Insight into drug resistance mechanisms and discovery of potential inhibitors against wild-type and L1196M mutant ALK from FDA-approved drugs

Anaplastic lymphoma kinase (ALK) plays a crucial role in multiple malignant cancers. It is known as a well-established target for the treatment of ALK-dependent cancers. Even though substantial efforts have been made to develop ALK inhibitors, only crizotinib, ceritinib, and alectinib had been approved by the U.S. Food and Drug Administration for patients with ALK-positive non-small cell lung cancer (NSCLC). The secondary mutations with drug-resistance bring up difficulties to develop effective drugs for ALK-positive cancers. To give a comprehensive understanding of molecular mechanism underlying inhibitor response to ALK tyrosine kinase mutations, we established an accurate assessment for the extensive profile of drug against ALK mutations by means of computational approaches. The molecular mechanics-generalized Born surface area (MM-GBSA) method based on molecular dynamics (MD) simulation was carried out to calculate relative binding free energies for receptor-drug systems. In addition, the structure-based virtual screening was utilized to screen effective inhibitors targeting wild-type ALK and the gatekeeper mutation L1196M from 3180 approved drugs. Finally, the mechanism of drug resistance was discussed, several novel potential wild-type and L1196M mutant ALK inhibitors were successfully identified.     

Theoretical insights into the stabilities,detonation performance,and electrostatic potentials of cocrystals containing α- or β-HMX and TATB,FOX-7, NTO,or DMF in various molar ratios

A molecular dynamics method was employed to study the binding energies associated with the cocrystallization (at selected crystal planes) of either 1,3,5-triamino-2,4,6-trinitro-benzene (TATB), 1,1-diamino-2,2-dinitroethylene, 3-nitro-1,2,4-triazol-5-one (TATB, FOX-7, and NTO, respectively, all of which are explosives), or N,N-dimethylformamide (DMF, a nonenergetic solvent) in various molar ratios with 1,3,5,7-tetranitro-1,3,5,7-tetrazacyclooctane in its α and β conformations (α-HMX and β-HMX, respectively). The results showed that the cocrystals with low molar ratios (2:1, 1:1, 1:2, and 1:3) were the most stable. The binding energies of HMX/NTO and HMX/DMF were larger than those of HMX/TATB and HMX/FOX-7. According to the calculated stabilities, HMX prefers to adopt its α form in HMX/TATB and its β form in HMX/NTO, whereas the two forms coexist in HMX/FOX-7. For HMX/TATB, HMX/NTO, and α-HMX/FOX-7, increasing the proportion of the cocrystal component with the highest detonation heat (HMX in the first two cases, FOX-7 in the latter) increases the detonation heat, velocity, and pressure of the cocrystal. However, increasing the proportion of the component with the highest detonation heat in β-HMX/FOX-7 and γ-CL-20/FOX-7 increases the detonation heat of the cocrystal but decreases its detonation velocity. An investigation of the surface electrostatic potential revealed how the sensitivity changes upon cocrystal formation.

Open image in new window

Graphical Abstract Surface electrostatic potential of HMX/TATB

     

A bispecific antibody effectively neutralizes all four serotypes of dengue virus by simultaneous blocking virus attachment and fusion

Although dengue virus (DENV) infection severely threatens the health of humans, no specific antiviral drugs are currently approved for clinical use against DENV infection. Attachment and fusion are 2 critical steps for the flavivirus infection, and the corresponding functional epitopes are located at E protein domain III (E-DIII) and domain II (E-DII), respectively. Here, we constructed a bispecific antibody (DVD-1A1D-2A10) based on the 2 well-characterized anti-DENV monoclonal antibodies 1A1D-2 (1A1D) and 2A10G6 (2A10). The 1A1D antibody binds E-DIII and can block the virus attaching to the cell surface, while the 2A10 antibody binds E-DII and is able to prevent the virus from fusing with the endosomal membrane. Our data showed that DVD-1A1D-2A10 retained the antigen-binding activity of both parental antibodies. Importantly, it was demonstrated to be significantly more effective at neutralizing DENV than its parental antibodies both in vitro and in vivo, even better than the combination of them. To eliminate the potential antibody-dependent enhancement (ADE) effect, this bispecific antibody was successfully engineered to prevent Fc-γ-R interaction. Overall, we generated a bispecific anti-DENV antibody targeting both attachment and fusion stages, and this bispecific antibody broadly neutralized all 4 serotypes of DENV without risk of ADE, suggesting that it has great potential as a novel antiviral strategy against DENV.     

Local proliferation initiates macrophage accumulation in adipose tissue during obesity

Obesity-associated chronic inflammation is characterized by an accumulation of adipose tissue macrophages (ATMs). It is generally believed that those macrophages are derived from peripheral blood monocytes. However, recent studies suggest that local proliferation of macrophages is responsible for ATM accumulation. In the present study, we revealed that both migration and proliferation contribute to ATM accumulation during obesity development. We show that there is a significant increase in ATMs at the early stage of obesity, which is largely due to an enhanced in situ macrophage proliferation. This result was obtained by employing fat-shielded irradiation and bone marrow reconstitution. Additionally, the production of CCL2, a pivotal chemoattractant of monocytes, was not found to be increased at this stage, corroborating with a critical role of proliferation. Nonetheless, as obesity proceeds, the role of monocyte migration into adipose tissue becomes more significant and those new immigrants further proliferate locally. These proliferating ATMs mainly reside in crown-like structures formed by macrophages surrounding dead adipocytes. We further showed that IL-4/STAT6 is a driving force for ATM proliferation. Therefore, we demonstrated that local proliferation of resident macrophages contributes to ATM accumulation during obesity development and has a key role in obesity-associated inflammation.The accumulation of adipose tissue macrophages (ATMs) is a significant characteristic of obesity-associated chronic inflammation. It is also critical in regulating obesity development. In lean animals, there is a low cellularity of resident ATMs interspersing among adipocytes, which are considered as M2 macrophages. During obesity, significantly increased macrophages accumulate in adipose tissue and form the so-called ‘crown-like structures'' (CLSs) around the dead adipocytes.^{1, 2} Those macrophages exhibit M1 phenotype and produce various types of inflammatory cytokines, such as TNF-α, resulting in the propagation of obesity-related inflammation and the development of metabolic disorders, such as insulin resistance.^{3, 4, 5}Traditionally, the accumulated ATMs are considered as a consequence of peripheral monocyte migration under inflammatory conditions. Recently, increasing evidences have shown that the maintenance of tissue macrophages is probably independent of the replenishment of circulating monocytes and even independent of precursors from bone marrow.⁶ Indeed, several kinds of tissue macrophages are capable of self-renewal and proliferate locally in naive state, such as microglia,^{7, 8} Kupffer cells,⁹ and Langerhans cells.¹⁰In acute inflammation status, for instance, during parasitic infection, local proliferation of macrophages is boosted and these macrophages exhibit phenotypes of alternatively activated macrophages, a process driven by Th2 cytokines.¹¹ In chronic inflammation conditions, such as atherosclerosis, local proliferation of macrophages also occurs and contributes to macrophage accumulation in arterial walls.¹² Most recently, it has been reported that local proliferation of macrophages could contribute to the ATM accumulation in obesity.^{13, 14}Given the potential contributions of monocyte migration and macrophage proliferation to ATM accumulation, an important question about the respective role of each event in ATM accumulation during obesity is raising. To address it, we first focus on the initiation of ATM accumulation in obesity. We found that, although there is no significant change in the level of chemokine (C-C motif) ligand 2 (CCL2) either in adipose tissue or in circulation, the cellularity of ATMs is dramatically elevated at the early stage of obesity. Interestingly, the increase of ATMs was accompanied with vigorous ATM proliferation. By inducing obesity in chimeric mice that were generated by fat-shielded irradiation and bone marrow transplantation, we demonstrated that in situ proliferation of resident macrophages dominates the initiation of ATM accumulation at early stage of obesity, and the recruited monocytes make contribution to ATM accumulation at a relatively late stage of obesity. This study sheds light on the dynamic process of ATM accumulation and provides insight on the initiation of obesity-associated inflammation.     

GADD45B mediates podocyte injury in zebrafish by activating the ROS-GADD45B-p38 pathway

GADD45 gene has been implicated in cell cycle arrest, cell survival or apoptosis in a cell type specific and context-dependent manner. Members of GADD45 gene family have been found differentially expressed in several podocyte injury models, but their roles in podocytes are unclear. Using an in vivo zebrafish model of inducible podocyte injury that we have previously established, we found that zebrafish orthologs of gadd45b were induced upon the induction of podocyte injury. Podocyte-specific overexpression of zebrafish gadd45b exacerbated edema, proteinuria and foot-process effacement, whereas knockdown of gadd45b by morpholino-oligos in zebrafish larvae ameliorated podocyte injury. We then explored the role of GADD45B induction in podocyte injury using in vitro podocyte culture. We confirmed that GADD45B was significantly upregulated during the early phase of podocyte injury in cultured human podocytes and that podocyte apoptosis induced by TGF-β and puromycin aminonucleoside (PAN) was aggravated by GADD45B overexpression but ameliorated by shRNA-mediated GADD45B knockdown. We also showed that ROS inhibitor NAC suppressed PAN-induced GADD45B expression and subsequent activation of p38 MAPK pathway in podocytes and that inhibition of GADD45B diminished PAN-induced p38 MAPK activation. Taken together, our findings demonstrated that GADD45B has an important role in podocyte injury and may be a therapeutic target for the management of podocyte injury in glomerular diseases.Podocyte dysfunction, injury or loss is a common and decisive cause of various glomerular diseases and understanding the molecular mechanism underlying podocyte response to stress will be very helpful to undermine the pathogenesis of podocyte injury and the targeted therapy for glomerular diseases.The members of Gadd45 gene family, Gadd45a, Gadd45b and Gadd45r have been commonly implicated in stress signaling in response to physiological or environmental stressors, resulting in cell cycle arrest, DNA damage repair, cell survival, senescence and apoptosis.¹ Recently, this gene family has been found differentially expressed in several podocyte injury models. Zhang et al.² observed an induction of GADD45β mRNA expression by lipopolysaccharide in the lung, kidney and spleen, which had the highest GADD45β mRNA expression among all of the tissues examined. Jeffrey W Pippin reported that protein expression of GADD45 was increased in glomeruli from passive Heymann nephritis rats and cultured podocytes exposed in vitro to C5b-9. ³ More recently, Shi et al.⁴ reported that Gadd45b was upregulated in glomeruli of mice with podocyte-specific deletion of Dicer, suggesting the involvement of Gadd45b in podocyte injury. However, no functional characterization of Gadd45 genes in podocytes has been conducted to date and the role of GADD45B in the context of podocyte injury remains unclear.Zebrafish has emerged as a new vertebrate model system for renal glomerular research. The podocytes and renal glomeruli in zebrafish kidney are structurally, molecularly and functionally conserved, rendering zebrafish a valuable and relevant model for podocyte studies. To characterize the role of GADD45b in podocyte injury, we therefore employed zebrafish as an in vivo model system and human podocytes as an in vitro model. We observed the upregulation of GADD45B on podocyte injury in zebrafish renal glomeruli as well as in cultured human podocytes treated with TGF-β and PAN. We further showed that podocyte-specific overexpression of zebrafish orthologs of gadd45b predisposed podocytes to injury, whereas inhibition of gadd45b expression in zebrafish larvae ameliorated podocyte injury and reduced proteinuria. Furthermore, we found that the ROS-GADD45B-p38 pathway was involved in the regulation of GADD45B expression and deleterious role in podocyte injury. Collectively, we have identified GADD45B as an important player in podocyte injury.     

Granzyme M has a critical role in providing innate immune protection in ulcerative colitis

Inflammatory bowel disease (IBD) is an immunoregulatory disorder, associated with a chronic and inappropriate mucosal immune response to commensal bacteria, underlying disease states such as ulcerative colitis (UC) and Crohn''s disease (CD) in humans. Granzyme M (GrzM) is a serine protease expressed by cytotoxic lymphocytes, in particular natural killer (NK) cells. Granzymes are thought to be involved in triggering cell death in eukaryotic target cells; however, some evidence supports their role in inflammation. The role of GrzM in the innate immune response to mucosal inflammation has never been examined. Here, we discover that patients with UC, unlike patients with CD, display high levels of GrzM mRNA expression in the inflamed colon. By taking advantage of well-established models of experimental UC, we revealed that GrzM-deficient mice have greater levels of inflammatory indicators during dextran sulfate sodium (DSS)-induced IBD, including increased weight loss, greater colon length reduction and more severe intestinal histopathology. The absence of GrzM expression also had effects on gut permeability, tissue cytokine/chemokine dynamics, and neutrophil infiltration during disease. These findings demonstrate, for the first time, that GrzM has a critical role during early stages of inflammation in UC, and that in its absence colonic inflammation is enhanced.Inflammatory bowel disease (IBD) is a gut-associated inflammatory disorder, which stems from a dysfunctional mucosal immune response to commensal bacteria.¹ As a multifactorial disease, IBD is the consequence of a complex interplay between environmental triggers, genetic susceptibility, and immunoregulatory defects, resulting in a pathogenesis that is still poorly understood.² These interactions result in the inability of an individual to control the normal inflammatory response to pathogens in the gut, leading to a chronic state of sustained and inappropriate inflammation. IBD underlies disease states such as ulcerative colitis (UC) and Crohn''s disease (CD), with symptoms including weight loss, abdominal pain, diarrhea, and rectal bleeding which often require intensive medical therapy and resective surgery.³ The pathogenesis of IBD, characterized by a defective mucosal immune response to microbial exposure in the gastrointestinal tract, is thought to be caused by a dysfunctional immune response to host microbiota, infection by specific pathogens, and/or a defective mucosal barrier to luminal pathogens.^{1, 2} IBD patients also have a high risk of developing colitis-associated colon cancer (CAC).⁴ Additionally, histological assessment of inflamed ileal and colonic segments from IBD patients typically shows increased infiltration of immune cells, particularly neutrophils, as well as crypt abscesses, mucin depletion, and ulcers—all correlating with the severity of small bowel and colonic tissue damage.⁵Cytotoxic pathways mediated by lymphocytes directly trigger cell death in target cells.⁶ These cytotoxic pathways are mediated by proteins such as perforin, which mediates pore formation in the target cell surface and allows granzyme (Grz)s to enter the intracellular compartment and induce cell death.⁷ To date, five different Grzs have been identified in humans (GrzA, GrzB, GrzH, GrzK, and GrzM), whereas mice express eleven Grzs (GrzA, GrzB, GrzC, GrzD, GrzE, GrzF, GrzG, GrzK, GrzL, GrzM, and GrzN).^{8, 9} Walch et al.¹⁰ recently demonstrated that Grzs (GrzA and GrzB) directly kill bacteria through granulysin-mediated delivery, suggesting that Grzs act as microbial modulating factors. Moreover, recently GrzA was shown to be increased in the colon biopsies of UC patients undergoing treatment with Etrolizumab, a monoclonal antibody targeting the β7 integrin subunit. Higher levels of GrzA could predict which patients were more likely to benefit from the therapy; however, the precise mechanism of action of GrzA in UC remains to be addressed.¹¹ GrzM was initially described as being constitutively expressed by natural killer (NK) cells,^{12, 13} and specifically associated with inflammation.¹⁴ This enzyme has been shown to preferentially cleave methionine and leucine residues in target cells, mediating direct, non-specific cell death.^{15, 16} More recently, GrzM was also shown to be an important mediator for the release of MIP-1α from NK cells, inducing NK cell and neutrophil recruitment during early microbial infection.¹⁷ We now observe that GrzM expression is increased in inflamed colon tissue samples from UC, but not CD patients. Further, GrzM-deficient (GrzM^−/−) mice are more sensitive to a mouse model of IBD and IBD-induced colorectal cancer (CRC). These findings demonstrate, for the first time, that GrzM has a critical role in mediating the early stages of the gut mucosal immune response.     

Highly Sensitive Detection of Melamine Using a One-Step Sample Treatment Combined with a Portable Ag Nanostructure Array SERS Sensor

There is an urgent need for rapid and reliable methods able to detect melamine in animal feed. In this study, a quick, simple, and sensitive method for the determination of melamine content in animal feed was developed using surface-enhanced Raman spectroscopy on fabricated Ag nanorod (AgNR) array substrates with a one-step sample extraction procedure. The AgNR array substrates washed by HNO₃ solvent (10⁻⁷ M) and methanol and showed the good stability within 6 months. The Raman shift at △ν = 682 cm⁻¹ was used as the characteristic melamine peak in the calculations. Sufficient linearity was obtained in the 2–200 μg·g⁻¹ range (R² = 0.926). The limits of detection and quantification were 0.9 and 2 μg·g⁻¹, respectively. The recovery rates were 89.7–93.3%, with coefficients of variation below 2.02%. The method showed good accuracy compared with the tradition GC-MS analysis. This new protocol only need 2 min to fininsh the detection which could be developed for rapid onsite screening of melamine contamination in quality control and market surveillance applications.