in silico screening of a library of compounds, a small compound, Cpd4 (1), which binds to a hydrophobic cavity located in the inner pocket on the tetramer interface of HBc proteins, was identified. In anti-HBV assays, this synthetic compound, Cpd4 (1) decreased the amount of HBV core related antigen (HBcrAg), which has been correlated with the proliferation of HBV, and decreased the amount of HBV surface antigen (HBsAg), which is correlated with the amount of cccDNA. Based on Cpd4 (1) as a lead compound, 20 derivatives of 1 were designed and synthesized and their structure-activity relationships were examined. As a result, specific interactions between each compound and amino acid residues of the target protein appeared to be unimportant but the shape/size of compounds which can bind to the hydrophobic cavity might be important in the expression of high anti-HBV activity, and a more potent derivative, TKB-HBV-CA-001 (3b), was discovered. These results will be useful in the development of novel anti-HBV agents for a complete cure of hepatitis B.

Structure-Activity Relationship Studies of SARS-CoV-2 Main Protease Inhibitors Containing 4-Fluorobenzothiazole-2-carbonyl Moieties.

The main protease (Mpro) of SARS-CoV-2 is an attractive target for the development of drugs to treat COVID-19. Here, we report the design, synthesis, and structure-activity relationship (SAR) studies of highly potent SARS-CoV-2 Mpro inhibitors including TKB245 (5)/TKB248 (6). Since we have previously developed Mpro inhibitors (3) and (4), several hybrid molecules of these previous compounds combined with nirmatrelvir (1) were designed and synthesized. Compounds such as TKB245 (5) and TKB248 (6), containing a 4-fluorobenzothiazole moiety at the P1' site, are highly effective in the blockade of SARS-CoV-2 replication in VeroE6 cells. Replacement of the P1-P2 amide with the thioamide surrogate in TKB248 (6) improved its PK profile in mice compared to that of TKB245 (5). A new diversity-oriented synthetic route to TKB245 (5) derivatives was also developed. The results of the SAR studies suggest that TKB245 (5) and TKB248 (6) are useful lead compounds for the further development of Mpro inhibitors.

Dimerized fusion inhibitor peptides targeting the HR1-HR2 interaction of SARS-CoV-2.

Membrane fusion is a critical and indispensable step in the replication cycles of viruses such as SARS-CoV-2 and human immunodeficiency virus type-1 (HIV-1). In this step, a trimer of the heptad repeat 1 (HR1) region interacts with the three HR2 regions and forms a 6-helix bundle (6-HB) structure to proceed with membrane fusion of the virus envelope and host cells. Recently, several researchers have developed potent peptidic SARS-CoV-2 fusion inhibitors based on the HR2 sequence and including some modifications. We have developed highly potent HIV-1 fusion inhibitors by dimerization of its HR2 peptides. Here, we report the development of dimerized HR2 peptides of SARS-CoV-2, which showed significantly higher antiviral activity than the corresponding monomers, suggesting that the dimerization strategy can facilitate the design of potent inhibitors of SARS-CoV-2.

Low-molecular-weight anti-HIV-1 agents targeting HIV-1 capsid proteins.

The HIV-1 capsid is a shell that encapsulates viral RNA, and forms a conical structure by assembling oligomers of capsid (CA) proteins. Since the CA proteins are highly conserved among many strains of HIV-1, the inhibition of the CA function could be an appropriate goal for suppression of HIV-1 replication, but to date, no drug targeting CA has been developed. Hydrophobic interactions between two CA molecules through Trp184 and Met185 in the protein are known to be indispensable for conformational stabilization of the CA multimer. In our previous study, a small molecule designed by in silico screening as a dipeptide mimic of Trp184 and Met185 in the interaction site was synthesized and found to have significant anti-HIV-1 activity. In the present study, molecules with different scaffolds based on a dipeptide mimic of Trp184 and Met185 have been designed and synthesized. Their significant anti-HIV activity and their advantages compared to the previous compounds were examined. The present results should be useful in the design of novel CA-targeting anti-HIV agents.

Potent and biostable inhibitors of the main protease of SARS-CoV-2.

Potent and biostable inhibitors of the main protease (Mpro) of SARS-CoV-2 were designed and synthesized based on an active hit compound 5h (2). Our strategy was based not only on the introduction of fluorine atoms into the inhibitor molecule for an increase of binding affinity for the pocket of Mpro and cell membrane permeability but also on the replacement of the digestible amide bond by a surrogate structure to increase the biostability of the compounds. Compound 3 is highly potent and blocks SARS-CoV-2 infection in vitro without a viral breakthrough. The derivatives, which contain a thioamide surrogate in the P2-P1 amide bond of these compounds (2 and 3), showed remarkably preferable pharmacokinetics in mice compared with the corresponding parent compounds. These data show that compounds 3 and its biostable derivative 4 are potential drugs for treating COVID-19 and that replacement of the digestible amide bond by its thioamide surrogate structure is an effective method.