SARS-CoV-2, known as severe acute respiratory syndrome coronavirus 2, is a new type of coronavirus responsible for 2019 pandemic of COVID-19. SARS-CoV-2 main protease (M^pro), also a 3C-like cysteine protease (3CL^pro), is one of the key enzymes of coronaviruses and plays a crucial role in mediating viral replication and transcription. Five non-covalent ligands were designed and grown for Southampton 3C-like protease (SV3CP) based on a hit from crystal-based fragment screening. These five ligands were crystalised with SARS-CoV-2 M^pro because of the similar active sites shared by SV3CP and SARS-CoV-2 M^pro, and we determined crystal structures of SARS-CoV-2 M^pro in complex with two ligands (S04 & S05). SARS-CoV-2 M^pro in complex with S05 is shown in Figure 1. We also developed a kinetic assay specific to SARS-CoV-2 M^pro showing Ki values of these five ligands range from 9.3 μM to 0.87 mM. These ligands show their potential as broad spectrum drug leads due to their inhibition activity in different 3CL proteases.

FmtA is a penicillin-recognizing protein (PRP) with novel hydrolytic activity toward the ester bond between d-Ala and the backbone of teichoic acids (TA), the polyol-phosphate polymers found in the S. aureus cell envelope. Two of the PRPs conserved motifs, namely SXXK and Y(S)XN, are involved in this hydrolysis, but its catalytic mechanism remains elusive. Here we determined the crystal structure of FmtA. FmtA shares the core structure of PRPs: an all α-helical domain and α/β domain sandwiched together. However, it does not have the typical PRPs active-site cleft. Its active site is shallow, solvent-exposed and wide. Furthermore, the SXXK and Y(S)XN motifs of FmtA offer all that is necessary for catalysis: the active-site nucleophile (serine) and the general base (lysine) required for acylation and deacylation steps and an anchor (tyrosine) to hold the active-site serine, and possibly the substrate, in an optimum conformation for catalysis [1]. Our study establishes that the FmtA esterase activity represents an expansion of the catalytic activity repertoire of the PRPs core structure, which typically displays peptidase activity. The structure of FmtA provides insights to the design of inhibitor molecules with the potential to serve as leads in the development of novel antibacterial chemotherapeutic agents.

[1] Dalal, V., Kumar, P., Rakhaminov, G., Qamar, A., Fan, X., Hunter, H., Tomar, S., Golemi-Kotra, D. and Kumar, P., (2019). Journal of molecular biology, 431(17), pp.3107-3123.

The antibiotic resistance incidence is alarmingly increasing in both human and veterinary medicine and is one of the major concerns of contemporary healthcare. Because most of the currently used antibiotics have natural analogs with similar native structures, AMR-associated genes are widely present in bacteria in the environment, and they can be easily distributed to clinically important strains through horizontal gene transfer. Such bacteria pose a threat primarily in hospitals, where infections of immunocompromised patients often end fatally. A small group of bacteria causing nosocomial infections are present in both the developed and developing world; it is called ESKAPE in abbreviation and is comprised of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and other Enterobacteriaceae species. These pathogens represent the most significant threat among the so-called superbugs, which can rapidly acquire resistance to several classes of antibiotics and can cause a variety of nosocomial infections, mostly in the respiratory or urinary tract, but bloodstream, wound, and skin infections are also frequent. Overall, nosocomial infections result in increased mortality and morbidity rate in the affected patients.

For endolysins targeting Gram-negative bacteria the specific activity against a broad host range is a known phenomenon, however, the molecular mechanisms specifying their broad spectrum of action are obscure. There is still no global understanding, whether bacterial cell lysis with endolysins is determined by the action of the enzymatic activity of the proteins or by additional functional domains containing in their structure, or the action of endolysins against Gram-negative microorganisms is determined by the joint interaction of several distinct domains.

Here we propose to investigate in details the structural aspects of LysSi3 endolysin mechanism of action. The LysSi3 is a peptidoglycan hydrolyzing, lysozyme-like enzyme with predicted muramidase activity (GH24 family) and broad bactericidal activity against ESKAPE pathogens.

Thalidomide (Thal) exerts adverse effects such as teratogenicity, but it is used for the therapy of multiple myeloma and other haematologic malignancies as immunomodulatory imide drugs (IMiDs). The molecular mechanism of thalidomide's pharmacological action has been gradually elucidated through the search for multiple target proteins that thalidomide acts on. Celebron (CRBN) is the intracellular receptor for Thal and induces Thal-dependent degradation of target protein (neosubstrate) as a component of an E3-ubiquitin ligase. Although C2H2 zinc finger (ZF) transcription factors, IKZF1 and SALL4, are concerned in immunomodulatory effects and teratogenicity of Thal, respectively, a primary Thal metabolite, 5-hydroxythalidomide (5HT), induces degradation of SALL4 but not IKZF1. Due to the action of the enzyme cytochrome P450 in the body, the administered thalidomide produces 5HT. Here, we focused on the molecular mechanism in which the selectivity of Thal toward C2H2 ZF-type neosubstrates is altered with its metabolism. First, we characterized the enantioselectivity of the formation in the SALL4-CRBN complex. The (S)-enantiomer of Thal and 5-HT showed more effect than the (R)-enantiomer, which is consistent to “Left-hand (S-form) theory of teratogenicity” of Thal. Based on the enantioselectivity, we determined the crystal structures of the ternary complexes of the Thal-binding domain (TBD) of human CRBN and the second ZF domain (ZF2) of human SALL4 induced by (S)-Thal and (S)-5HT. As a result, Thal and 5HT positioned between the interface of SALL4 ZF2 and CRBN TBD to mediate the protein-protein interaction as molecular glues. Although both compounds occupy at the same position in the SALL4-CRBN complex, the 5-hydroxy group of 5HT forms an additional hydrogen bond with CRBN TBD through a water molecule, which enhances the formation of the SALL4-CRBN complex. The 5-hydroxy group is also located near the 2nd and 9th residues of the β-hairpin structure in SALL4 ZF2, and these residues are different from IKZF1. The complex formation and proteasomal degradation experiments using the residue-swap mutants of SALL4 and IKZF1 elucidated the variation in the 2nd residue of β-hairpin structure defines the neosubstrate selectivity of 5HT. Thalidomide’s action on its target is altered through its metabolism in the body and if the hydroxylation of thalidomide found in this study is avoided, a new designed drug can be expected to reduce teratogenicity. Furthermore, our findings indicate that the structural differences found in C2H2 ZF-type transcription factors may be exploited to increase the efficiency of action of IMiDs, including thalidomide, on target proteins required for drug efficacy.

Antimitotic agents that target mitotic kinesins such as centromere associated protein E (CENP-E), are expected to be more likely to work on dividing cells but not non-dividing cells. Thus, antimitotic agents that inhibit the functions of the kinesin motor domains will minimize toxicities to non-dividing cells, causing lower side effects [1].

The motor domain, located at the N-terminus of CENP-E, is the active site of ATPase activity. Up to now, the only one crystal structure of CENP-E motor domain in complex with MgADP has been reported [2]. It is difficult to perform rational drug designing by fragment-based drug discovery (FBDD) or structure-based drug design (SBDD) due to the lack of structural information about CENP-E. Therefore, it is necessary to determine the crystal structure of CENP-E motor domain in complex with its inhibitors.

Here, in order to elucidate the mechanism how CENP-E motor domain binds to its inhibitor, we tried to cocrystallize CENP-E motor domain in complex with its ligand, 3-chloro-4-isopropoxyl benzoic acid (CIBA), one of the ATP-competitive inhibitors, or GSK923295, one of the ATP-uncompetitive inhibitors. First, we crystallized CENP-E motor domain in complex with CIBA, and determined the structure at 1.9 Å resolution (Figure 1). Endogenous ADP instead of CIBA was observed at the nucleotide-binding site, although ATP or ADP was not added. The determined structure of the CENP-E motor domain was compared with other kinesin motors. Based on the characteristic structure of CENP-E, the mechanism by which ADP is retained in CENP-E is discussed [3].

Next, in order to elucidate the structure in complex with an ATP analog, we tried to determine the structure of CENP-E motor domain in the presence of AMPPNP and Mg²⁺ at 1.8 Å resolution. Crystals belong to space group P2₁2₁2 with two molecules in the asymmetric unit. Structure refinement is now in progress.

[1] Sakowicz, R., Finer, J. T., Beraud, C., Crompton, A., Lewis, E., Fritsch, A., Lee, Y., Mak, J., Moody, R., Turincio, R., Chabala, J. C., Gonzales, P., Roth, S., Weitman, S. & Wood, K. W. (2004). Cancer Res. 64, 3276–3280.

[2] Garcia-Saez, I., Yen, T., Wade, R. H. & Kozielski, F. (2004). J. Mol. Biol. 340, 1107–1116.

[3] Shibuya, A., Ogo, N., Sawada, J., Asai, A., & Yokoyama, H. (2021). Acta Crystallographica Section D, D77, 280-287.

We would highly appreciate the cooperation and support rendered to us by all the staff members of High Energy Accelerator Research Organization in Tsukuba. We thank all involved in holding this congress.

Despite progress in vaccine development, antivirals targeting SARS-Co-2 are needed for those who are immunocompromised, and for future outbreaks. Proteases cleave peptide bonds of a very specific sequence making them strong drug targets. Antivirals that target proteases are already used clinically to treat HIV and Hepatitis C virus. We have developed inhibitors of the SARS-CoV-2 protease to prevent the main protease (M^pro or 3CL^pro) from cleaving the viral polypeptide and subsequent viral replication in cells. We developed novel α-acyloxymethylketone warhead peptidomimetic compounds with a 6-membered lactam glutamine mimic in P1. Compounds with potent SARS-CoV-2 3CL protease and in vitro viral replication inhibition were identified with low cytotoxicity and good plasma and glutathione stability. α-Acyloxymethylketone compounds also exhibited antiviral activity against an alpha- and non-SARS beta-coronavirus strains with similar potency and better selectivity index than remdesivir. X-ray crystallography revealed the mechanism of inhibition, and has helped the optimisation of new derivatives. Moving forward, these inhibitors will be tested with variant proteases, followed up by studies in animals to determine efficacy and pharmacokinetics in preparation for clinical trials.