2FA-Platform Generates Dual Fatty Acid-Conjugated GLP-1 Receptor Agonist TE-8105 with Enhanced Diabetes, Obesity, and NASH Efficacy Compared to Semaglutide.

Conjugating two fatty acids (2FAs) to peptide drugs can improve pharmacokinetics and therapeutic effects. However, optimizing FA spacing, chain combination, and attachment site to simultaneously enhance albumin binding and drug efficacy remains challenging. We introduce a multiarm linker technology enabling precise control of 2FA spacing, composition, and attachment.

PPI-hotspot for detecting protein-protein interaction hot spots from the free protein structure.

Experimental detection of residues critical for protein-protein interactions (PPI) is a time-consuming, costly, and labor-intensive process. Hence, high-throughput PPI-hot spot prediction methods have been developed, but they have been validated using relatively small datasets, which may compromise their predictive reliability. Here, we introduce PPI-hotspot, a novel method for identifying PPI-hot spots using the free protein structure, and validated it on the largest collection of experimentally confirmed PPI-hot spots to date.

Solution Ionic Strength Can Modulate Functional Loop Conformations in Dihydrofolate Reductase.

The observation of multiple conformations of a functional loop (termed M20) in the dihydrofolate reductase (DHFR) enzyme triggered the proposition that large-scale motions of protein structural elements contribute to enzyme catalysis. The transition of the M20 loop from a conformation to an conformation was thought to aid the rate-limiting release of the products. However, the influence of charged species in the solution environment on the observed M20 loop conformations, independent of charged ligands bound to the enzyme, had not been considered.

Using protein language models for protein interaction hot spot prediction with limited data.

Background: Protein language models, inspired by the success of large language models in deciphering human language, have emerged as powerful tools for unraveling the intricate code of life inscribed within protein sequences. They have gained significant attention for their promising applications across various areas, including the sequence-based prediction of secondary and tertiary protein structure, the discovery of new functional protein sequences/folds, and the assessment of mutational impact on protein fitness. However, their utility in learning to predict protein residue properties based on scant datasets, such as protein-protein interaction (PPI)-hotspots whose mutations significantly impair PPIs, remained unclear.

An Antibody-Drug Conjugate for Multiple Myeloma Prepared by Multi-Arm Linkers.

First-line treatment of multiple myeloma, a prevalent blood cancer lacking a cure, using anti-CD38 daratumumab antibody and lenalidomide is often inadequate due to relapse and severe side effects. To enhance drug safety and efficacy, an antibody-drug conjugate, TE-1146, comprising six lenalidomide drug molecules site-specifically conjugated to a reconfigured daratumumab to deliver cytotoxic lenalidomide to tumor cells is developed. TE-1146 is prepared using the HighDAR platform, which employs i) a maleimide-containing "multi-arm linker" to conjugate multiple drug molecules creating a drug bundle, and ii) a designed peptide with a Zn-binding cysteine at the C-termini of a reconfigured daratumumab for site-specific drug bundle conjugation.

System-Specific Parameter Optimization for Nonpolarizable and Polarizable Force Fields.

The accuracy of classical force fields (FFs) has been shown to be limited for the simulation of cation-protein systems despite their importance in understanding the processes of life. Improvements can result from optimizing the parameters of classical FFs or by extending the FF formulation by terms describing charge transfer (CT) and polarization (POL) effects. In this work, we introduce our implementation of the CTPOL model in OpenMM, which extends the classical additive FF formula by adding CT and POL.

Site-specific Conjugation of 6 DOTA Chelators to a CA19-9-targeting scFv-Fc Antibody for Imaging and Therapy.

Antibodies conjugated with diagnostic/therapeutic radionuclides are attractive options for inoperable cancers lacking accurate imaging methods and effective therapeutics, such as pancreatic cancer. Hence, we have produced an antibody radionuclide conjugate termed TE-1132 comprising a α-CA19-9 scFv-Fc that is site-specifically conjugated at each C-terminus to 3 DOTA chelators via a cysteine-containing peptide linker. The smaller scFv-Fc size facilitates diffusivity within solid tumors, whereas the chelator-to-antibody ratio of six enabled Lu-radiolabeled TE-1132 to exhibit high radioactivity up to 520 MBq/nmol.

Protein Ca-Sites Prone to Sr Substitution: Implications for Strontium Therapy.

Strontium (Sr), an alkali metal with properties similar to calcium, in the form of soluble salts is used to treat osteoporosis. Despite the information accumulated on the role of Sr as a Ca mimetic in biology and medicine, there is no systematic study of how the outcome of the competition between the two dications depends on the physicochemical properties of (i) the metal ions, (ii) the first- and second-shell ligands, and (iii) the protein matrix. Specifically, the key features of a Ca-binding protein that enable Sr to displace Ca remain unclear.

Efficient Strategy to Design Protease Inhibitors: Application to Enterovirus 71 2A Protease.

One strategy to counter viruses that persistently cause outbreaks is to design molecules that can specifically inhibit an essential multifunctional viral protease. Herein, we present such a strategy using well-established methods to first identify a region present only in viral (but human) proteases and find peptides that can bind specifically to this "unique" region by maximizing the protease-peptide binding free energy iteratively using single-point mutations starting with the substrate peptide. We applied this strategy to discover pseudosubstrate peptide inhibitors for the multifunctional 2A protease of enterovirus 71 (EV71), a key causative pathogen for hand-foot-and-mouth disease affecting young children, along with coxsackievirus A16.

Α γ-tubulin complex-dependent pathway suppresses ciliogenesis by promoting cilia disassembly.

The primary cilium, a microtubule-based sensory organelle, undergoes cycles of assembly and disassembly that govern the cell cycle progression critical to cell proliferation and differentiation. Although cilia assembly has been studied extensively, the molecular mechanisms underlying cilia disassembly are less well understood. Here, we uncover a γ-tubulin ring complex (γ-TuRC)-dependent pathway that promotes cilia disassembly and thereby prevents cilia formation.

Factors allowing small monovalent Li to displace Ca in proteins.

Because Li and Ca differ in both charge and size, the possibility that monovalent Li could dislodge the bulkier, divalent Ca in Ca proteins had not been considered. However, our recent density functional theory/continuum dielectric calculations predicted that Li could displace the native Ca from the C2 domain of cytosolic PKCα/γ. This would reduce electrostatic interactions between the Li-bound C2 domain and the membrane, consistent with experimental studies showing that Li can inhibit the translocation of cytoplasmic PKC to membranes.

From quantum-derived principles underlying cysteine reactivity to combating the COVID-19 pandemic.

The COVID-19 pandemic poses a challenge in coming up with quick and effective means to counter its cause, the SARS-CoV-2. Here, we show how the key factors governing cysteine reactivity in proteins derived from combined quantum mechanical/continuum calculations led to a novel multi-targeting strategy against SARS-CoV-2, in contrast to developing potent drugs/vaccines against a single viral target such as the spike protein. Specifically, they led to the discovery of reactive cysteines in evolutionary conserved Zn-sites in several SARS-CoV-2 proteins that are crucial for viral polypeptide proteolysis as well as viral RNA synthesis, proofreading, and modification.

Allosteric coupling between transmembrane segment 4 and the selectivity filter of TALK1 potassium channels regulates their gating by extracellular pH.

Opening of two-pore domain K channels (K2Ps) is regulated by various external cues, such as pH, membrane tension, or temperature, which allosterically modulate the selectivity filter (SF) gate. However, how these cues cause conformational changes in the SF of some K2P channels remains unclear. Herein, we investigate the mechanisms by which extracellular pH affects gating in an alkaline-activated K2P channel, TALK1, using electrophysiology and molecular dynamics (MD) simulations.

PPI-Hotspot: Database of Protein-Protein Interaction Hot Spots.

Single-point mutations of certain residues (so-called hot spots) impair/disrupt protein-protein interactions (PPIs), leading to pathogenesis and drug resistance. Conventionally, a PPI-hot spot is identified when its replacement decreased the binding free energy significantly, generally by ≥2 kcal/mol. The relatively few mutations with such a significant binding free energy drop limited the number of distinct PPI-hot spots.

Calcium in Signaling: Its Specificity and Vulnerabilities toward Biogenic and Abiogenic Metal Ions.

Divalent calcium ion (Ca) plays an indispensable role as a second messenger in a myriad of signal transduction processes. Of utmost importance for the faultless functioning of calcium-modulated signaling proteins is their binding selectivity of the native metal cation over rival biogenic/abiogenic metal ion contenders in the intra/extracellular fluids. In this Perspective, we summarize recent findings on the competition between the cognate Ca and other biogenic or abiogenic divalent cations for binding to Ca-signaling proteins or organic cofactors.

Trinuclear Calcium Site in the C2 Domain of PKCα/γ Is Prone to Lithium Attack.

Lithium (Li) is the first-line therapy for bipolar disorder and a candidate drug for various diseases such as amyotrophic lateral sclerosis, multiple sclerosis, and stroke. Despite being the captivating subject of many studies, the mechanism of lithium's therapeutic action remains unclear. To date, it has been shown that Li competes with Mg and Na to normalize the activity of inositol and neurotransmitter-related signaling proteins, respectively.

Influence of solution ionic strength on the stabilities of M20 loop conformations in apo E. coli dihydrofolate reductase.

Interactions among ions and their specific interactions with macromolecular solutes are known to play a central role in biomolecular stability. However, similar effects in the conformational stability of protein loops that play functional roles, such as binding ligands, proteins, and DNA/RNA molecules, remain relatively unexplored. A well-characterized enzyme that has such a functional loop is Escherichia coli dihydrofolate reductase (ecDHFR), whose so-called M20 loop has been observed in three ordered conformations in crystal structures.

Metal Affinity/Selectivity of Monophosphate-Containing Signaling/Lipid Molecules.

Monophosphate, an essential component of nucleic acids, as well as cell membranes and signaling molecules, is often bound to metal cations. Despite the biological importance of monophosphate-containing cell-signaling or lipid molecules, their propensity to bind the two most abundant cellular dications, Mg and Ca, in a particular mode (inner/outer shell, mono/bidentate) is not well understood. Whether they prefer binding to Mg than to Ca and if they can outcompete the carboxylates of excitatory Asp/Glu and inhibitory gamma-aminobutyric acid (GABA) neurotransmitters in binding to Mg/Ca remain unclear.

Sensitive and Specific Cadmium Biosensor Developed by Reconfiguring Metal Transport and Leveraging Natural Gene Repositories.

Whole-cell biosensors are useful for monitoring heavy metal toxicity in public health and ecosystems, but their development has been hindered by intrinsic trade-offs between sensitivity and specificity. Here, we demonstrated an effective engineering solution by building a sensitive, specific, and high-response biosensor for carcinogenic cadmium ions. We genetically programmed the metal transport system of to enrich intracellular cadmium ions and deprive interfering metal species.

Benchmarking polarizable and non-polarizable force fields for Ca-peptides against a comprehensive QM dataset.

Explicit description of atomic polarizability is critical for the accurate treatment of inter-molecular interactions by force fields (FFs) in molecular dynamics (MD) simulations aiming to investigate complex electrostatic environments such as metal-binding sites of metalloproteins. Several models exist to describe key monovalent and divalent cations interacting with proteins. Many of these models have been developed from ion-amino-acid interactions and/or aqueous-phase data on cation solvation.