Fragment-based ligand discovery (FBLD) is one of the most successful approaches to designing small-molecule protein–protein interaction (PPI) inhibitors. The incorporation of computational tools to FBLD allows the exploration of chemical space in a time- and cost-efficient manner. Herein, a computational protocol for the development of small-molecule PPI inhibitors using fragment hopping, a fragment-based de novo design approach, is described and a case study is presented to illustrate the efficiency of this protocol. Fragment hopping facilitates the design of PPI inhibitors from scratch solely based on key binding features in the PPI complex structure. This approach is an open system that enables the inclusion of different state-of-the-art programs and
softwares to improve its performances.
Protein–protein interactions (PPIs) play a crucial role in regulating biological pathways with more than 40,000 PPIs experimentally validated in the human body and even more predicted.The size, shape, and chemical characteristics of PPI interfaces differ from those of traditional drug targets, such as enzymes and receptors. The traditional targets display a substrate binding pocket with deep, well-defined shape (with an average of 260 Å3) containing residues that are ready for binding and/or catalyzing the substrate. In contrast, PPI binding sites have multiple flat, shallow pockets (typically 54 Å3) devoid of catalytic residues.These reflect on lacking the native small-molecule binding partner to serve as the starting point for hit discovery and optimization. When taken as a whole, these compounding factors have resulted in great challenges in developing small-molecule PPI inhibitors and such, few PPI inhibitors have advanced to clinical trials. Regardless of these problems, PPIs can greatly expand the druggable genome and represents a class of important potential targets. Hence, new technologies need to be developed to address these challenges.
The workflow of fragment hopping for designing small-molecule PPI inhibitors includes four major steps: 1) detection of minimal pharmacophoric elements, 2) fragment hopping, 3) scaffold construction, and 4) scaffold decoration and assessment. The starting point of fragment hopping is the PPI complex crystal or NMR structures. After separating the target protein from the ligand protein, fragment hopping is adopted to design small-molecule PPI inhibitors. In the transition between steps 1 and 2, the constructed basic fragment library will be used to identify fragments that match minimal pharmacophoric elements. To construct the PPI inhibitor scaffold, basic fragment, bioisostere, linker, and ADME/Tox libraries will be interrogated with the assistance of SciFinder® to design synthetically accessible molecules with appropriate metabolic stability and low toxicity. The same set of libraries will be examined with the assistance of SciFinder during the transition between steps 3 and 4. Below are the discussion on the preferred programs, softwares, and databases for the development of small-molecule PPI inhibitors and details on how to use specific programs. All default parameters and settings are used unless stated otherwise.
While the phenotypic screening and subsequent target identification is an exciting method to discover inhibitors for new PPI targets that drive the desired phenotype, macrocycles and natural product-inspired libraries are a rich source to approach PPI inhibitors, and targeted protein degradation, such as the use of proteolysis targeting chimeras (PROTACs)or molecular glues, has become an emerging field to downregulate aberrant PPIs, one strategy to overcome the challenges presented by PPIs is rational design by targeting PPI hot spots that often congregate into hot regions.A hot spot at the PPI interface is defined as a residue that exhibits the free energy of binding (ΔΔGbinding) > 1.5 kcal/mol when examined by alanine mutation studies. In a PPI complex structure, the hot region of the ligand protein packs against the hot region of the target protein. The hot spots in the target protein are organized into concave surface pockets which often help occlude solvent from hot spot pockets.The hot spots in the ligand protein function as protruding “anchor residues” to the target protein. Hence, hot spots provide an interesting and valuable starting point to design modulators for PPIs. In fact, hot spots have been successfully used in structure-based design, peptidomimetic design, and focused high-throughput screening including virtual screening of PPI inhibitors. Among them, the most successful approach is fragment-based ligand discovery (FBLD).
FBLD identifies low-molecular-weight fragments that bind to PPI target proteins.A fragment is defined as a small organic molecule that follows the Rule of Three (molecular weight < 300, ClogP ≤ 3, number of H-bond donors ≤ 3, number of H-bond acceptors ≤ 3, number of rotatable bonds ≤ 3, and polar surface area (PSA) ≤ 60 Å2). Yet, there have been challenges to this definition in particular for PPI targets, so this is not a firm restriction but a preliminary guideline. The starting point of FBLD is to obtain fragment hits. In experimental FBLD, nuclear magnetic resonance (such as SAR by NMR), X-ray crystallography, mass spectrometry, and surface plasmon resonance (SPR) spectroscopy methods are employed to identify fragment hits by screening fragment-size compound libraries. Fragment hits are then converted into a lead ligand via fragment growing, fragment linking, or in situ fragment assembly (in particular, tethering with extenders. Computational FBLD can also play important roles in discovering fragment hits.
They have developed a fragment hopping method to initiate discovery of fragment hits through fragment-based de novo design. Fragment hopping is a pharmacophore-driven strategy, with the core of this approach to derive minimal pharmacophoric elements for binding and then find fragments with different chemotypes to match the requirements of minimal pharmacophoric elements. In general, fragment hopping is advantageous as it can explore a wider swatch of chemical space than experimental FBLD leading to more effective fragment hits. After fragment growing and linking, the fragments in the lead molecule maintain the same spatial orientations as those in the minimal pharmacophoric elements, which can potentially lead to a higher success rate of fragment hit identification and optimization. As the pilot study, fragment hopping was successfully applied to develop highly potent and dual-selective small-molecule inhibitors for neuronal nitric oxide synthase (nNOS) Ever since, the concept of minimal pharmacophore has been adopted by FBLD laboratories in pharmaceutical companies.Fragment hopping expands to design new inhibitors for other enzymes.They have been applying fragment hopping to design PPI inhibitors using PPI hot spots as the starting point. Minimal pharmacophoric elements enables a new use of PPI hot spots and responding to the challenges presented by PPI targets. Herein, they disclose their fragment hopping protocol for the design of small-molecule PPI inhibitors.
Shelby R. Kell, Zhen Wang, Haitao Ji, Fragment hopping protocol for the design of small-molecule protein–protein interaction inhibitors, Bioorganic & Medicinal Chemistry, Volume 69, 2022, 116879,
ISSN 0968-0896, https://doi.org/10.1016/j.bmc.2022.116879.