Beyond sequences: structure-guided discovery of novel protein functions in plants.

The emergence of multidrug-resistant (MDR) strains of Salmonella typhimurium (S. typhimurium) causes a significant global health challenge and underscores the need to develop potential antimicrobial agents. Here, we considered RamR, the major transcriptional repressor of the AcrAB-TolC efflux pump system, to identify promising inhibitors that can restore antibiotic susceptibility. We adopted an integrated computational-experimental research strategy that involved in silico screening of a structurally diverse compound database. The top four candidates (144095451, 17515455, 26648946, and 26648774) were selected for detailed analysis, which included re-docking, molecular dynamics (MD) simulations, binding free energy calculations, and free energy landscape analysis mapping. Density functional theory (DFT) was employed to explain the electronic properties and chemical reactivity of these molecules. To enhance the predictive accuracy of inhibitory potency (pIC₅₀), a machine learning (ML) regression model was developed, in which the ExtraTrees algorithm demonstrated high performance (R2 = 0.975). Among the top-ranked compounds, 144095451 emerged as the most promising RamR inhibitor, as indicated by both computational predictions and ML modelling. Experimental verification with isothermal titration calorimetry (ITC) confirmed strong binding affinity (Ka = 5.43 × 10⁶ M⁻1; ΔH = -53.18kcal/mol; stoichiometry n = 1.74) of 144095451. Antimicrobial profiling also established its efficacy, with a minimum inhibitory concentration (MIC) of 121.65 ± 0.5µg/mL and a zone of inhibition of 18.54 ± 0.76. These results highlight compound 144095451 as a promising RamR-targeted antimicrobial lead. This research highlights the potential of the combinatorial approach, which utilizes computational screening, structural dynamics, machine learning-based biological activity prediction, and experimental confirmation of candidate molecules against multidrug-resistant S. typhimurium.

Using high concentration biochemical assays and fragment-based screening assisted by structure-guided design, we discovered a novel class of Rho-kinase inhibitors. Compound 18 was equipotent for ROCK1 (IC(50) = 650 nM) and ROCK2 (IC(50) = 670 nM), whereas compound 24 was more selective for ROCK2 (IC(50) = 100 nM) over ROCK1 (IC(50) = 1690 nM). The crystal structure of the compound 18-ROCK1 complex revealed that 18 is a type 1 inhibitor that binds the hinge region in the ATP binding site. Compounds 18 and 24 inhibited potently the phosphorylation of the ROCK substrate MLC2 in intact human breast cancer cells.

Structure-guided discovery and rational design of a new poly(ethylene terephthalate) hydrolase from AlphaFold protein structure database

Crystallography has made extraordinary contributions to our understanding of the biology and chemistry of HIV. Judicious applications of structure-based drug design against HIV-1 protease and reverse transcriptase (RT) has led to the discovery of key drugs that are used in combinations to treat HIV infection. Extensive research and development efforts by pharma, academia, and government have made it possible for an HIV-infected person to live a nearly normal life. I will summarize the elegant structures that have been determined of components of HIV, with an emphasis on the enzyme RT, which my laboratory has studied since 1987. HIV-1 RT is responsible for converting the viral 10-kilobase single-stranded RNA genome to double-stranded DNA. This fascinating and essential enzyme is the target of 13 approved anti-AIDS drugs: 8 nucleoside analog RT inhibitors (NRTIs) and 5 non-nucleoside RT inhibitors (NNRTIs). We have determined crystal structures of wild-type and drug-resistant RTs in complexes with nucleic acid and/or inhibitors. We participated in structure-guided discovery and development of two anti-AIDS drugs with exceptional potency against drug-resistant variants. Crystal structures combined with biochemical data help to elucidate intriguing molecular mechanisms by which HIV-1 develops resistance to different anti-AIDS drugs. Recent crystallographic fragment screening has revealed new allosteric inhibitory binding pockets for future drug discovery. I am very grateful to my many co-workers, colleagues, and friends for their contributions, synchrotron resources at CHESS, BNLS, and APS, and generous funding from NIH in support of research on HIV-1 RT.

FLT3 (FMS-like tyrosine kinase 3), a receptor tyrosine kinase, is frequently mutated in acute myeloid leukemia (AML), a hematologic malignancy marked by aggressive proliferation, poor prognosis, and high relapse rates. Although FDA-approved FLT3 inhibitors exist, their clinical efficacy is often undermined by resistance and off-target effects, underscoring the critical necessity for more effective and selective agents. Here, we employed a structure-based computational approach combining pharmacophore screening via Pharmit and the MolPort compound library to identify novel FLT3 inhibitors. Pharmacophore modeling, virtual screening, and docking identified two promising leads, MolPort-002-705-878 and MolPort-007-550-904, with binding affinities of –11.33 and –10.66 kcal/mol, correspondingly. These compounds were further evaluated using molecular dynamics (MD) simulations to assess binding stability, density functional theory (DFT) calculations to explore electronic reactivity, and ADMET profiling to examine pharmacokinetic and toxicity parameters. MD results, including principal component analysis (PCA) and free energy landscape (FEL) mapping, supported the integrity of the FLT3–lead complexes, with MM/GBSA binding free energies () of –39.23 kcal/mol and –27.03 kcal/mol for MolPort-002-705-878 and MolPort-007-550-904, respectively. DFT analysis indicated favorable frontier molecular orbital energies and reactivity indices, characterized by a low HOMO–LUMO energy gap and a reactive dipole moment. ADMET predictions indicated acceptable drug-likeness and low toxicity, pending further experimental confirmation. This integrated in silico pipeline highlights the therapeutic potential of these molecules as next-generation FLT3 inhibitors and offers a scalable strategy for targeted AML therapeutics.

The RAS-RAF-MEK-ERK signaling cascade is abnormally activated in various tumors, playing a crucial role in mediating tumor progression. As the key component at the terminal stage of this cascade, ERK1/2 emerges as a potential antitumor target and offers a promising therapeutic strategy for tumors harboring BRAF or RAS mutations. Here, we identified 36c with a (thiophen-3-yl)aminopyrimidine scaffold as a potent ERK1/2 inhibitor through structure-guided optimization for hit 18. In preclinical studies, 36c showed powerful ERK1/2 inhibitory activities (ERK1/2 IC50 = 0.11/0.08 nM) and potent antitumor efficacy both in vitro and in vivo against triple-negative breast cancer and colorectal cancer models harboring BRAF and RAS mutations. 36c could directly inhibit ERK1/2, significantly block the phosphorylation expression of their downstream substrates p90RSK and c-Myc, and induce cell apoptosis and incomplete autophagy-related cell death. Taken together, this work provides a promising ERK1/2 lead compound for multiple tumor-treatment drug discovery.

We report the structure-guided discovery, synthesis, and initial characterization of 3,5-diamino-piperidinyl triazines (DAPT), a novel translation inhibitor class that targets bacterial rRNA and exhibits broad-spectrum antibacterial activity. DAPT compounds were designed as structural mimetics of aminoglycoside antibiotics which bind to the bacterial ribosomal decoding site and thereby interfere with translational fidelity. We found that DAPT compounds bind to oligonucleotide models of decoding-site RNA, inhibit translation in vitro, and induce translation misincorporation in vivo, in agreement with a mechanism of action at the ribosomal decoding site. The novel DAPT antibacterials inhibit growth of gram-positive and gram-negative bacteria, including the respiratory pathogen Pseudomonas aeruginosa, and display low toxicity to human cell lines. In a mouse protection model, an advanced DAPT compound demonstrated efficacy against an Escherichia coli infection at a 50% protective dose of 2.4 mg/kg of body weight by single-dose intravenous administration.

Cystic Fibrosis Trust (Registered as a charity in England and Wales (1079049) and in Scotland (SC040196)

Supplementary material from "Structure-guided fragment-based drug discovery at the synchrotron: screening binding sites and correlations with hotspot mapping"

All viruses depend on host cell proteins for replication. Denying viruses' access to the function of critical host proteins can result in antiviral activity against multiple virus families. In particular, small-molecule drug candidates which inhibit the α-glucosidase enzymes of the endoplasmic reticulum (ER) translation quality control (QC) pathway have demonstrated broad-spectrum antiviral activities and low risk for development of viral resistance. However, antiviral drug discovery focused on the ERQC enzyme α-glucosidase I (α-GluI) has been hampered by difficulties in obtaining crystal structures of complexes with inhibitors. We report here the identification of an orthologous enzyme from a thermophilic fungus, Chaetomium thermophilum (Ct), as a robust surrogate for mammalian ER α-GluI and a platform for inhibitor design. Previously annotated only as a hypothetical protein, the Ct protein was validated as a bona fide α-glucosidase by comparing its crystal structure to that of mammalian α-GluI, by demonstrating enzymatic activity on the unusual α-d-Glcp-(1 → 2)-α-d-Glcp-(1 → 3) substrate glycan, and by showing that well-known inhibitors of mammalian α-GluI (1-DNJ, UV-4, UV-5) also inhibit Ct α-GluI. Crystal structures of Ct α-GluI in complex with three such inhibitors (UV-4, UV-5, EB-0159) revealed extensive interactions with all four enzyme subsites and provided insights into the catalytic mechanism. Identification of ER Ct α-GluI as a surrogate for mammalian α-GluI will accelerate the structure-guided discovery of broad-spectrum antivirals. This study also highlights Ct as a source of thermostable eukaryotic proteins, such as ER α-Glu I, that lack orthologs in bacterial or archaeal thermophiles.

In this study we explored the pharmaceutically underexploited ATPase domain of DNA gyrase (GyrB) as a potential platform for developing novel agents that target Mycobacterium tuberculosis. In this effort a combination of ligand- and structure-based pharmacophore modeling was used to identify structurally diverse small-molecule inhibitors of the mycobacterial GyrB domain based on the crystal structure of the enzyme with a pyrrolamide inhibitor (PDB ID: 4BAE). Pharmacophore modeling and subsequent in vitro screening resulted in an initial hit compound 5 [(E)-5-(5-(2-(1H-benzo[d]imidazol-2-yl)-2-cyanovinyl)furan-2-yl)isophthalic acid; IC50 =4.6±0.1 μm], which was subsequently tailored through a combination of molecular modeling and synthetic chemistry to yield the optimized lead compound 24 [(E)-3-(5-(2-cyano-2-(5-methyl-1H-benzo[d]imidazol-2-yl)vinyl)thiophen-2-yl)benzoic acid; IC50 =0.3±0.2 μm], which was found to display considerable in vitro efficacy against the purified GyrB enzyme and potency against the H37 Rv strain of M. tuberculosis. Structural handles were also identified that will provide a suitable foundation for further optimization of these potent analogues.

Aflatoxins (AFs) are highly carcinogenic metabolites produced by Aspergillus species that can contaminate critical food staples, leading to significant health and economic risks. The cytochrome P450 monooxygenase AflG catalyzes an early step in AF biosynthesis, resulting in the conversion of averantin (AVN) to 5'-hydroxy-averantin. However, the molecular mechanism underlying the AflG-AVN interaction remains unclear. Here, we sought to understand the structural features of AflG in complex with AVN to enable the identification of inhibitors targeting the AflG binding pocket. To achieve this goal, we employed a comprehensive approach combining computational and experimental methods. Structural modeling and microsecond-scale molecular dynamics (MD) simulations yielded new insights into AflG architecture and unveiled unique ligand binding conformations of the AflG-AVN complex. High-throughput virtual screening of more than 1.3 million compounds pinpointed specific subsets with favorable predicted docking scores. The resulting compounds were ranked based on binding free energy calculations and evaluated with MD simulations and in vitro experiments with Aspergillus flavus. Our results revealed two compounds significantly inhibited AF biosynthesis. Comprehensive structural analysis elucidated the binding sites of competitive inhibitors and demonstrated their regulation of AflG dynamics. This structure-guided pipeline successfully enabled the identification of novel AflG inhibitors and provided novel molecular insights that will guide future efforts to develop effective therapeutics that prevent AF contamination.

The cardiac voltage-gated sodium channel Nav1.5 is resistant to tetrodotoxin (TTXr). Here, we report a cryo-electron microscopy (cryo-EM) structure of wild-type human Nav1.5, coexpressed with the β1 auxiliary subunit and treated with high-concentration TTX, at 3.4 Å resolution. Structural comparison reveals the molecular determinants for the distinct responses to TTX as well as β subunits between TTXr and TTX-sensitive (TTXs) Nav channels. A conserved cation-π interaction between the guanidinium group of TTX and Tyr or Phe on the P2I helix in TTXs Nav channels is lost in all TTXr subtypes owing to the replacement by Cys/Ser at the corresponding locus, explaining their differential TTX sensitivities. The β1 subunit is invisible in the EM map. Comparison of Nav1.5 with Nav1.7 and Nav1.8, which are, respectively, TTXs and TTXr, identifies four sites on the extracellular loops (ECLs) that may account for their different β1-binding abilities. When the corresponding residues in TTXs Nav1.7 are replaced with those from Nav1.5, the modulatory effects of β1 on channel activation and inactivation are diminished. Consistently, β1 is absent in the 3D EM reconstruction of this Nav1.7 mutant. Together with our previous structure-guided discovery that TTXr channels lack a Cys on the ECLII for disulfide bond formation with β2 or β4, the structure-function relationship studies underscore the importance of the ECLs in the mechanistic distinctions between TTXs and TTXr Nav channels. The ECLs may be further explored for the development of subtype-specific drugs.

Structural biology is experiencing a paradigm shift from targeted structural determination to structure-guided discovery of previously uncharacterized bioentities. We employed cryoelectron microscopy (cryo-EM) to analyze filtered water samples collected from the Tsinghua Lotus Pond. Here, we report the structural determination and characterization of two highly similar helical fibrils, named TLP-1a and TLP-1b, each approximately 8 nm in diameter with a 15-Å wide tunnel. These fibrils are assembled from a similar protein protomer, whose structure was conveniently automodeled in CryoNet. The protomer structure does not match any available experimental structures, but shares the same fold as many predicted structures of unknown functions. The amino-terminal β strand of protomer n + 4 inserts into a cleft in protomer n to complete an immunoglobulin (Ig)-like domain. This packing mechanism, known as donor-strand exchange (DSE), has been observed in several bacterial pilus assemblies, wherein the donor is protomer n + 1. Despite distinct shape and thickness, this reminiscence suggests that TLP-1a/b fibrils may represent uncharacterized bacterial pili. Our study demonstrates an emerging paradigm in structural biology, where high-resolution structural determination precedes and drives the identification and characterization of completely unknown objects.

Structure-guided discovery of selective USP7 inhibitors with in vivo activity