Database-Driven Identification of Structurally Similar Protein-Protein Interfaces.

Analyzing the similarity of protein interfaces in protein-protein interactions gives new insights into protein function and assists in discovering new drugs. Usually, tools that assess the similarity focus on the interactions between two protein interfaces, while sometimes we only have one predicted interface. Herein, we present PiMine, a database-driven protein interface similarity search. It compares interface residues of one or two interacting chains by calculating and searching tetrahedral geometric patterns of α-carbon atoms and calculating physicochemical and shape-based similarity. On a dedicated, tailor-made dataset, we show that PiMine outperforms commonly used comparison tools in terms of early enrichment when considering interfaces of sequentially and structurally unrelated proteins. In an application example, we demonstrate its usability for protein interaction partner prediction by comparing predicted interfaces to known protein-protein interfaces.

Redocking the PDB.

Molecular docking is a standard technique in structure-based drug design (SBDD). It aims to predict the 3D structure of a small molecule in the binding site of a receptor (often a protein). Despite being a common technique, it often necessitates multiple tools and involves manual steps. Here, we present the JAMDA preprocessing and docking workflow that is easy to use and allows fully automated docking. We evaluate the JAMDA docking workflow on binding sites extracted from the complete PDB and derive key factors determining JAMDA's docking performance. With that, we try to remove most of the bias due to manual intervention and provide a realistic estimate of the redocking performance of our JAMDA preprocessing and docking workflow for any PDB structure. On this large PDBScan22 data set, our JAMDA workflow finds a pose with an RMSD of at most 2 Å to the crystal ligand on the top rank for 30.1% of the structures. When applying objective structure quality filters to the PDBScan22 data set, the success rate increases to 61.8%. Given the prepared structures from the JAMDA preprocessing pipeline, both JAMDA and the widely used AutoDock Vina perform comparably on this filtered data set (the PDBScan22-HQ data set).

User-centric design of a 3D search interface for protein-ligand complexes.

In this work, we present the frontend of GeoMine and showcase its application, focusing on the new features of its latest version. GeoMine is a search engine for ligand-bound and predicted empty binding sites in the Protein Data Bank. In addition to its basic text-based search functionalities, GeoMine offers a geometric query type for searching binding sites with a specific relative spatial arrangement of chemical features such as heavy atoms and intermolecular interactions. In contrast to a text search that requires simple and easy-to-formulate user input, a 3D input is more complex, and its specification can be challenging for users. GeoMine's new version aims to address this issue from the graphical user interface perspective by introducing an additional visualization concept and a new query template type. In its latest version, GeoMine extends its query-building capabilities primarily through input formulation in 2D. The 2D editor is fully synchronized with GeoMine's 3D editor and provides the same functionality. It enables template-free query generation and template-based query selection directly in 2D pose diagrams. In addition, the query generation with the 3D editor now supports predicted empty binding sites for AlphaFold structures as query templates. GeoMine is freely accessible on the ProteinsPlus web server ( https://proteins.plus ).

SpaceGrow: efficient shape-based virtual screening of billion-sized combinatorial fragment spaces.

The growing size of make-on-demand chemical libraries is posing new challenges to cheminformatics. These ultra-large chemical libraries became too large for exhaustive enumeration. Using a combinatorial approach instead, the resource requirement scales approximately with the number of synthons instead of the number of molecules. This gives access to billions or trillions of compounds as so-called chemical spaces with moderate hardware and in a reasonable time frame. While extremely performant ligand-based 2D methods exist in this context, 3D methods still largely rely on exhaustive enumeration and therefore fail to apply. Here, we present SpaceGrow: a novel shape-based 3D approach for ligand-based virtual screening of billions of compounds within hours on a single CPU. Compared to a conventional superposition tool, SpaceGrow shows comparable pose reproduction capacity based on RMSD and superior ranking performance while being orders of magnitude faster. Result assessment of two differently sized subsets of the eXplore space reveals a higher probability of finding superior results in larger spaces highlighting the potential of searching in ultra-large spaces. Furthermore, the application of SpaceGrow in a drug discovery workflow was investigated in four examples involving G protein-coupled receptors (GPCRs) with the aim to identify compounds with similar binding capabilities and molecular novelty.

ProteinsPlus: a comprehensive collection of web-based molecular modeling tools.

Upon the ever-increasing number of publicly available experimentally determined and predicted protein and nucleic acid structures, the demand for easy-to-use tools to investigate these structural models is higher than ever before. The ProteinsPlus web server (https://proteins.plus) comprises a growing collection of molecular modeling tools focusing on protein-ligand interactions. It enables quick access to structural investigations ranging from structure analytics and search methods to molecular docking. It is by now well-established in the community and constantly extended. The server gives easy access not only to experts but also to students and occasional users from the field of life sciences. Here, we describe its recently added new features and tools, beyond them a novel method for on-the-fly molecular docking and a search method for single-residue substitutions in local regions of a protein structure throughout the whole Protein Data Bank. Finally, we provide a glimpse into new avenues for the annotation of AlphaFold structures which are directly accessible via a RESTful service on the ProteinsPlus web server.

SiteMine: Large-scale binding site similarity searching in protein structure databases.

Drug discovery and design challenges, such as drug repurposing, analyzing protein-ligand and protein-protein complexes, ligand promiscuity studies, or function prediction, can be addressed by protein binding site similarity analysis. Although numerous tools exist, they all have individual strengths and drawbacks with regard to run time, provision of structure superpositions, and applicability to diverse application domains. Here, we introduce SiteMine, an all-in-one database-driven, alignment-providing binding site similarity search tool to tackle the most pressing challenges of binding site comparison. The performance of SiteMine is evaluated on the ProSPECCTs benchmark, showing a promising performance on most of the data sets. The method performs convincingly regarding all quality criteria for reliable binding site comparison, offering a novel state-of-the-art approach for structure-based molecular design based on binding site comparisons. In a SiteMine showcase, we discuss the high structural similarity between cathepsin L and calpain 1 binding sites and give an outlook on the impact of this finding on structure-based drug design. SiteMine is available at https://uhh.de/naomi.

Binding Site Detection Remastered: Enabling Fast, Robust, and Reliable Binding Site Detection and Descriptor Calculation with DoGSite3.

Binding site prediction on protein structures is a crucial step in early phase drug discovery whenever experimental or predicted structure models are involved. DoGSite belongs to the widely used tools for this task. It is a grid-based method that uses a Difference-of-Gaussian filter to detect cavities on the protein surface. We recently reimplemented the first version of this method, released in 2010, focusing on improved binding site detection in the presence of ligands and optimized parameters for more robust, reliable, and fast predictions and binding site descriptor calculations. Here, we introduce the new version, DoGSite3, compare it to its predecessor, and re-evaluate DoGSite on published data sets for a large-scale comparative performance evaluation.

SCOT: Rethinking the classification of secondary structure elements.

MOTIVATION: Secondary structure classification is one of the most important issues in structure-based analyses due to its impact on secondary structure prediction, structural alignment and protein visualization. There are still open challenges concerning helix and sheet assignments which are currently not addressed by a single multi-purpose software. RESULTS: We introduce SCOT (Secondary structure Classification On Turns) as a novel secondary structure element assignment software which supports the assignment of turns, right-handed α-, 310- and π-helices, left-handed α- and 310-helices, 2.27- and polyproline II helices, ß-sheets and kinks. We demonstrate that the introduction of helix Purity values enables a clear differentiation between helix classes. SCOT's unique strengths are highlighted by comparing it to six state-of-the-art methods (DSSP, STRIDE, ASSP, SEGNO, DISICL and SHAFT). The assignment approaches were compared concerning geometric consistency, protein structure quality and flexibility dependency and their impact on secondary structure element-based structural alignments. We show that only SCOT's combination of hydrogen bonds, geometric criteria and dihedral angles enables robust assignments independent of the structure quality and flexibility. We demonstrate that this combination and the elaborate kink detection lead to SCOT's clear superiority for protein alignments. As the resulting helices and strands are provided in a PDB conform output format, they can immediately be used for structure alignment algorithms. Taken together, the application of our new method and the straight-forward visualization using the accompanying PyMOL scripts enable the comprehensive analysis of regular backbone geometries in proteins. AVAILABILITY AND IMPLEMENTATION: https://this-group.rocks. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Optical Control of Transcription: Genetically Encoded Photoswitchable Variants of T7 RNA Polymerase.

Light-sensing protein domains that link an exogenous light signal to the activity of an enzyme have attracted much attention for the engineering of new regulatory mechanisms into proteins and for studying the dynamic behavior of intracellular reactions and reaction cascades. Light-oxygen-voltage (LOV) photoreceptors are blue-light-sensing modules that have been intensely characterized for this purpose and linked to several proteins of interest. For the successful application of these tools, it is crucial to identify appropriate fusion strategies for combining sensor and enzyme domains that sustain activity and light-induced responsivity. Terminal fusion of LOV domains is the natural strategy; however, this is not transferrable to T7 RNA polymerase because both of its termini are involved in catalysis. It is shown herein that it is possible to covalently insert LOV domains into the polymerase protein, while preserving its activity and generating new light-responsive allosteric coupling.

A benchmark driven guide to binding site comparison: An exhaustive evaluation using tailor-made data sets (ProSPECCTs).

The automated comparison of protein-ligand binding sites provides useful insights into yet unexplored site similarities. Various stages of computational and chemical biology research can benefit from this knowledge. The search for putative off-targets and the establishment of polypharmacological effects by comparing binding sites led to promising results for numerous projects. Although many cavity comparison methods are available, a comprehensive analysis to guide the choice of a tool for a specific application is wanting. Moreover, the broad variety of binding site modeling approaches, comparison algorithms, and scoring metrics impedes this choice. Herein, we aim to elucidate strengths and weaknesses of binding site comparison methodologies. A detailed benchmark study is the only possibility to rationalize the selection of appropriate tools for different scenarios. Specific evaluation data sets were developed to shed light on multiple aspects of binding site comparison. An assembly of all applied benchmark sets (ProSPECCTs-Protein Site Pairs for the Evaluation of Cavity Comparison Tools) is made available for the evaluation and optimization of further and still emerging methods. The results indicate the importance of such analyses to facilitate the choice of a methodology that complies with the requirements of a specific scientific challenge.

Significance of Individual Residues at the Regulatory Site of Yeast Pyruvate Decarboxylase for Allosteric Substrate Activation.

The catalytic activity of the allosteric enzyme pyruvate decarboxylase from yeast is strictly controlled by its own substrate pyruvate via covalent binding at a separate regulatory site. Kinetic studies, chemical modifications, cross-linking, small-angle X-ray scattering, and crystal structure analyses have led to a detailed understanding of the substrate activation mechanism at an atomic level with C221 as the core moiety of the regulatory site. To characterize the individual role of the residues adjacent to C221, we generated variants H92F, H225F, H310F, A287G, S311A, and C221A/C222A. The integrity of the protein structure of the variants was established by small-angle X-ray scattering measurements. The analyses of both steady state and transient kinetic data allowed the identification of the individual roles of the exchanged side chains during allosteric enzyme activation. In each case, the kinetic pattern of activation was modulated but not completely abolished. Despite the crucial role of C221, the covalent binding of pyruvate is not obligate for enzyme activation but is a requirement for a kinetically efficient transition from the inactive to the active state. Moreover, only one of the three histidines guiding the activator molecule to the binding pocket, H310, specifically interacts with C221. H310 stabilizes the thiolate form of C221, ensuring a rapid nucleophilic attack of the thiolate sulfur on C2 of the regulatory pyruvate, thus forming a regulatory dyad. The influence of the other two histidines is less pronounced. Substrate activation is slightly weakened for A287G and significantly retarded for S311A.

Small-Molecule Inhibition of the UNC119-Cargo Interaction.

N-Terminal myristoylation facilitates membrane binding and activity of proteins, in particular of Src family kinases, but the underlying mechanisms are only beginning to be understood. The chaperones UNC119A/B regulate the cellular distribution and signaling of N-myristoylated proteins. Selective small-molecule modulators of the UNC119-cargo interaction would be invaluable tools, but have not been reported yet. We herein report the development of the first UNC119-cargo interaction inhibitor, squarunkin A. Squarunkin A selectively inhibits the binding of a myristoylated peptide representing the N-terminus of Src kinase to UNC119A with an IC50 value of 10 nm. It binds to UNC119 proteins in cell lysate and interferes with the activation of Src kinase. Our results demonstrate that small-molecule inhibition of the UNC119-cargo interaction might provide new opportunities for modulating the activity of Src kinases that are independent of direct inhibition of the enzymatic kinase activity.

One-Pot N2C/C2C/N2N Ligation To Trap Weak Protein-Protein Interactions.

Weak transient protein-protein interactions (PPIs) play an essential role in cellular dynamics. However, it is challenging to obtain weak protein complexes owing to their short lifetime. Herein we present a general and facile method for trapping weak PPIs in an unbiased manner using proximity-induced ligations. To expand the chemical ligation spectrum, we developed novel N2N (N-terminus to N-terminus) and C2C (C-terminus to C-terminus) ligation approaches. By using N2C (N-terminus to C-terminus), N2N, and C2C ligations in one pot, the interacting proteins were linked. The weak Ypt1:GDI interaction drove C2C ligation with t1/2 of 4.8 min and near quantitative conversion. The Ypt1-GDI conjugate revealed that binding of Ypt1 G-domain causes opening of the lipid-binding site of GDI, which can accommodate one prenyl group, giving insights into Rab membrane recycling. Moreover, we used this strategy to trap the KRas homodimer, which plays an important role in Ras signaling.

Correction: A high-throughput effector screen identifies a novel small molecule scaffold for inhibition of ten-eleven translocation dioxygenase 2.

[This corrects the article DOI: 10.1039/D2MD00186A.].

Navigating large chemical spaces in early-phase drug discovery.

The size of actionable chemical spaces is surging, owing to a variety of novel techniques, both computational and experimental. As a consequence, novel molecular matter is now at our fingertips that cannot and should not be neglected in early-phase drug discovery. Huge, combinatorial, make-on-demand chemical spaces with high probability of synthetic success rise exponentially in content, generative machine learning models go hand in hand with synthesis prediction, and DNA-encoded libraries offer new ways of hit structure discovery. These technologies enable to search for new chemical matter in a much broader and deeper manner with less effort and fewer financial resources. These transformational developments require new cheminformatics approaches to make huge chemical spaces searchable and analyzable with low resources, and with as little energy consumption as possible. Substantial progress has been made in the past years with respect to computation as well as organic synthesis. First examples of bioactive compounds resulting from the successful use of these novel technologies demonstrate their power to contribute to tomorrow's drug discovery programs. This article gives a compact overview of the state-of-the-art.

LifeSoaks: a tool for analyzing solvent channels in protein crystals and obstacles for soaking experiments.

Due to the structural complexity of proteins, their corresponding crystal arrangements generally contain a significant amount of solvent-occupied space. These areas allow a certain degree of intracrystalline protein flexibility and mobility of solutes. Therefore, knowledge of the geometry of solvent-filled channels and cavities is essential whenever the dynamics inside a crystal are of interest. Especially in soaking experiments for structure-based drug design, ligands must be able to traverse the crystal solvent channels and reach the corresponding binding pockets. Unsuccessful screenings are sometimes attributed to the geometry of the crystal packing, but the underlying causes are often difficult to understand. This work presents LifeSoaks, a novel tool for analyzing and visualizing solvent channels in protein crystals. LifeSoaks uses a Voronoi diagram-based periodic channel representation which can be efficiently computed. The size and location of channel bottlenecks, which might hinder molecular diffusion, can be directly derived from this representation. This work presents the calculated bottleneck radii for all crystal structures in the PDB and the analysis of a new, hand-curated data set of structures obtained by soaking experiments. The results indicate that the consideration of bottleneck radii and the visual inspection of channels are beneficial for planning soaking experiments.

Fragtory: Pharmacophore-Focused Design, Synthesis, and Evaluation of an sp3-Enriched Fragment Library.

Fragment-based drug discovery has played an important role in medicinal chemistry and pharmaceutical research. Despite numerous demonstrated successes, the limited diversity and overrepresentation of planar, sp2-rich structures in commercial libraries often hamper the full potential of this approach. Hence, the thorough design of screening libraries inevitably determines the probability for meaningful hits and subsequent structural elaboration. Against this background, we present the generation of an exclusive fragment library based on iterative entry nomination by a specifically designed computational workflow: "Fragtory". Following a pharmacophore diversity-driven approach, we used Fragtory in an interdisciplinary academic setting to guide both tailored synthesis efforts and the implementation of in-house compounds to build a curated 288-member library of sp3-enriched fragments. Subsequent NMR screens against a model protein and hit validation by protein crystallography led to the identification of structurally novel ligands that were further characterized by isothermal titration calorimetry, demonstrating the applicability of our experimental approach.

Searching Geometric Patterns in Protein Binding Sites and Their Application to Data Mining in Protein Kinase Structures.

The ever-growing number of protein-ligand complex structures can give fundamental insights into protein functions and protein-ligand interactions, especially in the field of protein kinase research. The number of tools to mine this data for individually defined structural motifs is restricted due to the challenging task of developing efficient index structures for 3D data in relational databases. Herein we present GeoMine, a database system with web front-end mining of more than 900 000 binding sites. It enables database searches for geometric (interaction) patterns in protein-ligand interfaces by, for example, textual, numerical, substructure, similarity, and 3D searches. GeoMine processes reasonably selective user-defined queries within minutes. We demonstrate its usability for advancing protein kinase research with a special emphasis on unusual interactions, their use in designing selective kinase inhibitors, and the analysis of reactive cysteine residues that are amenable to covalent kinase inhibitors. GeoMine is freely available as part of our modeling support server at https://proteins.plus.

Navigating chemical reaction space - application to DNA-encoded chemistry.

Databases contain millions of reactions for compound synthesis, rendering selection of reactions for forward synthetic design of small molecule screening libraries, such as DNA-encoded libraries (DELs), a big data challenge. To support reaction space navigation, we developed the computational workflow Reaction Navigator. Reaction files from a large chemistry database were processed using the open-source KNIME Analytics Platform. Initial processing steps included a customizable filtering cascade that removed reactions with a high probability to be incompatible with DEL, as they would e.g. damage the genetic barcode, to arrive at a comprehensive list of transformations for DEL design with applicability potential. These reactions were displayed and clustered by user-defined molecular reaction descriptors which are independent of reaction core substitution patterns. Thanks to clustering, these can be searched manually to identify reactions for DEL synthesis according to desired reaction criteria, such as ring formation or sp3 content. The workflow was initially applied for mapping chemical reaction space for aromatic aldehydes as an exemplary functional group often used in DEL synthesis. Exemplary reactions have been successfully translated to DNA-tagged substrates and can be applied to library synthesis. The versatility of the Reaction Navigator was then shown by mapping reaction space for different reaction conditions, for amines as a second set of starting materials, and for data from a second database.

A high-throughput effector screen identifies a novel small molecule scaffold for inhibition of ten-eleven translocation dioxygenase 2.

Ten-eleven translocation dioxygenases (TETs) are the erasers of 5-methylcytosine (mC), the central epigenetic regulator of mammalian DNA. TETs convert mC to three oxidized derivatives with unique physicochemical properties and inherent regulatory potential, and it initializes active demethylation by the base excision repair pathway. Potent small molecule inhibitors would be useful tools to study TET functions by conditional control. To facilitate the discovery of such tools, we here report a high-throughput screening pipeline and its application to screen and validate 31.5k compounds for inhibition of TET2. Using a homogenous fluorescence assay, we discover a novel quinoline-based scaffold that we further validate with an orthogonal semi-high throughput MALDI-MS assay for direct monitoring of substrate turnover. Structure-activity relationship (SAR) studies involving >20 derivatives of this scaffold led to the identification of optimized inhibitors, and together with computational studies suggested a plausible model for its mode of action.