Structure-guided identification and characterization of potent inhibitors targeting PhoP and MtrA to combat mycobacteria.

Mycobacteria are causative agents of tuberculosis (TB), which is a global health concern. Drug-resistant TB strains are rapidly emerging, thereby necessitating the urgent development of new drugs. Two-component signal transduction systems (TCSs) are signaling pathways involved in the regulation of various bacterial behaviors and responses to environmental stimuli. Applying specific inhibitors of TCSs can disrupt bacterial signaling, growth, and virulence, and can help combat drug-resistant TB. We conducted a comprehensive pharmacophore-based inhibitor screening and biochemical and biophysical examinations to identify, characterize, and validate potential inhibitors targeting the response regulators PhoP and MtrA of mycobacteria. The constructed pharmacophore model Phar-PR-n4 identified effective inhibitors of formation of the PhoP-DNA complex: ST132 (IC50 = 29 ± 1.6 µM) and ST166 (IC50 = 18 ± 1.3 µM). ST166 (KD = 18.4 ± 4.3 µM) and ST132 (KD = 14.5 ± 0.1 µM) strongly targeted PhoP in a slow-on, slow-off manner. The inhibitory potency and binding affinity of ST166 and ST132 for MtrAC were comparable to those of PhoP. Structural analyses and molecular dynamics simulations revealed that ST166 and ST132 mainly interact with the α8-helix and C-terminal ß-hairpin of PhoP, with functionally essential residue hotspots for structure-based inhibitor optimization. Moreover, ST166 has in vitro antibacterial activity against Macrobacterium marinum. Thus, ST166, with its characteristic 1,2,5,6-tetrathiocane and terminal sulphonic groups, has excellent potential as a candidate for the development of novel antimicrobial agents to combat pathogenic mycobacteria.

Discovery of a potent inhibitor, D-132, targeting AsfvPolX, via protein-DNA complex-guided pharmacophore screening and in vitro molecular characterizations.

The heightened transmissibility and capacity of African swine fever virus (ASFV) induce fatal diseases in domestic pigs and wild boars, posing significant economic repercussions and global threats. Despite extensive research efforts, the development of potent vaccines or treatments for ASFV remains a persistent challenge. Recently, inhibiting the AsfvPolX, a key DNA repair enzyme, emerges as a feasible strategy to disrupt viral replication and control ASFV infections. In this study, a comprehensive approach involving pharmacophore-based inhibitor screening, coupled with biochemical and biophysical analyses, were implemented to identify, characterize, and validate potential inhibitors targeting AsfvPolX. The constructed pharmacophore model, Phar-PolX-S, demonstrated efficacy in identifying a potent inhibitor, D-132 (IC50 = 2.8 ± 0.2 µM), disrupting the formation of the AsfvPolX-DNA complex. Notably, D-132 exhibited strong binding to AsfvPolX (KD = 6.9 ± 2.2 µM) through a slow-on-fast-off binding mechanism. Employing molecular modeling, it was elucidated that D-132 predominantly binds in-between the palm and finger domains of AsfvPolX, with crucial residues (R42, N48, Q98, E100, F102, and F116) identified as hotspots for structure-based inhibitor optimization. Distinctively characterized by a 1,2,5,6-tetrathiocane with modifications at the 3 and 8 positions involving ethanesulfonates, D-132 holds considerable promise as a lead compound for the development of innovative agents to combat ASFV infections.

Klebsiella pneumoniae K2 capsular polysaccharide degradation by a bacteriophage depolymerase does not require trimer formation.

K2-capsular Klebsiella pneumoniae is a hypervirulent pathogen that causes fatal infections. Here, we describe a phage tailspike protein, named K2-2, that specifically depolymerizes the K2 capsular polysaccharide (CPS) of K. pneumoniae into tetrasaccharide repeating units. Nearly half of the products contained O-acetylation, which was thought crucial to the immunogenicity of CPS. The product-bound structures of this trimeric enzyme revealed intersubunit carbohydrate-binding grooves, each accommodating three tetrasaccharide units of K2 CPS. The catalytic residues and the key interactions responsible for K2 CPS recognition were identified and verified by site-directed mutagenesis. Further biophysical and functional characterization, along with the structure of a tetrameric form of K2-2, demonstrated that the formation of intersubunit catalytic center does not require trimerization, which could be nearly completely disrupted by a single-residue mutation in the C-terminal domain. Our findings regarding the assembly and catalysis of K2-2 provide cues for the development of glycoconjugate vaccines against K. pneumoniae infection. IMPORTANCE: Generating fragments of capsular polysaccharides from pathogenic bacteria with crucial antigenic determinants for vaccine development continues to pose challenges. The significance of the C-terminal region of phage tailspike protein (TSP) in relation to its folding and trimer formation remains largely unexplored. The polysaccharide depolymerase described here demonstrates the ability to depolymerize the K2 CPS of K. pneumoniae into tetrasaccharide fragments while retaining the vital O-acetylation modification crucial for immunogenicity. By carefully characterizing the enzyme, elucidating its three-dimensional structures, conducting site-directed mutagenesis, and assessing the antimicrobial efficacy of the mutant enzymes against K2 K. pneumoniae, we offer valuable insights into the mechanism by which this enzyme recognizes and depolymerizes the K2 CPS. Our findings, particularly the discovery that trimer formation is not required for depolymerizing activity, challenge the current understanding of trimer-dependent TSP activity and highlight the catalytic mechanism of the TSP with an intersubunit catalytic center.

Pharmacophore-driven identification of human glutaminyl cyclase inhibitors from foods, plants and herbs unveils the bioactive property and potential of Azaleatin in the treatment of Alzheimer's disease.

Alzheimer's disease (AD) is the leading cause of disabilities in old age and a rapidly growing condition in the elderly population. AD brings significant burden and has a devastating impact on public health, society and the global economy. Thus, developing new therapeutics to combat AD is imperative. Human glutaminyl cyclase (hQC), which catalyzes the formation of neurotoxic pyroglutamate (pE)-modified ß-amyloid (Aß) peptides, is linked to the amyloidogenic process that leads to the initiation of AD. Hence, hQC is an essential target for developing anti-AD therapeutics. Here, we systematically screened and identified hQC inhibitors from natural products by pharmacophore-driven inhibitor screening coupled with biochemical and biophysical examinations. We employed receptor-ligand pharmacophore generation to build pharmacophore models and Phar-MERGE and Phar-SEN for inhibitor screening through ligand-pharmacophore mapping. About 11 and 24 hits identified from the Natural Product and Traditional Chinese Medicine databases, respectively, showed diverse hQC inhibitory abilities. Importantly, the inhibitors TCM1 (Azaleatin; IC50 = 1.1 µM) and TCM2 (Quercetin; IC50 = 4.3 µM) found in foods and plants exhibited strong inhibitory potency against hQC. Furthermore, the binding affinity and molecular interactions were analyzed by surface plasmon resonance (SPR) and molecular modeling/simulations to explore the possible modes of action of Azaleatin and Quercetin. Our study successfully screened and characterized the foundational biochemical and biophysical properties of Azaleatin and Quercetin toward targeting hQC, unveiling their bioactive potential in the treatment of AD.

Correction: Discovery of 8-prenylnaringenin from hop (Humulus lupulus L.) as a potent monoacylglycerol lipase inhibitor for treatments of neuroinflammation and Alzheimer's disease.

[This corrects the article DOI: 10.1039/D1RA05311F.].

Antigen-Antibody Complex-Guided Exploration of the Hotspots Conferring the Immune-Escaping Ability of the SARS-CoV-2 RBD.

The COVID-19 pandemic resulting from the spread of SARS-CoV-2 spurred devastating health and economic crises around the world. Neutralizing antibodies and licensed vaccines were developed to combat COVID-19, but progress was slow. In addition, variants of the receptor-binding domain (RBD) of the spike protein confer resistance of SARS-CoV-2 to neutralizing antibodies, nullifying the possibility of human immunity. Therefore, investigations into the RBD mutations that disrupt neutralization through convalescent antibodies are urgently required. In this study, we comprehensively and systematically investigated the binding stability of RBD variants targeting convalescent antibodies and revealed that the RBD residues F456, F490, L452, L455, and K417 are immune-escaping hotspots, and E484, F486, and N501 are destabilizing residues. Our study also explored the possible modes of actions of emerging SARS-CoV-2 variants. All results are consistent with experimental observations of attenuated antibody neutralization and clinically emerging SARS-CoV-2 variants. We identified possible immune-escaping hotspots that could further promote resistance to convalescent antibodies. The results provide valuable information for developing and designing novel monoclonal antibody drugs to combat emerging SARS-CoV-2 variants.

Discovery of 8-prenylnaringenin from hop (Humulus lupulus L.) as a potent monoacylglycerol lipase inhibitor for treatments of neuroinflammation and Alzheimer's disease.

Monoacylglycerol lipase (MAGL), a serine hydrolase, converts endocannabinoid 2-arachidonoylglycerol (2-AG) to arachidonic acid (AA) and glycerol in the brain and plays a bidirectional role in controlling nueroinflammation. MAGL, involved in Alzheimer's and Parkinson's diseases, is a promising target for treatment of neurodegenerative disorders. However, the irreversible inhibitors of MAGL lead to the desensitization of CB1 receptors further impairing the benefits associated with the indirect CB1 stimulation. Therefore, development of potent reversible inhibitors from natural products (NPs) and traditional chinese medicines (TCMs) are safer and free from adverse side effects and feasible to avoid drawbacks which irreversible inhibitors cause. Here, we employed pharmacophore-based screening of drug candidates coupled with molecular docking, biochemical assay and Ligplot analyses to identify and characterize inhibitors targeting human MAGL (hMAGL). The built pharmacophore model, Phar-MAGL successfully identified inhibitors NP-2 (IC50 = 9.5 ± 1.2 µM), NP-5 (IC50 = 14.5 ± 1.3 µM), and NP-3 (IC50 = 15.2 ± 1.4 µM), which apparently attenuated the activities of hMAGL in vitro. The evident activities of the identified inhibitors against hMAGL showed that the pharmacophore model, Phar-MAGL is reliable and efficient in screening inhibitors against hMAGL. Our study successfully identified a natrual product inhibitor, NP-2 (8-PN), from the plant Humulus lupulus L. (hops) and its positive effects in neurogenesis and neurodifferentiation along with the evident inhibitory potency against hMAGL revealed the potential for further optimizing and developing into drugs to treat neuroinflammation, Alzheimer's and Parkinson's diseases.

Characterizing the structure-activity relationships of natural products, tanshinones, reveals their mode of action in inhibiting spleen tyrosine kinase.

The cytosolic non-receptor protein kinase, spleen tyrosine kinase (SYK), is an attractive drug target in autoimmune, inflammatory disorder, and cancers indications. Here, we employed pharmacophore-based drug screening combined with biochemical assay and molecular dynamics (MD) simulations to identify and characterize inhibitors targeting SYK. The built pharmacophore model, phar-TanI, successfully identified tanshinone (TanI (IC50 = 1.72 µM)) and its analogs (TanIIA (IC50 = 3.2 µM), ST32da (IC50 = 46 µM), and ST32db (IC50 = 51 µM)) which apparently attenuated the activities of SYK in vitro. Additionally, the MD simulations followed by Ligplot analyses revealed that TanI and TanIIA interfered SYK activity through binding deeply into the active site. Besides, TanI and TanIIA mainly interact with residues L377, A400, V433, M448, M450, A451, E452, L453, G454, P455, and L501, which are functional hotspots for structure-based inhibitor optimization against SYK. The structure-activity relationships (SAR) study of the identified SYK inhibitors demonstrated that the pharmacophore model, phar-TanI is reliable and precise in screening inhibitors against SYK. This study disclosed the structure-function relationships of tanshinones from Traditional Chinese Medicine (Danshen), revealing their binding site and mode of action in inhibiting SYK and provides applicability in developing new therapeutic agents.

Characterization of the structure-function relationship of a novel salt-resistant antimicrobial peptide, RR12.

Antimicrobial peptides (AMPs) are potential candidates in designing new anti-infective agents. However, many AMPs show poor bactericidal activities in physical salt and serum solutions. Here, we disclosed the structure-function relationships of a novel salt-resistant antimicrobial peptide, RR12, which could further explain its mode of action and show its applicability in developing new antibacterial agents.

Complete Genome Sequence of Xanthomonas campestris pv. campestris Strain 17 from Taiwan.

Xanthomonas campestris pv. campestris 17 is a Gram-negative bacterium that is phytopathogenic to cruciferous plants in Taiwan. The 4,994,426-bp-long genome consists of 24 contigs with 4,050 protein-coding genes, 1 noncoding RNA (ncRNA) gene, 6 rRNA genes, and 55 tRNA genes.

A novel tetrameric PilZ domain structure from xanthomonads.

PilZ domain is one of the key receptors for the newly discovered secondary messenger molecule cyclic di-GMP (c-di-GMP). To date, several monomeric PilZ domain proteins have been identified. Some exhibit strong c-di-GMP binding activity, while others have barely detectable c-di-GMP binding activity and require an accessory protein such as FimX to indirectly respond to the c-di-GMP signal. We now report a novel tetrameric PilZ domain structure of XCC6012 from the plant pathogen Xanthomonas campestris pv. campestris (Xcc). It is one of the four PilZ domain proteins essential for Xcc pathogenicity. Although the monomer adopts a structure similar to those of the PilZ domains with very weak c-di-GMP binding activity, it is nevertheless interrupted in the middle by two extra long helices. Four XCC6012 proteins are thus self-assembled into a tetramer via the extra heptad repeat α3 helices to form a parallel four-stranded coiled-coil, which is further enclosed by two sets of inclined α2 and α4 helices. We further generated a series of XCC6012 variants and measured the unfolding temperatures and oligomeric states in order to investigate the nature of this novel tetramer. Discovery of this new PilZ domain architecture increases the complexity of c-di-GMP-mediated regulation.