RESUMEN
The G12D mutation is among the most common KRAS mutations associated with cancer, in particular, pancreatic cancer. Here, we have developed monobodies, small synthetic binding proteins, that are selective to KRAS(G12D) over KRAS(wild type) and other oncogenic KRAS mutations, as well as over the G12D mutation in HRAS and NRAS. Crystallographic studies revealed that, similar to other KRAS mutant-selective inhibitors, the initial monobody bound to the S-II pocket, the groove between switch II and α3 helix, and captured this pocket in the most widely open form reported to date. Unlike other G12D-selective polypeptides reported to date, the monobody used its backbone NH group to directly recognize the side chain of KRAS Asp12, a feature that closely resembles that of a small-molecule inhibitor, MTRX1133. The monobody also directly interacted with H95, a residue not conserved in RAS isoforms. These features rationalize the high selectivity toward the G12D mutant and the KRAS isoform. Structure-guided affinity maturation resulted in monobodies with low nM KD values. Deep mutational scanning of a monobody generated hundreds of functional and nonfunctional single-point mutants, which identified crucial residues for binding and those that contributed to the selectivity toward the GTP- and GDP-bound states. When expressed in cells as genetically encoded reagents, these monobodies engaged selectively with KRAS(G12D) and inhibited KRAS(G12D)-mediated signaling and tumorigenesis. These results further illustrate the plasticity of the S-II pocket, which may be exploited for the design of next-generation KRAS(G12D)-selective inhibitors.
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Neoplasias Pancreáticas , Proteínas Proto-Oncogénicas p21(ras) , Humanos , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Mutación , Transformación Celular Neoplásica/genética , Carcinogénesis , Neoplasias Pancreáticas/genéticaRESUMEN
Antibody-drug conjugates (ADCs) are a class of targeted therapeutics used to selectively kill cancer cells. It is important that they remain intact in the bloodstream and release their payload in the target cancer cell for maximum efficacy and minimum toxicity. The development of effective ADCs requires the study of factors that can alter the stability of these therapeutics at the atomic level. Here, we present a general strategy that combines synthesis, bioconjugation, linker technology, site-directed mutagenesis, and modeling to investigate the influence of the site and microenvironment of the trastuzumab antibody on the stability of the conjugation and linkers. Trastuzumab is widely used to produce targeted ADCs because it can target with high specificity a receptor that is overexpressed in certain breast cancer cells (HER2). We show that the chemical environment of the conjugation site of trastuzumab plays a key role in the stability of linkers featuring acid-sensitive groups such as acetals. More specifically, Lys-207, located near the reactive Cys-205 of a thiomab variant of the antibody, may act as an acid catalyst and promote the hydrolysis of acetals. Mutation of Lys-207 into an alanine or using a longer linker that separates this residue from the acetal group stabilizes the conjugates. Analogously, Lys-207 promotes the beneficial hydrolysis of the succinimide ring when maleimide reagents are used for conjugation, thus stabilizing the subsequent ADCs by impairing the undesired retro-Michael reactions. This work provides new insights for the design of novel ADCs with improved stability properties.
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Antineoplásicos , Inmunoconjugados , Acetales , Antineoplásicos/química , Antineoplásicos/farmacología , Inmunoconjugados/química , Maleimidas/química , Mutación , Compuestos de Sulfhidrilo/química , Trastuzumab/químicaRESUMEN
Monoclonal antibodies have emerged as an important class of therapeutics in oncological and autoimmune diseases due to their several attractive properties, such as high binding affinity and specificity. However, it has recently become clear that antibodies recovered from serum show a significantly decreased potency owing to various reasons, including deamidation, oxidation, fragment antigen binding (Fab) exchange, and disulfide shuffling. Fab exchange and disulfide shuffling result because of the instability of disulfides in serum. Herein, we reported a 'one-pot' stapling strategy using isobutylene motifs to stabilise the interchain disulfides of antibodies. This general method was applied to a Fab fragment of the anti-HER2 antibody. The stapled Fab was completely stable in the presence of biological thiols. The approach was further applied to two different full-length IgGs, trastuzumab and rituximab, under mild and biocompatible conditions. The binding affinity of the antibody was enhanced, relative to its native form, after being stapled. The stapled structure maintained its effector functions and behaved similarly to its native form in vivo. This work provides a straightforward and scalable method for the stabilisation of antibodies in various formats.
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Chemical modification of proteins is essential for a variety of important diagnostic and therapeutic applications. Many strategies developed to date lack chemo- and regioselectivity as well as result in non-native linkages that may suffer from instability in vivo and adversely affect the protein's structure and function. We describe here the reaction of N-nucleophiles with the amino acid dehydroalanine (Dha) in a protein context. When Dha is chemically installed in proteins, the addition of a wide-range N-nucleophiles enables the rapid formation of amine linkages (secondary and tertiary) in a chemoselective manner under mild, biocompatible conditions. These new linkages are stable at a wide range of pH values (pH 2.8 to 12.8), under reducing conditions (biological thiols such as glutathione) and in human plasma. This method is demonstrated for three proteins and is shown to be fully compatible with disulfide bridges, as evidenced by the selective modification of recombinant albumin that displays 17 structurally relevant disulfides. The practicability and utility of our approach is further demonstrated by the construction of a chemically modified C2A domain of Synaptotagmin-I protein that retains its ability to preferentially bind to apoptotic cells at a level comparable to the native protein. Importantly, the method was useful for building a homogeneous antibody-drug conjugate with a precise drug-to-antibody ratio of 2. The kinase inhibitor crizotinib was directly conjugated to Dha through its piperidine motif, and its antibody-mediated intracellular delivery results in 10-fold improvement of its cancer cell-killing efficacy. The simplicity and exquisite site-selectivity of the aza-Michael ligation described herein allows the construction of stable secondary and tertiary amine-linked protein conjugates without affecting the structure and function of biologically relevant proteins.
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Alanina/análogos & derivados , Albúminas/química , Aminas/química , Anexina A5/química , Sinaptotagmina I/química , Alanina/química , Animales , Anticuerpos/química , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Crizotinib , Disulfuros/química , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Cinética , Ratones , Modelos Moleculares , Estructura Molecular , Pirazoles/química , Pirazoles/farmacología , Piridinas/química , Piridinas/farmacología , Teoría CuánticaRESUMEN
Antisense oligonucleotides (ASOs) are promising therapeutics for treating various neurological disorders. However, ASOs are unable to readily cross the mammalian blood-brain barrier (BBB) and therefore need to be delivered intrathecally to the central nervous system (CNS). Here, we engineered a human transferrin receptor 1 (TfR1) binding molecule, the oligonucleotide transport vehicle (OTV), to transport a tool ASO across the BBB in human TfR knockin (TfRmu/hu KI) mice and nonhuman primates. Intravenous injection and systemic delivery of OTV to TfRmu/hu KI mice resulted in sustained knockdown of the ASO target RNA, Malat1, across multiple mouse CNS regions and cell types, including endothelial cells, neurons, astrocytes, microglia, and oligodendrocytes. In addition, systemic delivery of OTV enabled Malat1 RNA knockdown in mouse quadriceps and cardiac muscles, which are difficult to target with oligonucleotides alone. Systemically delivered OTV enabled a more uniform ASO biodistribution profile in the CNS of TfRmu/hu KI mice and greater knockdown of Malat1 RNA compared with a bivalent, high-affinity TfR antibody. In cynomolgus macaques, an OTV directed against MALAT1 displayed robust ASO delivery to the primate CNS and enabled more uniform biodistribution and RNA target knockdown compared with intrathecal dosing of the same unconjugated ASO. Our data support systemically delivered OTV as a potential platform for delivering therapeutic ASOs across the BBB.
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Barrera Hematoencefálica , Oligonucleótidos Antisentido , ARN Largo no Codificante , Receptores de Transferrina , Animales , Oligonucleótidos Antisentido/farmacocinética , Oligonucleótidos Antisentido/administración & dosificación , Barrera Hematoencefálica/metabolismo , Receptores de Transferrina/metabolismo , Humanos , ARN Largo no Codificante/metabolismo , ARN Largo no Codificante/genética , Ratones , Transporte Biológico , Macaca fascicularis , Técnicas de Silenciamiento del Gen , Distribución TisularRESUMEN
Intracellular oncoproteins can be inhibited with targeted therapy, but responses are not durable. Immune therapies can be curative, but most oncogene-driven tumors are unresponsive to these agents. Fragments of intracellular oncoproteins can act as neoantigens presented by the major histocompatibility complex (MHC), but recognizing minimal differences between oncoproteins and their normal counterparts is challenging. We have established a platform technology that exploits hapten-peptide conjugates generated by covalent inhibitors to create distinct neoantigens that selectively mark cancer cells. Using the FDA-approved covalent inhibitors sotorasib and osimertinib, we developed "HapImmune" antibodies that bind to drug-peptide conjugate/MHC complexes but not to the free drugs. A HapImmune-based bispecific T-cell engager selectively and potently kills sotorasib-resistant lung cancer cells upon sotorasib treatment. Notably, it is effective against KRASG12C-mutant cells with different HLA supertypes, HLA-A*02 and A*03/11, suggesting loosening of MHC restriction. Our strategy creates targetable neoantigens by design, unifying targeted and immune therapies. SIGNIFICANCE: Targeted therapies against oncoproteins often have dramatic initial efficacy but lack durability. Immunotherapies can be curative, yet most tumors fail to respond. We developed a generalizable technology platform that exploits hapten-peptides generated by covalent inhibitors as neoantigens presented on MHC to enable engineered antibodies to selectively kill drug-resistant cancer cells. See related commentary by Cox et al., p. 19. This article is highlighted in the In This Issue feature, p. 1.
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Neoplasias Pulmonares , Complejo Mayor de Histocompatibilidad , Humanos , Neoplasias Pulmonares/patología , Antígenos de Histocompatibilidad Clase I , Antígenos de Neoplasias , Péptidos/farmacología , Antígenos de Histocompatibilidad , HaptenosRESUMEN
Brain exposure of systemically administered biotherapeutics is highly restricted by the blood-brain barrier (BBB). Here, we report the engineering and characterization of a BBB transport vehicle targeting the CD98 heavy chain (CD98hc or SLC3A2) of heterodimeric amino acid transporters (TVCD98hc). The pharmacokinetic and biodistribution properties of a CD98hc antibody transport vehicle (ATVCD98hc) are assessed in humanized CD98hc knock-in mice and cynomolgus monkeys. Compared to most existing BBB platforms targeting the transferrin receptor, peripherally administered ATVCD98hc demonstrates differentiated brain delivery with markedly slower and more prolonged kinetic properties. Specific biodistribution profiles within the brain parenchyma can be modulated by introducing Fc mutations on ATVCD98hc that impact FcγR engagement, changing the valency of CD98hc binding, and by altering the extent of target engagement with Fabs. Our study establishes TVCD98hc as a modular brain delivery platform with favorable kinetic, biodistribution, and safety properties distinct from previously reported BBB platforms.
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Barrera Hematoencefálica , Encéfalo , Animales , Ratones , Distribución Tisular , Anticuerpos , Ingeniería , Macaca fascicularisRESUMEN
The development of novel antimicrobial agents having high selectivity toward bacterial cells over mammalian cells is urgently required to curb the widespread emergence of infectious diseases caused by pathogenic bacteria. Toward this end, we have developed a set of cationic dimeric amphiphiles (bearing cleavable amide linkages between the headgroup and the hydrocarbon tail with different methylene spacers) that showed high antibacterial activity against human pathogenic bacteria (Escherichia coli and Staphylococcus aureus) and low cytotoxicity. The Minimum Inhibitory Concentrations (MIC) were found to be very low for the dimeric amphiphiles and were lower or comparable to the monomeric counterpart. In the case of dimeric amphiphiles, MIC was found to decrease with the increase in the spacer chain length (n = 2 to 6) and again to increase at higher spacer length (n > 6). It was found that the compound with six methylene spacers was the most active among all of the amphiphiles (MICs = 10-13 µM). By fluorescence spectroscopy, fluorescence microscopy, and field-emission scanning electron microscopy (FESEM), it was revealed that these cationic amphiphiles interact with the negatively charged bacterial cell membrane and disrupt the membrane integrity, thus killing the bacteria. All of the cationic amphiphiles showed low hemolytic activity (HC(50)) and high selectivity against both gram-positive and gram-negative bacteria. The most active amphiphile (n = 6) had a 10-13-fold higher HC(50) than did the MIC. Also, this amphiphile did not show any cytotoxicity against mammalian cells (HeLa cells) even at a concentration above the MIC (20 µM). The critical micellar concentration (CMC) values of gemini surfactants were found to be very low (CMC = 0.30-0.11 mM) and were 10-27 times smaller than the corresponding monomeric analogue (CMC = 2.9 mM). Chemical hydrolysis and thermogravimetric analysis (TGA) proved that these amphiphiles are quite stable under both acidic and thermal conditions. Collectively, these properties make the newly synthesized amphiphiles potentially superior disinfectants and antiseptics for various biomedical and biotechnological applications.
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Antibacterianos/farmacología , Antibacterianos/toxicidad , Hidrocarburos/farmacología , Hidrocarburos/toxicidad , Interacciones Hidrofóbicas e Hidrofílicas , Amidas/química , Antibacterianos/síntesis química , Antibacterianos/química , Permeabilidad de la Membrana Celular , Técnicas de Química Sintética , Dimerización , Escherichia coli/citología , Escherichia coli/efectos de los fármacos , Células HeLa , Hemólisis/efectos de los fármacos , Humanos , Hidrocarburos/síntesis química , Hidrocarburos/química , Micelas , Pruebas de Sensibilidad Microbiana , Solubilidad , Staphylococcus aureus/citología , Staphylococcus aureus/efectos de los fármacosRESUMEN
Despite increased investment and technological advancement, new drug approvals have not proportionally increased. Low drug approval rates, particularly for new targets, are linked to insufficient target validation at early stages. Thus, there remains a strong need for effective target validation techniques. Here, we review the use of synthetic binding proteins as tools for drug target validation, with focus on the monobody platform among several advanced synthetic binding protein platforms. Monobodies with high affinity and high selectivity can be rapidly developed against challenging targets, such as KRAS mutants, using protein engineering technologies. They have strong tendency to bind to functional sites and thus serve as drug-like molecules, and they can serve as targeting ligands for constructing bio-PROTACs. Genetically encoded monobodies are effective "tool biologics" for validating intracellular targets. They promote crystallization and help reveal the atomic structures of the monobody-target interface, which can inform drug design. Using case studies, we illustrate the potential of the monobody technology in accelerating target validation and small-molecule drug discovery.
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T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with a dismal prognosis in patients with resistant or relapsed disease. Although NOTCH is a known driver in T-ALL, its clinical inhibition has significant limitations. Our previous studies suggested that NRARP, a negative regulator of Notch signaling, could have a suppressive role in T-ALL. Here, we report that NRARP levels are significantly increased in primary T-ALL cells suggesting that NRARP is not sufficient to block NOTCH oncogenic signals. Interestingly, although NRARP overexpression blocks NOTCH1 signaling and delays the proliferation of T-ALL cells that display high levels of Notch1 signaling, it promotes the expansion of T-ALL cells with lower levels of Notch1 activity. We found that NRARP interacts with lymphoid enhancer-binding factor 1 (LEF1) and potentiates Wnt signaling in T-ALL cells with low levels of Notch. Together these results indicate that NRARP plays a dual role in T-ALL pathogenesis, regulating both Notch and Wnt pathways, with opposite functional effects depending on Notch activity. Consistent with this hypothesis, mice transplanted with T-cells co-expressing NOTCH1 and NRARP develop leukemia later than mice transplanted with T-NOTCH1 cells. Importantly, mice transplanted with T-cells overexpressing NRARP alone developed leukemia with similar kinetics to those transplanted with T-NOTCH1 cells. Our findings uncover a role for NRARP in T-ALL pathogenesis and indicate that Notch inhibition may be detrimental for patients with low levels of Notch signaling, which would likely benefit from the use of Wnt signaling inhibitors. Importantly, our findings may extend to other cancers where Notch and Wnt play a role.
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Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Receptores Notch/metabolismo , Vía de Señalización Wnt , Línea Celular Tumoral , Humanos , Regulación hacia ArribaRESUMEN
Interleukin 7 (IL-7) and its receptor (IL-7R, a heterodimer of IL-7Rα and γc) are essential for normal lymphoid development. In their absence, severe combined immunodeficiency occurs. By contrast, excessive IL-7/IL-7R-mediated signaling can drive lymphoid leukemia development, disease acceleration and resistance to chemotherapy. IL-7 and IL-7R activate three main pathways: STAT5, PI3K/Akt/mTOR and MEK/Erk, ultimately leading to the promotion of leukemia cell viability, cell cycle progression and growth. However, the contribution of each of these pathways towards particular functional outcomes is still not completely known and appears to differ between normal and malignant states. For example, IL-7 upregulates Bcl-2 in a PI3K/Akt/mTOR-dependent and STAT5-independent manner in T-ALL cells. This is a 'symmetric image' of what apparently happens in normal lymphoid cells, where PI3K/Akt/mTOR does not impact on Bcl-2 and regulates proliferation rather than survival. In this review, we provide an updated summary of the knowledge on IL-7/IL-7R-mediated signaling in the context of cancer, focusing mainly on T-cell acute lymphoblastic leukemia, where this axis has been more extensively studied.
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Ciclo Celular , Subunidad alfa del Receptor de Interleucina-7/metabolismo , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Transducción de Señal , Animales , Supervivencia Celular , Humanos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologíaRESUMEN
There is considerable interest in the development of chemical methods for the precise, site-selective modification of antibodies for therapeutic applications. In this protocol, we describe a strategy for the irreversible and selective modification of cysteine residues on antibodies, using functionalized carbonylacrylic reagents. This protocol is based on a thiol-Michael-type addition of native or engineered cysteine residues to carbonylacrylic reagents equipped with functional compounds such as cytotoxic drugs. This approach is a robust alternative to the conventional maleimide technique; the reaction is irreversible and uses synthetically accessible reagents. Complete conversion to the conjugates, with improved quality and homogeneity, is often achieved using a minimal excess (typically between 5 and 10 equiv.) of the carbonylacrylic reagent. Potential applications of this method cover a broad scope of cysteine-tagged antibodies in various formats (full-length IgGs, nanobodies) for the site-selective incorporation of cytotoxic drugs without loss of antigen-binding affinity. Both the synthesis of the carbonylacrylic reagent armed with a synthetic molecule of interest and the subsequent preparation of the chemically defined, homogeneous antibody conjugate can be achieved within 48 h and can be easily performed by nonspecialists. Importantly, the conjugates formed are stable in human plasma. The use of liquid chromatography-mass spectrometry (LC-MS) analysis is recommended for monitoring the progression of the bioconjugation reactions on protein and antibody substrates with accurate resolution.
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Acrilatos/química , Métodos Analíticos de la Preparación de la Muestra , Cisteína/química , Inmunoconjugados/química , Inmunoglobulinas/química , Anticuerpos de Dominio Único/química , Cromatografía Liquida , Reactivos de Enlaces Cruzados/química , Humanos , Inmunoconjugados/aislamiento & purificación , Inmunoglobulinas/aislamiento & purificación , Maleimidas/química , Anticuerpos de Dominio Único/aislamiento & purificación , Espectrometría de Masas en TándemRESUMEN
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological cancer for which treatment options often result in incomplete therapeutic efficacy and long-term side-effects. Interleukin 7 (IL-7) and its receptor IL-7Rα promote T-ALL development and mutational activation of IL-7Rα associates with very high risk in relapsed disease. Using combinatorial phage-display libraries and antibody reformatting, we generated a fully human IgG1 monoclonal antibody (named B12) against both wild-type and mutant human IL-7Rα, predicted to form a stable complex with IL-7Rα at a different site from IL-7. B12 impairs IL-7/IL-7R-mediated signaling, sensitizes T-ALL cells to treatment with dexamethasone and can induce cell death per se. The antibody also promotes antibody-dependent natural killer-mediated leukemia cytotoxicity in vitro and delays T-cell leukemia development in vivo, reducing tumor burden and promoting mouse survival. B12 is rapidly internalized and traffics to the lysosome, rendering it an attractive vehicle for targeted intracellular delivery of cytotoxic cargo. Consequently, we engineered a B12-MMAE antibody-drug conjugate and provide proof-of-concept evidence that it has increased leukemia cell killing abilities as compared with the naked antibody. Our studies serve as a stepping stone for the development of novel targeted therapies in T-ALL and other diseases where IL-7Rα has a pathological role.
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Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/economía , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Receptores de Interleucina-7/metabolismo , Animales , Línea Celular , Humanos , Inmunoglobulina G/metabolismo , Interleucina-7/metabolismo , Ratones , Transducción de Señal/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
Using machine learning, targets were identified for ß-lapachone. Resorting to biochemical assays, ß-lapachone was validated as a potent, ligand efficient, allosteric and reversible modulator of 5-lipoxygenase (5-LO). Moreover, we provide a rationale for 5-LO modulation and show that inhibition of 5-LO is relevant for the anticancer activity of ß-lapachone. This work demonstrates the power of machine intelligence to deconvolute complex phenotypes, as an alternative and/or complement to chemoproteomics and as a viable general approach for systems pharmacology studies.
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T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological cancer that arises from clonal expansion of transformed T-cell precursors. In this review we summarize the current knowledge on the external stimuli and cell-intrinsic lesions that drive aberrant activation of pivotal, pro-tumoral intracellular signaling pathways in T-cell precursors, driving transformation, leukemia expansion, spread or resistance to therapy. In addition to their pathophysiological relevance, receptors and kinases involved in signal transduction are often attractive candidates for targeted drug development. As such, we discuss also the potential of T-ALL signaling players as targets for therapeutic intervention.
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Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Transducción de Señal , Animales , Humanos , Modelos BiológicosRESUMEN
Bacterial colonization and subsequent formation of biofilms onto surfaces of medical devices and implants is a major source of nosocomial infections. Most antibacterial coatings to combat infections are either metal-based or nondegradable-polymer-based and hence limited by their nondegradability and unpredictable toxicity. Moreover, to combat infections effectively, the coatings are required to display simultaneous antibacterial and antibiofilm activity. Herein we report biocompatible and biodegradable coatings based on organo-soluble quaternary chitin polymers which were immobilized noncovalently onto surfaces as bactericidal paint. The polycationic paint was found to be active against both drug-sensitive and -resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), and ß-lactam-resistant Klebsiella pneumoniae. The cationic polymers were shown to interact with the negatively charged bacterial cell membrane and disrupt the membrane integrity, thereby causing leakage of intracellular constituents and cell death upon contact. Importantly, surfaces coated with the polymers inhibited formation of biofilms against both Gram-positive S. aureus and Gram-negative E. coli, two of the most clinically important bacteria that form biofilms. Surfaces coated with the polymers displayed negligible toxicity against human erythrocytes and embryo kidney cells. Notably, the polymers were shown to be susceptible toward lysozyme. Furthermore, subcutaneous implantation of polymer discs in rats led to 15-20% degradation in 4 weeks thereby displaying their biodegradability. In a murine model of subcutaneous infection, polymer-coated medical-grade catheter reduced MRSA burden by 3.7 log compared to that of noncoated catheter. Furthermore, no biofilm development was observed on the coated catheters under in vivo conditions. The polycationic materials thus developed herein represent a novel class of safe and effective coating agents for the prevention of device-associated infections.
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Pintura , Animales , Antibacterianos , Biopelículas , Escherichia coli , Humanos , Ratones , Pruebas de Sensibilidad Microbiana , Ratas , Staphylococcus aureusRESUMEN
The emergence of drug resistance along with a declining pipeline of clinically useful antibiotics has made it vital to develop more effective antimicrobial therapeutics, particularly against difficult-to-treat Gram-negative pathogens (GNPs). Many antibacterial agents, including glycopeptide antibiotics such as vancomycin, are inherently inactive toward GNPs because of their inability to cross the outer membrane of these pathogens. Here, we demonstrate, for the first time, lipophilic cationic (permanent positive charge) vancomycin analogues were able to permeabilize the outer membrane of GNPs and overcome the inherent resistance of GNPs toward glycopeptides. Unlike vancomycin, these analogues were shown to have a high activity against a variety of multidrug-resistant clinical isolates such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. In the murine model of carbapenem-resistant A. baumannii infection, the optimized compound showed potent activity with no observed toxicity. The notable activity of these compounds is attributed to the incorporation of new membrane disruption mechanisms (cytoplasmic membrane depolarization along with outer and inner (cytoplasmic) membrane permeabilization) into vancomycin. Therefore, our results indicate the potential of the present vancomycin analogues to be used against drug-resistant GNPs, thus strengthening the antibiotic arsenal for combating Gram-negative bacterial infections.
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Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Infecciones por Bacterias Gramnegativas/microbiología , Vancomicina/farmacología , Acinetobacter baumannii/efectos de los fármacos , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/microbiología , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/efectos de los fármacos , Relación Estructura-Actividad , Vancomicina/análogos & derivadosRESUMEN
Systemic chemotherapy, the current standard of care for the treatment of cancer, is rarely curative and is often accompanied by debilitating side effects. Targeted drug delivery stands as an alternative to chemotherapy, with the potential to improve upon its low efficacy and systemic toxicity. Among targeted therapeutic options, antibody-drug conjugates (ADCs) have emerged as the most promising. These conjugates represent a new class of biopharmaceuticals that selectively deliver potent cytotoxic drugs to cancer cells, sparing healthy tissue throughout the body. Despite this promise, early heterogenous ADCs suffered from stability, pharmacokinetic, and efficacy issues that hindered clinical development. Recent advances in antibody engineering, linkers for drug-release, and chemical site-selective antibody conjugation have led to the creation of homogenous ADCs that have proven to be more efficacious than their heterogeneous predecessors both in vitro and in vivo. In this minireview, we focus on and discuss recent advances in chemical site-selective modification strategies for the conjugation of drugs to antibodies and the resulting potential for the development of a new generation of homogenous ADCs.
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Microbial attachment and subsequent colonization onto surfaces lead to the spread of deadly community-acquired and hospital-acquired (nosocomial) infections. Noncovalent immobilization of water insoluble and organo-soluble cationic polymers onto a surface is a facile approach to prevent microbial contamination. In the present study, we described the synthesis of water insoluble and organo-soluble polymeric materials and demonstrated their structure-activity relationship against various human pathogenic bacteria including drug-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and beta lactam-resistant Klebsiella pneumoniae as well as pathogenic fungi such as Candida spp. and Cryptococcus spp. The polymer coated surfaces completely inactivated both bacteria and fungi upon contact (5 log reduction with respect to control). Linear polymers were more active and found to have a higher killing rate than the branched polymers. The polymer coated surfaces also exhibited significant activity in various complex mammalian fluids such as serum, plasma, and blood and showed negligible hemolysis at an amount much higher than minimum inhibitory amounts (MIAs). These polymers were found to have excellent compatibility with other medically relevant polymers (polylactic acid, PLA) and commercial paint. The cationic hydrophobic polymer coatings disrupted the lipid membrane of both bacteria and fungi and thus showed a membrane-active mode of action. Further, bacteria did not develop resistance against these membrane-active polymers in sharp contrast to conventional antibiotics and lipopeptides, thus the polymers hold great promise to be used as coating materials for developing permanent antimicrobial paint.