Preventing osteoporotic bone loss in mice by promoting balanced bone remodeling through M-CSF, a dual antagonist to c-FMS and αvβ3 receptors.

Osteoporosis is a common, age-related disease caused by imbalanced bone remodeling. Current treatments either shut down bone resorption or robustly stimulate bone formation. Here, we describe a novel compound that inhibits osteoclast activity without causing apparent disruptions to bone formation by targeting both c-FMS (i.

Mapping the sclerostin-LRP4 binding interface identifies critical interaction hotspots in loops 1 and 3 of sclerostin.

The interaction of sclerostin (Scl) with the low-density lipoprotein receptor-related protein 4 (LRP4) leads to a marked reduction in bone formation by inhibiting the Wnt/β-catenin pathway. To characterize the Scl-LRP4 binding interface, we sorted a combinatorial library of Scl variants and isolated variants with reduced affinity to LRP4. We identified Scl single-mutation variants enriched during the sorting process and verified their reduction in affinity toward LRP4-a reduction that was not a result of changes in the variants' secondary structure or stability.

Deep neural networks for predicting the affinity landscape of protein-protein interactions.

Studies determining protein-protein interactions (PPIs) by deep mutational scanning have focused mainly on a narrow range of affinities within complexes and thus include only partial coverage of the mutation space of given proteins. By inserting an affinity-reducing N-terminal alanine in the N-terminal domain of the tissue inhibitor of metalloproteinases-2 (N-TIMP2), we overcame the limitation of its narrow affinity range for matrix metalloproteinase 9 (MMP9). We trained deep neural networks (DNNs) to quantitatively predict the binding affinity of unobserved wild-type variants and variants carrying an N-terminal alanine.

PSMA-reactive NB7 single domain antibody fragment: A potential scaffold for developing prostate cancer theranostics.

Introduction: Single domain antibody fragments (sdAbs) are an appealing scaffold for radiopharmaceutical development due to their small size (~15 kDa), high solubility, high stability, and excellent tumor penetration. Previously, we developed NB7 sdAb, which has very high affinity for an epitope on PSMA that is different from those targeted by small molecule PSMA inhibitors. Herein, we evaluated NB7 after radioiodination using [*I]SGMIB (1,3,4-isomer) and iso-[*I]SGMIB (1,3,5-isomer), as well as their At-labeled analogues.

Potent inhibition of MMP-9 by a novel sustained-release platform attenuates left ventricular remodeling following myocardial infarction.

Sustained drug-release systems prolong the retention of therapeutic drugs within target tissues to alleviate the need for repeated drug administration. Two major caveats of the current systems are that the release rate and the timing cannot be predicted or fine-tuned because they rely on uncontrolled environmental conditions and that the system must be redesigned for each drug and treatment regime because the drug is bound via interactions that are specific to its structure and composition. We present a controlled and universal sustained drug-release system, which comprises minute spherical particles in which a therapeutic protein is affinity-bound to alginate sulfate (AlgS) through one or more short heparin-binding peptide (HBP) sequence repeats.

Serine protease inhibitors decrease metastasis in prostate, breast, and ovarian cancers.

Targeted therapies for prostate, breast, and ovarian cancers are based on their activity against primary tumors rather than their anti-metastatic activity. Consequently, there is an urgent need for new agents targeting the metastatic process. Emerging evidence correlates in vitro and in vivo cancer invasion and metastasis with increased activity of the proteases mesotrypsin (prostate and breast cancer) and kallikrein 6 (KLK6; ovarian cancer).

APPI-Derived Cyclic Peptide Enhances Aβ42 Aggregation and Reduces Aβ42-Mediated Membrane Destabilization and Cytotoxicity.

An amyloid precursor protein inhibitor (APPI) and amyloid beta 42 (Aβ42) are both subdomains of the human transmembrane amyloid precursor protein (APP). In the brains of patients with Alzheimer's disease (AD), Aβ42 oligomerizes into aggregates of various sizes, with intermediate, low-molecular-weight Aβ42 oligomers currently being held to be the species responsible for the most neurotoxic effects associated with the disease. Strategies to ameliorate the toxicity of these intermediate Aβ42 oligomeric species include the use of short, Aβ42-interacting peptides that either inhibit the formation of the Aβ42 oligomeric species or promote their conversion to high-molecular-weight aggregates.

Developing a dual VEGF/PDL1 inhibitor based on high-affinity scFv heterodimers as an anti-cancer therapeutic strategy.

Cancer progression is enhanced by the interaction of programmed death-ligand 1 (PDL1), which is associated with inhibition of the immune response against tumors, and vascular endothelial growth factor (VEGF), which inhibits immune cell activity while inducing angiogenesis and proliferation of cancer cells. Dual inhibition of PDL1 and VEGF may therefore confer a synergistic anti-cancer therapeutic effect. We present a novel strategy for developing a therapeutic that simultaneously binds and inhibits both PDL1 and VEGF.

Utilizing genetic code expansion to modify N-TIMP2 specificity towards MMP-2, MMP-9, and MMP-14.

Matrix metalloproteinases (MMPs) regulate the degradation of extracellular matrix (ECM) components in biological processes. MMP activity is controlled by natural tissue inhibitors of metalloproteinases (TIMPs) that non-selectively inhibit the function of multiple MMPs via interaction with the MMPs' Zn-containing catalytic pocket. Recent studies suggest that TIMPs engineered to confer MMP specificity could be exploited for therapeutic purposes, but obtaining specific TIMP-2 inhibitors has proved to be challenging.

Utilizing genetic code expansion to modify N-TIMP2 specificity towards MMP-2, MMP-9, and MMP-14.

Matrix metalloproteinases (MMPs) regulate the degradation of extracellular matrix (ECM) components in biological processes. MMP activity is controlled by natural tissue inhibitors of metalloproteinases (TIMPs) that non-selectively inhibit the function of multiple MMPs via interaction with the MMPs' Zn -containing catalytic pocket. Recent studies suggest that TIMPs engineered to confer MMP specificity could be exploited for therapeutic purposes, but obtaining specific TIMP-2 inhibitors has proved to be challenging.

Yeast-based directed-evolution for high-throughput structural stabilization of G protein-coupled receptors (GPCRs).

The immense potential of G protein-coupled receptors (GPCRs) as targets for drug discovery is not fully realized due to the enormous difficulties associated with structure elucidation of these profoundly unstable membrane proteins. The existing methods of GPCR stability-engineering are cumbersome and low-throughput; in addition, the scope of GPCRs that could benefit from these techniques is limited. Here, we present a yeast-based screening platform for a single-step isolation of GRCR variants stable in the presence of short-chain detergents, a feature essential for their successful crystallization using vapor diffusion method.

In vitro inhibition of cancer angiogenesis and migration by a nanobody that targets the orphan receptor Tie1.

The human signaling molecules Tie1 and Tie2 receptor tyrosine kinases (RTKs) play important pathophysiological roles in many diseases, including different cancers. The activity of Tie1 is mediated mainly through the downstream angiopoietin-1 (Ang1)-dependent activation of Tie2, rendering both Tie 1 and the Tie1/Tie2/Ang1 axis attractive putative targets for therapeutic intervention. However, the development of inhibitors that target Tie1 and an understanding of their effect on Tie2 and on the Tie1/Tie2/Ang1 axis remain unfulfilled tasks, due, largely, to the facts that Tie1 is an orphan receptor and is difficult to produce and use in the quantities required for immune antibody library screens.

Bioorthogonal PEGylation Prolongs the Elimination Half-Life of N-TIMP2 While Retaining MMP Inhibition.

Tissue inhibitors of metalloproteinases (TIMPs) are natural inhibitors of the matrix metalloproteinase (MMP) family of proteins, whose members are key regulators of the proteolysis of extracellular matrix components and hence of multiple biological processes. In particular, imbalanced activity of matrix metalloproteinase-14 (MMP-14) may lead to the development of cancer and cardiovascular and other diseases. This study aimed to engineer TIMP2, one of the four homologous TIMPs, as a potential therapeutic by virtue of its ability to bind to the active-site Zn of MMP-14.

Modifying pH-sensitive PCSK9/LDLR interactions as a strategy to enhance hepatic cell uptake of low-density lipoprotein cholesterol (LDL-C).

LDL-receptor (LDLR)-mediated uptake of LDL-C into hepatocytes is impaired by lysosomal degradation of LDLR, which is promoted by proprotein convertase subtilisin/kexin type 9 (PCSK9). Cell surface binding of PCSK9 to LDLR produces a complex that translocates to an endosome, where the acidic pH strengthens the binding affinity of PCSK9 to LDLR, preventing LDLR recycling to the cell membrane. We present a new approach to inhibit PCSK9-mediated LDLR degradation, namely, targeting the PCSK9/LDLR interface with a PCSK9-antagonist, designated Flag-PCSK9PH, which prevents access of WT PCSK9 to LDLR.

Competitive blocking of LRP4-sclerostin binding interface strongly promotes bone anabolic functions.

Induction of bone formation by Wnt ligands is inhibited when sclerostin (Scl), an osteocyte-produced antagonist, binds to its receptors, the low-density lipoprotein receptor-related proteins 5 or 6 (LRP5/6). Recently, it was shown that enhanced inhibition is achieved by Scl binding to the co-receptor LRP4. However, it is not clear if the binding of Scl to LRP4 facilitates Scl binding to LRP5/6 or inhibits the Wnt pathway in an LRP5/6-independent manner.

Avidity observed between a bivalent inhibitor and an enzyme monomer with a single active site.

Although myriad protein-protein interactions in nature use polyvalent binding, in which multiple ligands on one entity bind to multiple receptors on another, to date an affinity advantage of polyvalent binding has been demonstrated experimentally only in cases where the target receptor molecules are clustered prior to complex formation. Here, we demonstrate cooperativity in binding affinity (i.e.

Bcl-2-Homology-Only Proapoptotic Peptides Modulate β-Amyloid Aggregation and Toxicity.

Aggregation of the β-Amyloid (Aβ) peptide in brain tissues is the hallmark of Alzheimer's disease (AD). While Aβ is presumed to be insidiously involved in the disease's pathophysiology, concrete mechanisms accounting for the role of Aβ in AD are yet to be deciphered. While Aβ has been primarily identified in the extracellular space, the peptide also accumulates in cellular compartments such as mitochondria and lysosomes and impairs cellular functions.

Amyloid β structural polymorphism, associated toxicity and therapeutic strategies.

A review of the multidisciplinary scientific literature reveals a large variety of amyloid-β (Aβ) oligomeric species, differing in molecular weight, conformation and morphology. These species, which may assemble via either on- or off-aggregation pathways, exhibit differences in stability, function and neurotoxicity, according to different experimental settings. The conformations of the different Aβ species are stabilized by intra- and inter-molecular hydrogen bonds and by electrostatic and hydrophobic interactions, all depending on the chemical and physical environment (e.

Climbing Up and Down Binding Landscapes through Deep Mutational Scanning of Three Homologous Protein-Protein Complexes.

Protein-protein interactions (PPIs) have evolved to display binding affinities that can support their function. As such, cognate and noncognate PPIs could be highly similar structurally but exhibit huge differences in binding affinities. To understand this phenomenon, we study three homologous protease-inhibitor PPIs that span 9 orders of magnitude in binding affinity.

Kallikrein-Related Peptidase 6 Is Associated with the Tumour Microenvironment of Pancreatic Ductal Adenocarcinoma.

As cancer-associated factors, kallikrein-related peptidases (KLKs) are components of the tumour microenvironment, which represents a rich substrate repertoire, and considered attractive targets for the development of novel treatments. Standard-of-care therapy of pancreatic cancer shows unsatisfactory results, indicating the need for alternative therapeutic approaches. We aimed to investigate the expression of KLKs in pancreatic cancer and to inhibit the function of KLK6 in pancreatic cancer cells.

A KLK4 proteinase substrate capture approach to antagonize PAR1.

Proteinase-activated receptor-1 (PAR1), triggered by thrombin and other serine proteinases such as tissue kallikrein-4 (KLK4), is a key driver of inflammation, tumor invasiveness and tumor metastasis. The PAR1 transmembrane G-protein-coupled receptor therefore represents an attractive target for therapeutic inhibitors. We thus used a computational design to develop a new PAR1 antagonist, namely, a catalytically inactive human KLK4 that acts as a proteinase substrate-capture reagent, preventing receptor cleavage (and hence activation) by binding to and occluding the extracellular R41-S42 canonical PAR1 proteolytic activation site.

Predicting mutant outcome by combining deep mutational scanning and machine learning.

Deep mutational scanning provides unprecedented wealth of quantitative data regarding the functional outcome of mutations in proteins. A single experiment may measure properties (eg, structural stability) of numerous protein variants. Leveraging the experimental data to gain insights about unexplored regions of the mutational landscape is a major computational challenge.

A Mechanism for the Inhibition of Tau Neurotoxicity: Studies with Artificial Membranes, Isolated Mitochondria, and Intact Cells.

It is currently believed that molecular agents that specifically bind to and neutralize the toxic proteins/peptides, amyloid β (Aβ42), tau, and the tau-derived peptide PHF6, hold the key to attenuating the progression of Alzheimer's disease (AD). We thus tested our previously developed nonaggregating Aβ42 double mutant (Aβ42) as a multispecific binder for three AD-associated molecules, wild-type Aβ42, the tau mutant, and a synthetic PHF6 peptide. Aβ42 acted as a functional inhibitor of these molecules in assays and in neuronal cell-based models of AD.

Simultaneous targeting of CD44 and MMP9 catalytic and hemopexin domains as a therapeutic strategy.

Crosstalk of the oncogenic matrix metalloproteinase-9 (MMP9) and one of its ligands, CD44, involves cleavage of CD44 by the MMP9 catalytic domain, with the CD44-MMP9 interaction on the cell surface taking place through the MMP9 hemopexin domain (PEX). This interaction promotes cancer cell migration and invasiveness. In concert, MMP9-processed CD44 induces the expression of MMP9, which degrades ECM components and facilitates growth factor release and activation, cancer cell invasiveness, and metastasis.

Engineering Stem Cell Factor Ligands with Different c-Kit Agonistic Potencies.

Receptor tyrosine kinases (RTKs) are major players in signal transduction, regulating cellular activities in both normal regeneration and malignancy. Thus, many RTKs, c-Kit among them, play key roles in the function of both normal and neoplastic cells, and as such constitute attractive targets for therapeutic intervention. We thus sought to manipulate the self-association of stem cell factor (SCF), the cognate ligand of c-Kit, and hence its suboptimal affinity and activation potency for c-Kit.