Homogeneous antibody-drug conjugates (ADCs) that use a highly reactive buried lysine (Lys) residue embedded in a dual variable domain (DVD)-IgG1 format can be assembled with high precision and efficiency under mild conditions. Here we show that replacing the Lys with an arginine (Arg) residue affords an orthogonal ADC assembly that is site-selective and stable. X-ray crystallography confirmed the location of the reactive Arg residue at the bottom of a deep pocket. As the Lys-to-Arg mutation is confined to a single residue in the heavy chain of the DVD-IgG1, heterodimeric assemblies that combine a buried Lys in one arm, a buried Arg in the other arm, and identical light chains, are readily assembled. Furthermore, the orthogonal conjugation chemistry enables the loading of heterodimeric DVD-IgG1s with two different cargos in a one-pot reaction and thus affords a convenient platform for dual-warhead ADCs and other multifaceted antibody conjugates.
Bioterrorism is an ongoing threat to global peace and health because weaponized agents are available to potential terrorist groups either directly or through scientists who may be willing to cooperate with terrorists. Biological weapons would have larger disastrous global health effects compared to chemical weapons. Therefore, there is a need for merging endeavors of the scientific community on the tasks of developing methodologies for prevention of biological attack/threat, for biological weapons control and for completing a biodefence system. This monograph is a result of such endeavors. 1.1 Control of Biological Weapons The scientific community should focus on two strategies: 1.1.1 Strategy of Intelligence There are no easily identifiable footprints marking bioweapons development as it is nearly indistinguishable from legitimate biological science and biotechnology, and such efforts are easily hidden in plain sight. Gathering intelligence on national level and limiting the illicit transfer of materials, technologies and knowledge, are not easy tasks. Furhter, the intelligence community should develop capability of tactical warning of a planned bioterrorist attack. In the aftermath of a bioattack the authorities want to know who the attacker was, how the attack was carried out and how the next attack could be stopped? Some of the multibillion dollar biodefence systems, such as Bio Watch in the U.S.A. that has been developed for years does not provide information on who and how attacked, V. Radosavljevic (*) Military Medical Headquarter, Ministry of Defence, Crnotravska 17, Belgrade, Serbia University of Defence, Belgrade, Serbia e-mail: vladanr4@gmail.com
The AMP-activated protein kinase (AMPK) alpha beta gamma het-erotrimer regulates cellular energy homeostasis with tissue-specific isoform distribution. Smallmolecule activation of skeletal muscle alpha 2 beta 2 AMPK complexes may prove a valuable treatment strategy for type 2 diabetes and insulin resistance. Herein, we report the small-molecule SC4 is a potent, direct AMPK activator that preferentially activates alpha 2 complexes and stimulates skeletal muscle glucose uptake. In parallel with the term secretagog, we propose "importagog" to define a substance that induces or augments cellular uptake of another substance. Three-dimensional structures of the glucose importagog SC4 bound to activated alpha 2 beta 2 gamma 1 and alpha 2 beta 1 gamma 1 complexes reveal binding determinants, in particular a key interaction between the SC4 imidazopyridine 4'-nitrogen and beta 2-Asp111, which provide a design paradigm for beta 2-AMPK therapeutics. The alpha 2 beta 2 gamma 1/SC4 structure reveals an interaction between a beta 2 N-terminal alpha helix and the alpha 2 autoinhibitory domain. Our results provide a structurefunction guide to accelerate development of potent, but importantly tissue-specific, beta 2-AMPK therapeutics.
This book describes the latest molecular insights needed to understand the chemical and biological (CB) agents and their associated biotechnologies. Its primary focus is to present and discuss molecul
This book highlights the current state-of-the-art regarding the application of applied crystallographic methodologies for understanding, predicting and controlling the transformation from the molecula
This book comprises 25 contributions focussed on nanotechnology for sensor applications. They stem from presentations at the NATO Advanced Research Workshop "Nanomaterials for Security". The chapters
Tuberculosis (TB) is a recurring threat to contemporary civilization. It affects not only those within developing countries, but has also appeared again in places where it was once considered eradicated. TB co-infection in patients infected by HIV is, at the time of writing, the most common cause of death. In the field of searching for new antimycobacterial drug leads, compounds of natural origin still remain a promising source. The review is intended to gather information about natural products (metabolites of plants, fungi, bacteria, and marine sponges) that show activity against mycobacterial enzymes. Here, natural metabolites are presented as being inhibitors/activators of the mycobacterial enzymes involved in mycobacterial growth in vitro (ClpC1, ClpP, MurE ligase, mycothiol S-conjugate amidase, beta-ketoacyl-ACP synthase, InhA) and in vivo, as regards the host cell (PtpB). Each enzyme is briefly described so as to generate an understanding of its role in mycobacterial growth and engender a perception of the mechanism of action of the studied natural compounds. Furthermore, after the introduction of the enzyme, its inhibitors are listed and exactly characterized.
In this issue of Chemistry & Biology, Zhou et al. demonstrate the possibility of effective multiple targeting of HIV infection by using a multifunctional molecule in which an anti-CCR5 receptor aptamer (G-3) is conjugated to an anti-TNPO3 siRNA.
Proteopathies are a large and diverse group of human diseases that are caused by protein misfolding. Well-known examples of proteopathies are Alzheimer's and Parkinson's disease, which are both linked to amyloid fibril formation. In this issue of Chemistry & Biology, Castellano et al. (2015) describe the way to harness the power of a protein from baker's yeast, Hsp104, to disaggregate the fibrils.
In this issue of Chemistry & Biology , Cognetta et al. (2015) describe new pharmacological tools, including N -hydroxyhydantoin-containing carbamate inhibitors and an activity-based probe, for palmitoyl protein thioesterase 1 and alpha, beta-hydrolase domain-4 that expand the toolkit for the serine hydrolases.
Accumulating evidence has linked G-quadruplex structures to a number of biological processes in vivo, including DNA replication, transcription, and genomic maintenance. However, the precise function and mechanism of G-quadruplex formation in mammalian cells remains poorly defined. Therefore, the development of small-molecule G-quadruplex probes has attracted significant attention in recent years. This review highlights examples of G-quadruplex probes that have been reported over the last few years, some of which have been employed for cellular imaging or for use in the construction of G-quadruplex-based analytical sensing platforms.
The heat shock protein HSP70 plays antiapoptotic and oncogenic roles, and thus its inhibition has been recognized as a potential avenue for anticancer therapy. Here we describe the small molecule, apoptozole (Az), which inhibits the ATPase activity of HSP70 by binding to its ATPase domain and, as a result, induces an array of apoptotic phenotypes in cancer cells. Affinity chromatography provides evidence that Az binds HSP70 but not other types of heat shock proteins including HSP40, HSP60, and HSP90. We also demonstrate that Az induces cancer cell death via caspase-dependent apoptosis by disrupting the interaction of HSP70 with APAF-1. Animal studies indicate that Az treatment retards tumor growth in a xenograft mouse model without affecting mouse viability. These studies suggest that Az will aid the development of new cancer therapies and serve as a chemical probe to gain a better understanding of the diverse functions of HSP70.
Inhibitors of histone deacetylases (HDACi) hold considerable therapeutic promise as clinical anticancer therapies. However, currently known HDACi exhibit limited isoform specificity, off-target activity, and undesirable pharmaceutical properties. Thus, HDACi with new chemotypes are needed to overcome these limitations. Here, we identify a class of HDACi with a previously undescribed benzoylhydrazide scaffold that is selective for the class I HDACs. These compounds are competitive inhibitors with a fast-on/slow-off HDAC-binding mechanism. We show that the lead compound, UF010, inhibits cancer cell proliferation via class I HDAC inhibition. This causes global changes in protein acetylation and gene expression, resulting in activation of tumor suppressor pathways and concurrent inhibition of several oncogenic pathways. The isotype selectivity coupled with interesting biological activities in suppressing tumor cell proliferation support further preclinical development of the UF010 class of compounds for potential therapeutic applications.
Targeting TrkB signaling could represent a good therapeutic strategy to prevent obesity. In this issue of Chemistry & Biology, Chan et al. report the efficacy of 7,8-DHT, a TrkB agonist, in preventing obesity in female mice. The underlying molecular mechanisms behind this activity seem to involve increased energy expenditure in skeletal muscle.
Thiostrepton (TSR) is an archetypal thiopeptide antibiotic possessing a quinaldic acid (QA) moiety in the side ring system. According to the mechanism of TSR previously known to target bacterial ribosome, we recently designed and biosynthesized several TSR derivatives that varied in QA substitution. Utilizing these thiopeptide antibiotics to treat the intracellular pathogen Mycobacterium marinum, we herein report a novel mode of action of TSRs, which induce ER stress-mediated autophagy to enhance host cell defense. This intracellular response, which is sensitive to the modification of the QA group, serves as an indirect but unignorable mechanism for eliminating intracellular pathogens. TSRs are thus the only type of antibiotics, to our knowledge, with the dual action on both the parasitic bacteria and the infected host cells. The newly observed mechanism of TSRs may inspire the future change in the treatment of intracellular pathogens, by taking host response into account.
Human β-defensins play a pivotal role in the innate immune response. Although expressed by and acting at epithelial surfaces, little is known about their specific interaction with epithelial structures. Here, we identify the transmembrane protein CD98 as a cell surface receptor involved in the internalization of human β-defensin 3 (hBD3) in human epithelial A549 cells. CD98 and hBD3 extensively colocalize on the basolateral domain of A549. While verifying their direct binding by fluorescence resonance energy transfer and surface plasmon resonance, we mapped the interaction to CD98 residues 304–414, i.e. to the region known to interact with the proteins of intestinal bacteria during colonic invasion. Treatment of A549 cells with hBD3 dramatically reduces CD98 expression and conversely, knockdown of CD98 expression impairs hBD3 cell surface binding and internalization. Competition for bacterial binding to CD98 and downregulation of CD98 expression may represent novel mechanisms for the antibacterial activity of hBD3.
In this issue of Chemistry & Biology, Duan et al. (2015) report the use of a light-inducible protein-protein interaction system to dynamically control the movement of intracellular organelles with spatial and temporal precision in living cells.
Staphylococcus aureus produces peptide toxins that it uses to respond to environmental cues. We previously characterized PepA1, a peptide toxin from S. aureus, that induces lytic cell death of both bacterial and host cells. That led us to suggest that PepA1 has an antibacterial activity. Here, we demonstrate that exogenously provided PepA1 has activity against both Gram-positive and Gram-negative bacteria. We also see that PepA1 is significantly hemolytic, thus limiting its use as an antibacterial agent. To overcome these limitations, we converted PepA1 into nonhemolytic derivatives. Our most promising derivative is a cyclic heptapseudopeptide with inconsequential toxicity to human cells, enhanced stability in human sera, and sharp antibacterial activity. Mechanistically, linear and helical PepA1 derivatives form pores at the bacterial and erythrocyte surfaces, while the cyclic peptide induces bacterial envelope reorganization, with insignificant action on the erythrocytes. Our work demonstrates that bacterial toxins might be an attractive starting point for antibacterial drug development.
The inflammatory response is a critical component of the immune system that is activated by stimuli such as cytokines, foreign DNA, RNA, or other harmful substances. Krukenberg et al. (2015) identify poly(ADP-ribose) as a new signaling molecule that activates inflammation, thus providing yet another mechanism by which PARPs are involved in cellular stress responses.
Elucidating how and to what extent lipid metabolism is remodeled under changing conditions is essential for understanding cellular physiology. Here, we analyzed proteome and lipidome dynamics to investigate how regulation of lipid metabolism at the global scale supports remodeling of cellular architecture and processes during physiological adaptations in yeast. Our results reveal that activation of cardiolipin synthesis and remodeling supports mitochondrial biogenesis in the transition from fermentative to respiratory metabolism, that down-regulation of de novo sterol synthesis machinery prompts differential turnover of lipid droplet-associated triacylglycerols and sterol esters during respiratory growth, that sphingolipid metabolism is regulated in a previously unrecognized growth stage-specific manner, and that endogenous synthesis of unsaturated fatty acids constitutes an in vivo upstream activator of peroxisomal biogenesis, via the heterodimeric Oaf1/Pip2 transcription factor. Our work demonstrates the pivotal role of lipid metabolism in adaptive processes and provides a resource to investigate its regulation at the cellular level.
