Fc-GDF15 glyco-engineering and receptor binding affinity optimization for body weight regulation

AbstractGDF15 is a distant TGF-β family member that induces anorexia and weight loss. Due to its function, GDF15 has attracted attention as a potential therapeutic for the treatment of obesity and its associated metabolic diseases. However, the pharmacokinetic and physicochemical properties of GDF15 present several challenges for its development as a therapeutic, including a short half-life, high aggregation propensity, and protease susceptibility in serum. Here, we report the design, characterization and optimization of GDF15 in an Fc-fusion protein format with improved therapeutic properties. Using a structure-based engineering approach, we combined knob-into-hole Fc technology and N-linked glycosylation site mutagenesis for half-life extension, improved solubility and protease resistance. In addition, we identified a set of mutations at the receptor binding site of GDF15 that show increased GFRAL binding affinity and led to significant half-life extension. We also identified a single point mutation that increases p-ERK signaling activity and results in improved weight loss efficacy in vivo. Taken together, our findings allowed us to develop GDF15 in a new therapeutic format that demonstrates better efficacy and potential for improved manufacturability.

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A Fungal Defensin Targets the SARS−CoV−2 Spike Receptor−Binding Domain

Journal of Fungi ◽

10.3390/jof7070553 ◽

2021 ◽

Vol 7 (7) ◽

pp. 553

Author(s):

Bin Gao ◽

Shunyi Zhu

Keyword(s):

Receptor Binding ◽

Viral Entry ◽

Single Point ◽

Binding Domain ◽

Host Cells ◽

Single Point Mutation ◽

Binding Motif ◽

Microsporum Canis ◽

Receptor Binding Domain ◽

Fungal Defensin

Coronavirus Disease 2019 (COVID−19) elicited by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS−CoV−2) is calling for novel targeted drugs. Since the viral entry into host cells depends on specific interactions between the receptor−binding domain (RBD) of the viral Spike protein and the membrane−bound monocarboxypeptidase angiotensin converting enzyme 2 (ACE2), the development of high affinity RBD binders to compete with human ACE2 represents a promising strategy for the design of therapeutics to prevent viral entry. Here, we report the discovery of such a binder and its improvement via a combination of computational and experimental approaches. The binder micasin, a known fungal defensin from the dermatophytic fungus Microsporum canis with antibacterial activity, can dock to the crevice formed by the receptor−binding motif (RBM) of RBD via an extensive shape complementarity interface (855.9 Å2 in area) with numerous hydrophobic and hydrogen−bonding interactions. Using microscale thermophoresis (MST) technique, we confirmed that micasin and its C−terminal γ−core derivative with multiple predicted interacting residues exhibited a low micromolar affinity to RBD. Expanding the interface area of micasin through a single point mutation to 970.5 Å2 accompanying an enhanced hydrogen bond network significantly improved its binding affinity by six−fold. Our work highlights the naturally occurring fungal defensins as an emerging resource that may be suitable for the development into antiviral agents for COVID−19.

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Structural basis for a complex I mutation that blocks pathological ROS production

Nature Communications ◽

10.1038/s41467-021-20942-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Zhan Yin ◽

Nils Burger ◽

Duvaraka Kula-Alwar ◽

Dunja Aksentijević ◽

Hannah R. Bridges ◽

...

Keyword(s):

Point Mutation ◽

Complex I ◽

Structural Changes ◽

Ischemia Reperfusion ◽

Single Point ◽

Structural Basis ◽

Single Point Mutation ◽

Ros Production

AbstractMitochondrial complex I is central to the pathological reactive oxygen species (ROS) production that underlies cardiac ischemia–reperfusion (IR) injury. ND6-P25L mice are homoplasmic for a disease-causing mtDNA point mutation encoding the P25L substitution in the ND6 subunit of complex I. The cryo-EM structure of ND6-P25L complex I revealed subtle structural changes that facilitate rapid conversion to the “deactive” state, usually formed only after prolonged inactivity. Despite its tendency to adopt the “deactive” state, the mutant complex is fully active for NADH oxidation, but cannot generate ROS by reverse electron transfer (RET). ND6-P25L mitochondria function normally, except for their lack of RET ROS production, and ND6-P25L mice are protected against cardiac IR injury in vivo. Thus, this single point mutation in complex I, which does not affect oxidative phosphorylation but renders the complex unable to catalyse RET, demonstrates the pathological role of ROS production by RET during IR injury.

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Sulfated non-anticoagulant heparin derivative modifies intracellular hemoglobin, inhibits cell sickling in vitro, and prolongs survival of sickle cell mice under hypoxia

Haematologica ◽

10.3324/haematol.2020.272393 ◽

2021 ◽

Author(s):

Osheiza Abdulmalik ◽

Noureldien H. E. Darwish ◽

Vandhana Muralidharan-Chari ◽

Maii Abu Taleb ◽

Shaker A. Mousa

Keyword(s):

Sickle Cell ◽

Inflammatory Responses ◽

Anticoagulant Activity ◽

Single Point ◽

Bleeding Complications ◽

Single Point Mutation ◽

Repeated Dosing ◽

Heparin Derivative

Sickle cell disease (SCD) is an autosomal recessive genetic disease caused by a single point mutation, resulting in abnormal sickle hemoglobin (HbS). During hypoxia or dehydration, HbS polymerizes to form insoluble aggregates and induces sickling of red blood cells (RBCs). RBC sickling increases adhesiveness of RBCs to alter the rheological properties of the blood and triggers inflammatory responses, leading to hemolysis and vaso-occlusive crisis sequelae. Unfractionated heparin (UFH) and low-molecular weight heparins (LMWH) have been suggested as treatments to relieve coagulation complications in SCD. However, they are associated with bleeding complications after repeated dosing. An alternative sulfated nonanticoagulant heparin derivative (S-NACH) was previously reported to have none to low systemic anticoagulant activity and no bleeding side effects, and it interfered with P-selectindependent binding of sickle cells to endothelial cells, with concomitant decrease in the levels of adhesion biomarkers in SCD mice. S-NACH has been further engineered and structurally enhanced to bind with and modify HbS to directly inhibit sickling, thus employing a multimodal approach. Here, we show that S-NACH can (i) directly engage in Schiff-base reactions with HbS to decrease RBC sickling under both normoxia and hypoxia in vitro, ii) prolong the survival of SCD mice under hypoxia, and (iii) regulate the altered steady state levels of pro- and antiinflammatory cytokines. Thus, our proof of concept in vitro and in vivo preclinical studies demonstrate that the multimodal S-NACH is a highly promising candidate for development into an improved and optimized alternative to LMWHs for the treatment of patients with SCD.

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FtsZ-Dependent Elongation of a Coccoid Bacterium

mBio ◽

10.1128/mbio.00908-16 ◽

2016 ◽

Vol 7 (5) ◽

Cited By ~ 13

Author(s):

Ana R. Pereira ◽

Jen Hsin ◽

Ewa Król ◽

Andreia C. Tavares ◽

Pierre Flores ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Shape ◽

Single Point ◽

Spherical Shape ◽

Single Point Mutation ◽

Wild Type ◽

Dead End ◽

Dynamics Simulations

ABSTRACT A mechanistic understanding of the determination and maintenance of the simplest bacterial cell shape, a sphere, remains elusive compared with that of more complex shapes. Cocci seem to lack a dedicated elongation machinery, and a spherical shape has been considered an evolutionary dead-end morphology, as a transition from a spherical to a rod-like shape has never been observed in bacteria. Here we show that a Staphylococcus aureus mutant (M5) expressing the ftsZ G193D allele exhibits elongated cells. Molecular dynamics simulations and in vitro studies indicate that FtsZ G193D filaments are more twisted and shorter than wild-type filaments. In vivo , M5 cell wall deposition is initiated asymmetrically, only on one side of the cell, and progresses into a helical pattern rather than into a constricting ring as in wild-type cells. This helical pattern of wall insertion leads to elongation, as in rod-shaped cells. Thus, structural flexibility of FtsZ filaments can result in an FtsZ-dependent mechanism for generating elongated cells from cocci. IMPORTANCE The mechanisms by which bacteria generate and maintain even the simplest cell shape remain an elusive but fundamental question in microbiology. In the absence of examples of coccus-to-rod transitions, the spherical shape has been suggested to be an evolutionary dead end in morphogenesis. We describe the first observation of the generation of elongated cells from truly spherical cocci, occurring in a Staphylococcus aureus mutant containing a single point mutation in its genome, in the gene encoding the bacterial tubulin homologue FtsZ. We demonstrate that FtsZ-dependent cell elongation is possible, even in the absence of dedicated elongation machinery.

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Single point mutation in adeno-associated viral vectors -DJ capsid leads to improvement for gene delivery in vivo

BMC Biotechnology ◽

10.1186/s12896-015-0230-0 ◽

2016 ◽

Vol 16 (1) ◽

Cited By ~ 14

Author(s):

Yingying Mao ◽

Xuejun Wang ◽

Renhe Yan ◽

Wei Hu ◽

Andrew Li ◽

...

Keyword(s):

Gene Delivery ◽

Point Mutation ◽

Viral Vectors ◽

Single Point ◽

Single Point Mutation

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In Vitro Androgen Receptor Binding Affinity and in Vivo Inhibitory Activity of 5?-Pregnane-3, 20-Dione

Annals of the New York Academy of Sciences ◽

10.1111/j.1749-6632.1988.tb51470.x ◽

1988 ◽

Vol 529 (1 Fourth Colloq) ◽

pp. 239-241

Author(s):

SAUDHAMINI PARTHASARATHY ◽

ANDREA CHIN ◽

VIRGINIA MALLOY ◽

JONATHAN MATIAS

Keyword(s):

Androgen Receptor ◽

Binding Affinity ◽

Receptor Binding ◽

Inhibitory Activity ◽

Receptor Binding Affinity

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Engineering of Long-Circulating Peptidoglycan Hydrolases Enables Efficient Treatment of Systemic Staphylococcus aureus Infection

mBio ◽

10.1128/mbio.01781-20 ◽

2020 ◽

Vol 11 (5) ◽

Cited By ~ 1

Author(s):

Anna M. Sobieraj ◽

Markus Huemer ◽

Léa V. Zinsli ◽

Susanne Meile ◽

Anja P. Keller ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Human Blood ◽

Half Life ◽

Multidrug Resistant ◽

Life Extension ◽

Drug Resistant ◽

Content Type ◽

Peptidoglycan Hydrolases ◽

Life Threatening

ABSTRACT Staphylococcus aureus is a human pathogen causing life-threatening diseases. The increasing prevalence of multidrug-resistant S. aureus infections is a global health concern, requiring development of novel therapeutic options. Peptidoglycan-degrading enzymes (peptidoglycan hydrolases, PGHs) have emerged as a highly effective class of antimicrobial proteins against S. aureus and other pathogens. When applied to Gram-positive bacteria, PGHs hydrolyze bonds within the peptidoglycan layer, leading to rapid bacterial death by lysis. This activity is highly specific and independent of the metabolic activity of the cell or its antibiotic resistance patterns. However, systemic application of PGHs is limited by their often low activity in vivo and by an insufficient serum circulation half-life. To address this problem, we aimed to extend the half-life of PGHs selected for high activity against S. aureus in human serum. Half-life extension and increased serum circulation were achieved through fusion of PGHs to an albumin-binding domain (ABD), resulting in high-affinity recruitment of human serum albumin and formation of large protein complexes. Importantly, the ABD-fused PGHs maintained high killing activity against multiple drug-resistant S. aureus strains, as determined by ex vivo testing in human blood. The top candidate, termed ABD_M23, was tested in vivo to treat S. aureus-induced murine bacteremia. Our findings demonstrate a significantly higher efficacy of ABD_M23 than of the parental M23 enzyme. We conclude that fusion with ABD represents a powerful approach for half-life extension of PGHs, expanding the therapeutic potential of these enzybiotics for treatment of multidrug-resistant bacterial infections. IMPORTANCE Life-threatening infections with Staphylococcus aureus are often difficult to treat due to the increasing prevalence of antibiotic-resistant bacteria and their ability to persist in protected niches in the body. Bacteriolytic enzymes are promising new antimicrobials because they rapidly kill bacteria, including drug-resistant and persisting cells, by destroying their cell wall. However, when injected into the bloodstream, these enzymes are not retained long enough to clear an infection. Here, we describe a modification to increase blood circulation time of the enzymes and enhance treatment efficacy against S. aureus-induced bloodstream infections. This was achieved by preselecting enzyme candidates for high activity in human blood and coupling them to serum albumin, thereby preventing their elimination by kidney filtration and blood vessel cells.

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The Carboxy-Terminal Fragment of Nucleolin Interacts with the Nucleocapsid Domain of Retroviral Gag Proteins and Inhibits Virion Assembly

Journal of Virology ◽

10.1128/jvi.74.23.11027-11039.2000 ◽

2000 ◽

Vol 74 (23) ◽

pp. 11027-11039 ◽

Cited By ~ 36

Author(s):

Eran Bacharach ◽

Jason Gonsky ◽

Kimona Alin ◽

Marianna Orlova ◽

Stephen P. Goff

Keyword(s):

Leukemia Virus ◽

Single Point ◽

Host Protein ◽

Single Point Mutation ◽

Gag Protein ◽

Virion Assembly ◽

Gag Proteins ◽

Carboxy Terminal

ABSTRACT A yeast two-hybrid screen for cellular proteins that interact with the murine leukemia virus (MuLV) Gag protein resulted in the identification of nucleolin, a host protein known to function in ribosome assembly. The interacting fusions contained the carboxy-terminal 212 amino acids of nucleolin [Nuc(212)]. The nucleocapsid (NC) portion of Gag was necessary and sufficient to mediate the binding to Nuc(212). The interaction of Gag with Nuc(212) could be demonstrated in vitro and was manifested in vivo by the NC-dependent incorporation of Nuc(212) inside MuLV virions. Overexpression of Nuc(212), but not full-length nucleolin, potently and specifically blocked MuLV virion assembly and/or release. A mutant of MuLV, selected to specifically disrupt the binding to Nuc(212), was found to be severely defective for virion assembly. This mutant harbors a single point mutation in capsid (CA) adjacent to the CA-NC junction, suggesting a role for this region in Moloney MuLV assembly. These experiments demonstrate that selection for proteins that bind assembly domain(s) can yield potent inhibitors of virion assembly. These experiments also raise the possibility that a nucleolin-Gag interaction may be involved in virion assembly.

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A single point mutation in TFIIA suppresses NC2 requirement in vivo

The EMBO Journal ◽

10.1093/emboj/19.4.672 ◽

2000 ◽

Vol 19 (4) ◽

pp. 672-682 ◽

Cited By ~ 40

Author(s):

Jun Xie ◽

Martine Collart ◽

Marc Lemaire ◽

Gertraud Stelzer ◽

Michael Meisterernst

Keyword(s):

Point Mutation ◽

Single Point ◽

Single Point Mutation

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Neoplastic transformation induced by an activated lymphocyte-specific protein tyrosine kinase (pp56lck).

Molecular and Cellular Biology ◽

10.1128/mcb.8.2.540 ◽

1988 ◽

Vol 8 (2) ◽

pp. 540-550 ◽

Cited By ~ 137

Author(s):

J D Marth ◽

J A Cooper ◽

C S King ◽

S F Ziegler ◽

D A Tinker ◽

...

Keyword(s):

Tyrosine Kinases ◽

Cell Transformation ◽

Single Point ◽

Normal Growth ◽

Protein Tyrosine Kinases ◽

Single Point Mutation ◽

Tyrosine Residue ◽

Protein Tyrosine ◽

Carboxy Terminal

The lck proto-oncogene encodes a lymphocyte-specific member of the src family of protein tyrosine kinases. Here we demonstrate that pp56lck is phosphorylated in vivo at a carboxy-terminal tyrosine residue (Tyr-505) analogous to Tyr-527 of pp60c-src. Substitution of phenylalanine for tyrosine at this position resulted in increased phosphorylation of a second tyrosine residue (Tyr-394) and was associated with an increase in apparent kinase activity. In addition, this single point mutation unmasked the oncogenic potential of pp56lck in NIH 3T3 cell transformation assays. Viewed in the context of similar results obtained with pp60c-src, it is likely that the enzymatic activity and transforming ability of all src-family protein tyrosine kinases can be regulated by carboxy-terminal tyrosine phosphorylation. We further demonstrate that overexpression of pp56lck in the murine T-cell lymphoma LSTRA as a result of a retroviral insertion event produces a kinase protein that despite wild-type primary structure is nevertheless hypophosphorylated at Tyr-505. Thus, control of normal growth in this lymphoid cell line may have been abrogated through acquisition of a posttranslationally activated version of pp56lck.

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