Hirokazu Tamamura

Synthesized Plk1 PBD-binding peptides with N -methylation exhibited up to 4-fold improved PBD affinities and with lipidation demonstrated up to 260-fold improved PBD affinities as compared with the parent peptide.

Antibody‐drug conjugates (ADCs) that equip multiple cytotoxic drugs on an antibody have been developed, particularly in cancer chemotherapy. In the treatment of viral infectious diseases, there are dominantly fewer examples of ADCs. Recently, we developed double‐warhead ADCs targeting the entry of human immunodeficiency virus type 1 (HIV‐1) into host cells. One is a small molecule CD4 mimic, which is a competitive inhibitor against the interaction between a viral envelop protein, gp120, and a primary receptor, CD4, and the other is neutralizing antibodies, which recognize the regions of gp120, exposed by its conformational change after the interaction between gp120 and CD4. The conformational changes are also triggered by the binding of gp120 and a CD4 mimic, and therefore, the ADCs show positive effects on anti‐HIV‐1 activity compared to the combinational use of CD4 mimics with neutralizing antibodies. Herein, we synthesized novel ADCs containing a CD4 mimic and a neutralizing antibody, KD‐247, using tCAP chemistry, which is based on a site‐specific modification method for IgG antibodies, and evaluated their anti‐HIV‐1 and antibody‐dependent cellular cytotoxicity (ADCC) activities. As a result, the KD‐247‐adopted ADCs demonstrated enhanced anti‐HIV‐1 activities, whereas all of the ADCs reduced their ADCC activities.

The SARS-CoV-2 main protease (M^pro) remains a prime antiviral target because its inhibition halts viral replication. To probe how subtle atomic changes influence drug performance, we carried out a systematic halogen scan on a potent ketoamide scaffold, replacing a single fluorine with chlorine, bromine, or iodine. Enzymatic assays revealed that the F- and Cl-substituted analogues inhibit M^pro at nanomolar levels, whereas Br and I variants are 10- to 20-fold weaker. Cell-based antiviral tests mirrored this trend, yet uptake studies showed the opposite: iodine markedly enhances intracellular accumulation. High-resolution X-ray structures (1.6–1.8 Å) explain the dichotomy: small halogens fit snugly in the S1′ σ-hole pocket, maximizing hydrogen-bond geometry, while bulkier atoms distort binding but create a lipophilic patch that boosts permeability. These data yield the first fluorine-to-iodine structure–activity map for SARS-CoV-2 M^pro inhibitors. These findings highlight the critical role of halogen selection in antiviral inhibitor design.

The development of new anti-HIV drugs with a novel mechanism of action is still required. A human cell surface protein, CD4, is a primary receptor for HIV-1 entry, and we have developed small-molecule CD4-competitive HIV-1 entry inhibitors, including CD4 mimics. CD4 mimics bind to a viral envelope protein, gp120, and cause a conformational change. Combinational use of CD4 mimics with CD4-induced antibodies, which recognize the exposed regions of gp120 induced by the CD4/CD4 mimic binding, shows a synergistic effect of anti-HIV activity. Therefore, we envisioned that antibody-drug conjugates (ADCs) of CD4 mimics and CD4-induced antibodies would effectively inhibit virus entry. Herein, ADCs of CD4 mimics and CD4-induced antibodies were designed and synthesized, and their anti-HIV-1 activities were evaluated. As a result, several synthesized ADCs exhibited 7- to 10-fold higher anti-HIV-1 activity than that of the parent antibodies, although there remains significant room for improvement in these ADCs.

Proteolysis targeting chimeras (PROTACs) have become a new modality for drug development of particular importance for cancer chemotherapy. PROTACs are composed of a ligand that binds to the protein of interest (POI) tethered by a linker to a ubiquitin E3 ligase-binding motif. These molecules can degrade the POI by ubiquitination and subsequent digestion using the ubiquitin-proteasome system (UPS). Although more than six hundred E3 ligases are encoded in human genome, only a small number are currently utilized by PROTACs. Because the expression levels and activities of E3 ligases vary among the cell lines, it can be advantageous to develop PROTACs that utilize new E3 ligase-binding components. In our current work we report new E3 ligase-binding ligands that employ viral protein R (Vpr), an accessory protein of the human immunodeficiency virus type-1 (HIV-1). Vpr can bind to both the E3 ligase complex, Cul4A-DDB1-DCAF1 and host proteins, such as UNG2 and facilitate host protein degradation via the UPS. We envisioned that Vpr fragments may function in PROTACs as new E3 ligase-binding ligands. Herein, we designed, synthesized and evaluated bromodomain 4 (BRD4)-targeting PROTACs (BRD4-PROTACs) that employ a well-known BRD4 inhibitor (JQ1) as a warhead and Vpr-derived peptides as the E3 ligase-binding ligands. We successfully demonstrate that the Vpr-derived peptides can function as E3 ligase-targeting ligands for PROTAC development. We also evaluated PROTACs based on the HIV-1 latency-reversing activity of JQ-1. The chemical degraders are less effective than the parent inhibitor as a latency-reversing agent (LRA). However, the low cytotoxicity of the new peptidic PROTACs allowed the compounds to be tolerated at high does, leading to potent LRA activity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative virus of COVID-19, remains a worldwide health threat. SARS-CoV-2 possesses two types of proteases: a main protease (M^pro, 3C-like) and a papain-like protease (PL^pro). These proteases cleave two translated nonstructural proteins, pp1a and pp1ab, into viral functional proteins. In the present study, we developed new inhibitors targeting the PL^pro of SARS-CoV-2 based on GRL-0048 (2) as a lead compound. Structure-activity relationship (SAR) researches on GRL-0048 (2) led to the finding of compound 9r, which showed significantly enhanced inhibitory activity against PL^pro. This enhancement was accomplished by the introduction of a halogenated aromatic carbonyl moiety into the parent structure of GRL-0048 (2).

Development of anti-SARS-CoV-2 agents is still required because of the appearance of drug-resistant strains, and targeting the main protease (M^pro) of SARS-CoV-2 is a powerful way to develop therapeutics against COVID-19. To date, we have developed several M^pro inhibitors including compounds 3, 4, TKB245 (6), and TKB248 (7), which have a 4-fluorobezothiazole ketone moiety as a warhead structure. Further studies led to the development of a more potent M^pro inhibitor, TKB272 (8), which has a 5-fluorobenzothiazole ketone moiety. The slight difference in the introduction position of a fluorine atom enhanced its antiviral activity approximately 3-fold for VeroE6 cells and 15-fold for HeLa^{hACE2‑TMPRSS2} cells when TKB272 (8) was compared to TKB245 (6). Herein, we report the synthesis and structure–activity relationship (SAR) studies of TKB272 (8). TKB272 (8) is a promising drug candidate for COVID-19 therapy, and the present data of the SAR studies are useful for the further development of M^pro inhibitors.

Since the membrane fusion step is the last chance to block the virus extracellularly, membrane fusion is an important target for anti‐human immunodeficiency virus (HIV) agents. Previously, the dimeric derivatives of C34, which are contained in the HIV‐1 envelope protein gp41 are found, linked by a disulfide bridge or a pegylated linker at its C ‐terminus have more potent anti‐HIV activity than the C34 peptide monomer, and that bivalent inhibitors crosslinking two peptidomimetic small compounds have more potent anti‐HIV activity than the parent small compounds. In the present study, the hybrids of small compounds ( 7 – 9 ) and peptides (SC34 ( 2 ) and SC22EK ( 3 )) are designed as heterodimeric molecules ( 10 – 15 ) to compensate for the drawbacks of the above homodimeric molecules. Some hybrid molecules of small compounds ( 7 – 9 ) and peptide SC22EK ( 3 ) have remarkably higher anti‐HIV activity than peptide SC22EK ( 3 ). Crosslinking small compounds and peptides ( 3 ) is found to be critical for an increase in anti‐HIV activity. Hybrid molecules with small compounds and peptides are useful HIV‐1 fusion inhibitors.

A diastereo-controlled synthetic method for ( Z )-fluoroalkene dipeptide isosteres (FADIs) was developed utilizing our established methodology for chloroalkene dipeptide isosteres (CADIs) to construct two chiral centers.