Structure-Based De Novo Design for the Discovery of Miniprotein Inhibitors Targeting Oncogenic Mutant BRAF.

Being a component of the Ras/Raf/MEK/ERK signaling pathway crucial for cellular responses, the VRAF murine sarcoma viral oncogene homologue B1 (BRAF) kinase has emerged as a promising target for anticancer drug discovery due to oncogenic mutations that lead to pathway hyperactivation. Despite the discovery of several small-molecule BRAF kinase inhibitors targeting oncogenic mutants, their clinical utility has been limited by challenges such as off-target effects and suboptimal pharmacological properties. This study focuses on identifying miniprotein inhibitors for the oncogenic V600E mutant BRAF, leveraging their potential as versatile drug candidates. Using a structure-based de novo design approach based on binding affinity to V600E mutant BRAF and hydration energy, 39 candidate miniprotein inhibitors comprising three helices and 69 amino acids were generated from the substructure of the endogenous ligand protein (14-3-3). Through in vitro binding and kinase inhibition assays, two miniproteins (63 and 76) were discovered as novel inhibitors of V600E mutant BRAF with low-micromolar activity, with miniprotein 76 demonstrating a specific impediment to MEK1 phosphorylation in mammalian cells. These findings highlight miniprotein 76 as a potential lead compound for developing new cancer therapeutics, and the structural features contributing to its biochemical potency against V600E mutant BRAF are discussed in detail.

Protein purification, crystallization, and structure determination of transcription factor YhaJ in complex with DNT metabolites.

The microbial transcription factor YhaJ responds to 2,4-dinitrotoluene (DNT) derivatives. Here, we describe steps for overexpression and purification of the protein, characterization for the binding of a DNT derivative methylhydroquinone, and crystallization by using a random seeding technique. We then detail procedures for structure determination by employing the crystal-twin resolving processes. This protocol can also be performed using other DNT derivatives. For complete details on the use and execution of this protocol, please refer to Kim et al.1.

Structural basis of transcription factor YhaJ for DNT detection.

Detection of landmines without harming personnel is a global issue. The bacterial transcription factor YhaJ selectively detects metabolites of explosives, and it can be used as a key component of DNT biosensors. However, the wild-type YhaJ has a binding affinity that is not sufficient for the detection of trace amounts of explosives leaked from landmines buried in the soil. Here, we report crystal structures of the effector-binding domain of YhaJ in both the apo- and effector-bound forms. A structural comparison of the two forms revealed that the loop above the primary effector-binding site significantly switches its conformation upon effector binding. The primary effector-binding site involves hydrophobic and polar interactions, having specificity to hydroxyl-substituted benzene compounds. The structures explain the mechanism of activity-enhancing mutations and provide information for the rational engineering of YhaJ biosensors for the sensitive detection of explosives.

Crystal structure of the catalytic domain of human RPTPH.

Receptor-type protein tyrosine phosphatases (RPTPs) receive extracellular stimuli and transfer them into cells. They regulate cell growth, differentiation and death via specific signals. They have also been implicated in cancer, diabetes and neurological diseases. RPTPH, a member of the type 3 RPTP (R3-PTP) family, is an important regulator of colorectal cancer and hepatic carcinoma. Despite its importance in drug development, the structure of RPTPH has not yet been resolved. Here, the crystal structure of the catalytic domain of RPTPH was determined at 1.56 Šresolution. Despite similarities to other R3-PTPs in its overall structure, RPTPH exhibited differences in its loop regions and side-chain conformations. Compared with other R3-PTPs, RPTPH has unique side chains near its active site that may confer specificity for inhibitor binding. Therefore, detailed information on the structure of RPTPH provides clues for the development of specific inhibitors.

A Dynamic Substrate Pool Revealed by cryo-EM of a Lipid-Preserved Respiratory Supercomplex.

Aims: Mitochondrial respiratory supercomplexes mediate redox electron transfer, generating a proton gradient for ATP synthesis. To provide structural information on the function of supercomplexes in physiologically relevant conditions, we conducted cryoelectron microscopy studies with supercomplexes in a lipid-preserving state. Results: Here, we present cryoelectron microscopy structures of bovine respiratory supercomplex I1III2IV1 by using a lipid-preserving sample preparation. The preparation greatly enhances the intercomplex quinone transfer activity. The structures reveal large intercomplex motions that result in different shapes and sizes of the intercomplex space between complexes I and III, forming a dynamic substrate pool. Biochemical and structural analyses indicated that intercomplex phospholipids mediate the intercomplex motions. An analysis of the different classes of focus-refined complex I showed that structural switches due to quinone reduction led to the formation of a novel channel that could transfer reduced quinones to the intercomplex substrate pool. Innovation and Conclusion: Our results indicate potential mechanism for the facilitated electron transfer involving a dynamic substrate pool and intercomplex movement by which supercomplexes play an active role in the regulation of metabolic flux and reactive oxygen species.

Synthesis and biological evaluation of acylthiourea against DUSP1 inhibition.

Structure based virtual screening attempts to discover DUSP1 inhibitors have yielded a scaffold featuring benzoxazole and acylthiourea pharmacophore. A series of its analogues were synthesized to explore structure activity relationship (SAR) of DUSP1 inhibition.

Two intermediate states of the conformational switch in dual specificity phosphatase 13a.

Dual specificity phosphatases (DUSPs) include MAP kinase phosphatases and atypical dual specificity phosphatases and mediate cell growth and differentiation, brain function, and immune responses. They serve as targets for drug development against cancers, diabetes and depression. Several DUSPs have non-canonical conformation of the central ß-sheet and active site loops, suggesting that they may have conformational switch that is related to the regulation of enzyme activity. Here, we determined the crystal structure of DUSP13a, and identified two different structures that represent intermediates of the postulated conformational switch. Amino acid sequence of DUSP13a is not significantly homologous to DUSPs with conformational switch, indicating that the conformational switch is not sequence-dependent, but rather determined by ligand interaction. The sequence-independency suggests that other DUSPs with canonical conformation may have the conformational switch during specific cellular regulation. The conformational switch leads to significant changes in the protein surface, including a hydrophobic surface and pockets, which can be exploited for development of allosteric modulators of drug target DUSPs.

Structural and biochemical analysis of atypically low dephosphorylating activity of human dual-specificity phosphatase 28.

Dual-specificity phosphatases (DUSPs) constitute a subfamily of protein tyrosine phosphatases, and are intimately involved in the regulation of diverse parameters of cellular signaling and essential biological processes. DUSP28 is one of the DUSP subfamily members that is known to be implicated in the progression of hepatocellular and pancreatic cancers, and its biological functions and enzymatic characteristics are mostly unknown. Herein, we present the crystal structure of human DUSP28 determined to 2.1 Å resolution. DUSP28 adopts a typical DUSP fold, which is composed of a central ß-sheet covered by α-helices on both sides and contains a well-ordered activation loop, as do other enzymatically active DUSP proteins. The catalytic pocket of DUSP28, however, appears hardly accessible to a substrate because of the presence of nonconserved bulky residues in the protein tyrosine phosphatase signature motif. Accordingly, DUSP28 showed an atypically low phosphatase activity in the biochemical assay, which was remarkably improved by mutations of two nonconserved residues in the activation loop. Overall, this work reports the structural and biochemical basis for understanding a putative oncological therapeutic target, DUSP28, and also provides a unique mechanism for the regulation of enzymatic activity in the DUSP subfamily proteins.