Understanding the interaction of 14-3-3 proteins with hDMX and hDM2: a structural and biophysical study

p53 plays a critical role in regulating diverse biological processes: DNA repair, cell cycle arrest, apoptosis, and senescence. The p53 pathway has therefore served as the focus for drug-discovery efforts. p53 is negatively regulated by hDMX and hDM2; prior studies have identified 14-3-3 proteins as hDMX and hDM2 client proteins. 14-3-3 proteins are adaptor proteins that modulate localisation, degradation and interactions of their targets in response to phosphorylation. Thus 14-3-3 proteins may indirectly modulate the interaction between hDMX or hDM2 and p53 and represent potential targets for modulation of the p53 pathway. In this manuscript we report on the biophysical and structural characterization of peptide/protein interactions that are representative of the interaction between 14-3-3 and hDMX or hDM2. The data establish that proximal phosphosites spaced ~20-25 residues apart in both hDMX and hDM2 co-operate to facilitate high-affinity 14-3-3 binding and provide structural insight that can be utilized in future stabilizer/inhibitor discovery efforts.

BRET-based self-cleaving biosensors for SARS-CoV-2 3CLpro Inhibitor Discovery

The 3C-like protease (3CLpro) of SARS-CoV-2 is an attractive drug target for developing antivirals against SARS-CoV-2. A few small molecule inhibitors of 3CLpro are in clinical trials for COVID-19 treatments and more inhibitors are being developed. One limiting factor for 3CLpro inhibitors development is that the cellular activities of such inhibitors have to be evaluated in a Biosafety Level 3 (BSL-3) or BSL-4 laboratory. Here, we design genetically encoded biosensors that can be used in BSL-2 laboratories to set up cell-based assays for 3CLpro inhibitor discovery. The biosensors were constructed by linking a green fluorescent protein (GFP2) to the N-terminus and a Renilla luciferase (RLuc8) to the C-terminus of SARS-CoV-2 3CLpro, with the linkers derived from the cleavage sequences of 3CLpro. After over-expression of the biosensors in HEK293 cells, 3CLpro can be released from GFP2 and RLuc by self-cleavage, resulting in a decrease of the bioluminescence resonance energy transfer (BRET) signal. Using one of these biosensors, pBRET-10, we evaluated the cellular activities of several 3CLpro inhibitors. These inhibitors restored the BRET signal by blocking the proteolysis of pBRET-10, and their relative activities measured using pBRET-10 were consistent with their anti-SARS-CoV-2 activities reported previously. We conclude that the biosensor pBRET-10 is a useful tool for SARS-CoV-2 3CLpro inhibitor discovery. Furthermore, our strategy can be used to design biosensors for other viral proteases that share the same activation mechanism as 3CLpro, such as HIV protease PR and HCV protease NS3.

Virtual Evolution of HVEM Segment for Checkpoint Inhibitor Discovery

Immune therapy has emerged as an effective treatment against cancers. Inspired by the PD-1/PD-L1 antibodies, which have achieved great success in clinical, other immune checkpoint proteins have drawn increasing attention in cancer research. B and T lymphocyte attenuator (BTLA) and herpes virus entry mediator (HVEM) are potential targets for drug development. The co-crystal structure of BTLA/HVEM have revealed that HVEM (26–38) fragment is the core sequence which directly involved on the interface. Herein, we conducted virtual evolution with this sequence by using saturation mutagenesis in silico and mutants with lower binding energy were selected. Wet-lab experiments confirmed that several of them possessed higher affinity with BTLA. Based on the best mutant of the core sequence, extended peptides with better efficacy were obtained. Furthermore, the mechanism of the effects of mutations was revealed by computational analysis. The mutated peptide discovered here can be a potent inhibitor to block BTLA/HVEM interaction and its mechanism may extend people’s view on inhibitor discovery for the checkpoint pair.

Small Molecule HIV-1 Attachment Inhibitors: Discovery, Mode of Action and Structural Basis of Inhibition

Viral entry into host cells is a critical step in the viral life cycle. HIV-1 entry is mediated by the sole surface envelope glycoprotein Env and is initiated by the interaction between Env and the host receptor CD4. This interaction, referred to as the attachment step, has long been considered an attractive target for inhibitor discovery and development. Fostemsavir, recently approved by the FDA, represents the first-in-class drug in the attachment inhibitor class. This review focuses on the discovery of temsavir (the active compound of fostemsavir) and analogs, mechanistic studies that elucidated the mode of action, and structural studies that revealed atomic details of the interaction between HIV-1 Env and attachment inhibitors. Challenges associated with emerging resistance mutations to the attachment inhibitors and the development of next-generation attachment inhibitors are also highlighted.

Norstictic acid is a selective allosteric transcriptional regulator

Inhibitors of transcriptional protein-protein interactions (PPIs) have high value both as tools and for therapeutic applications. The PPI network mediated by the transcriptional coactivator Med25, for example, regulates stress-response and motility pathways and dysregulation of the PPI networks contributes to oncogenesis and metastasis. The canonical transcription factor binding sites within Med25 are large (~900 square angstroms) and have little topology, and thus do not present an array of attractive small-molecule binding sites for inhibitor discovery. Here we demonstrate that the depsidone natural product norstictic acid functions through an alternative binding site to block Med25-transcriptional activator PPIs in vitro and in cell culture. Norstictic acid targets a binding site comprised of a highly dynamic loop flanking one canonical binding surface and in doing so, it both orthosterically and allosterically alters Med25-driven transcription in a patient-derived model of triple negative breast cancer. These results highlight the potential of Med25 as a therapeutic target as well as the inhibitor discovery opportunities presented by structurally dynamic loops within otherwise challenging proteins.