Exploring switch II pocket conformation of KRAS(G12D) with mutant-selective monobody inhibitors.

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.

Mutations in the α4-α5 allosteric lobe of RAS do not significantly impair RAS signaling or self-association.

Mutations in one of the three RAS genes (HRAS, KRAS, and NRAS) are present in nearly 20% of all human cancers. These mutations shift RAS to the GTP-loaded active state due to impairment in the intrinsic GTPase activity and disruption of GAP-mediated GTP hydrolysis, resulting in constitutive activation of effectors such as RAF. Because activation of RAF involves dimerization, RAS dimerization has been proposed as an important step in RAS-mediated activation of effectors. The α4-α5 allosteric lobe of RAS has been proposed as a RAS dimerization interface. Indeed, the NS1 monobody, which binds the α4-α5 region within the RAS G domain, inhibits RAS-dependent signaling and transformation as well as RAS nanoclustering at the plasma membrane. Although these results are consistent with a model in which the G domain dimerizes through the α4-α5 region, the isolated G domain of RAS lacks intrinsic dimerization capacity. Furthermore, prior studies analyzing α4-α5 point mutations have reported mixed effects on RAS function. Here, we evaluated the activity of a panel of single amino acid substitutions in the α4-α5 region implicated in RAS dimerization. We found that these proposed "dimerization-disrupting" mutations do not significantly impair self-association, signaling, or transformation of oncogenic RAS. These results are consistent with a model in which activated RAS protomers cluster in close proximity to promote the dimerization of their associated effector proteins (e.g., RAF) without physically associating into dimers mediated by specific molecular interactions. Our findings suggest the need for a nonconventional approach to developing therapeutics targeting the α4-α5 region.

Helicobacter pylori employs a general protein glycosylation system for the modification of outer membrane adhesins.

Helicobacter pylori infection is associated with the development of several gastric diseases including gastric cancer. To reach a long-term colonization in the host stomach, H. pylori employs multiple outer membrane adhesins for binding to the gastric mucosa. However, due to the redundancy of adhesins that complement the adhesive function of bacteria, targeting each individual adhesin alone usually achieves nonideal outcomes for preventing bacterial adhesion. Here, we report that key adhesins AlpA/B and BabA/B in H. pylori are modified by glycans and display a two-step molecular weight upshift pattern from the cytoplasm to the inner membrane and from the inner membrane to the outer membrane. Nevertheless, this upshift pattern is missing when the expression of some enzymes related to lipopolysaccharide (LPS) biosynthesis, including the LPS O-antigen assembly and ligation enzymes WecA, Wzk, and WaaL, is disrupted, indicating that the underlying mechanisms and the involved enzymes for the adhesin glycosylation are partially shared with the LPS biosynthesis. Loss of the adhesin glycosylation not only reduces the protease resistance and the stability of the tested adhesins but also changes the adhesin-binding ability. In addition, mutations in the LPS biosynthesis cause a significant reduction in bacterial adhesion in the in vitro cell-line model. The current findings reveal that H. pylori employs a general protein glycosylation system related to LPS biosynthesis for adhesin modification and its biological significance. The enzymes required for adhesin glycosylation rather than the adhesins themselves are potentially better drug targets for preventing or treating H. pylori infection.

Inhibition of RAS-driven signaling and tumorigenesis with a pan-RAS monobody targeting the Switch I/II pocket.

RAS mutants are major therapeutic targets in oncology with few efficacious direct inhibitors available. The identification of a shallow pocket near the Switch II region on RAS has led to the development of small-molecule drugs that target this site and inhibit KRAS(G12C) and KRAS(G12D). To discover other regions on RAS that may be targeted for inhibition, we have employed small synthetic binding proteins termed monobodies that have a strong propensity to bind to functional sites on a target protein. Here, we report a pan-RAS monobody, termed JAM20, that bound to all RAS isoforms with nanomolar affinity and demonstrated limited nucleotide-state specificity. Upon intracellular expression, JAM20 potently inhibited signaling mediated by all RAS isoforms and reduced oncogenic RAS-mediated tumorigenesis in vivo. NMR and mutation analysis determined that JAM20 bound to a pocket between Switch I and II, which is similarly targeted by low-affinity, small-molecule inhibitors, such as BI-2852, whose in vivo efficacy has not been demonstrated. Furthermore, JAM20 directly competed with both the RAF(RBD) and BI-2852. These results provide direct validation of targeting the Switch I/II pocket for inhibiting RAS-driven tumorigenesis. More generally, these results demonstrate the utility of tool biologics as probes for discovering and validating druggable sites on challenging targets.

Identification of the nucleotide-free state as a therapeutic vulnerability for inhibition of selected oncogenic RAS mutants.

RAS guanosine triphosphatases (GTPases) are mutated in nearly 20% of human tumors, making them an attractive therapeutic target. Following our discovery that nucleotide-free RAS (apo RAS) regulates cell signaling, we selectively target this state as an approach to inhibit RAS function. Here, we describe the R15 monobody that exclusively binds the apo state of all three RAS isoforms in vitro, regardless of the mutation status, and captures RAS in the apo state in cells. R15 inhibits the signaling and transforming activity of a subset of RAS mutants with elevated intrinsic nucleotide exchange rates (i.e., fast exchange mutants). Intracellular expression of R15 reduces the tumor-forming capacity of cancer cell lines driven by select RAS mutants and KRAS(G12D)-mutant patient-derived xenografts (PDXs). Thus, our approach establishes an opportunity to selectively inhibit a subset of RAS mutants by targeting the apo state with drug-like molecules.

The Effects of HP0044 and HP1275 Knockout Mutations on the Structure and Function of Lipopolysaccharide in Helicobacter pylori Strain 26695.

Helicobacter pylori infection is associated with several gastric diseases, including gastritis, peptic ulcer, gastric adenocarcinoma and mucosa-associated lymphatic tissue (MALT) lymphoma. Due to the prevalence and severeness of H. pylori infection, a thorough understanding of this pathogen is necessary. Lipopolysaccharide, one of the major virulence factors of H. pylori, can exert immunomodulating and immunostimulating functions on the host. In this study, the HP0044 and HP1275 genes were under investigation. These two genes potentially encode GDP-D-mannose dehydratase (GMD) and phosphomannomutase (PMM)/phosphoglucomutase (PGM), respectively, and are involved in the biosynthesis of fucose. HP0044 and HP1275 knockout mutants were generated; both mutants displayed a truncated LPS, suggesting that the encoded enzymes are not only involved in fucose production but are also important for LPS construction. In addition, these two gene knockout mutants exhibited retarded growth, increased surface hydrophobicity and autoaggregation as well as being more sensitive to the detergent SDS and the antibiotic novobiocin. Furthermore, the LPS-defective mutants also had significantly reduced bacterial infection, adhesion and internalization in the in vitro cell line model. Moreover, disruptions of the HP0044 and HP1275 genes in H. pylori altered protein sorting into outer membrane vesicles. The critical roles of HP0044 and HP1275 in LPS biosynthesis, bacterial fitness and pathogenesis make them attractive candidates for drug inventions against H. pylori infection.

Helicobacter pylori GmhB enzyme involved in ADP-heptose biosynthesis pathway is essential for lipopolysaccharide biosynthesis and bacterial virulence.

Helicobacter pylori infection is linked to serious gastric-related diseases including gastric cancer. However, current therapies for treating H. pylori infection are challenged by the increased antibiotic resistance of H. pylori. Therefore, it is in an urgent need to identify novel targets for drug development against H. pylori infection. In this study, HP0860 gene from H. pylori predicted to encode a D-glycero-D-manno-heptose-1,7-bisphosphate phosphatase (GmhB) involved in the synthesis of ADP-L-glycero-D-manno-heptose for the assembly of lipopolysaccharide (LPS) in the inner core region was cloned and characterized. We reported HP0860 protein is monomeric and functions as a phosphatase by converting D-glycero-D-manno-heptose-1,7-bisphosphate into D-glycero-D-manno-heptose-1-phosphate with a preference for the ß-anomer over the α-anomer of sugar phosphate substrates. Subsequently, a HP0860 knockout mutant and its complementary mutant were constructed and their phenotypic properties were examined. HP0860 knockout mutant contained both mature and immature forms of LPS and could still induce significant IL-8 secretion after gastric AGS cell infection, suggesting other enzymatic activities in HP0860 knockout mutant might be able to partially compensate for the loss of HP0860 activity. In addition, HP0860 knockout mutant was much more sensitive to antibiotic novobiocin, had decreased adherence abilities, and caused less classic hummingbird phenotype on the infected AGS cells, indicating H. pylori lacking HP0860 is less virulent. Furthermore, the disruption of HP0860 gene altered the sorting of cargo proteins into outer membrane vesicles (OMVs). The above findings confirm the importance of HP0860 in LPS core biosynthesis and shed light on therapeutic intervention against H. pylori infection.

Selective and noncovalent targeting of RAS mutants for inhibition and degradation.

Activating mutants of RAS are commonly found in human cancers, but to date selective targeting of RAS in the clinic has been limited to KRAS(G12C) through covalent inhibitors. Here, we report a monobody, termed 12VC1, that recognizes the active state of both KRAS(G12V) and KRAS(G12C) up to 400-times more tightly than wild-type KRAS. The crystal structures reveal that 12VC1 recognizes the mutations through a shallow pocket, and 12VC1 competes against RAS-effector interaction. When expressed intracellularly, 12VC1 potently inhibits ERK activation and the proliferation of RAS-driven cancer cell lines in vitro and in mouse xenograft models. 12VC1 fused to VHL selectively degrades the KRAS mutants and provides more extended suppression of mutant RAS activity than inhibition by 12VC1 alone. These results demonstrate the feasibility of selective targeting and degradation of KRAS mutants in the active state with noncovalent reagents and provide a starting point for designing noncovalent therapeutics against oncogenic RAS mutants.

SHP2 inhibition diminishes KRASG12C cycling and promotes tumor microenvironment remodeling.

KRAS is the most frequently mutated human oncogene, and KRAS inhibition has been a longtime goal. Recently, inhibitors were developed that bind KRASG12C-GDP and react with Cys-12 (G12C-Is). Using new affinity reagents to monitor KRASG12C activation and inhibitor engagement, we found that an SHP2 inhibitor (SHP2-I) increases KRAS-GDP occupancy, enhancing G12C-I efficacy. The SHP2-I abrogated RTK feedback signaling and adaptive resistance to G12C-Is in vitro, in xenografts, and in syngeneic KRASG12C-mutant pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC). SHP2-I/G12C-I combination evoked favorable but tumor site-specific changes in the immune microenvironment, decreasing myeloid suppressor cells, increasing CD8+ T cells, and sensitizing tumors to PD-1 blockade. Experiments using cells expressing inhibitor-resistant SHP2 showed that SHP2 inhibition in PDAC cells is required for PDAC regression and remodeling of the immune microenvironment but revealed direct inhibitory effects on tumor angiogenesis and vascularity. Our results demonstrate that SHP2-I/G12C-I combinations confer a substantial survival benefit in PDAC and NSCLC and identify additional potential combination strategies.

Evaluation of Polycaprolactone/Gelatin/Chitosan Electrospun Membrane for Peritoneal Adhesion Reduction.

In this study, a novel antiadhesion membrane made of polycaprolactone, gelatin, and chitosan was fabricated using the electrospinning technique. A series of polycaprolactone/gelatin/chitosan (PGC) electrospun membranes with different amounts of chitosan (0%, 0.5%, 1%, and 2% in weight percentage) was synthesized. The physicochemical properties and biocompatibility of the fabricated membranes were examined and compared with the aim to select an effective antiadhesion membrane. Scanning electron microscopy showed that these 4 electrospun membranes had similar fiber diameter and pore area, with no statistical differences between them. Furthermore, the contact angle decreased with increased chitosan content, indicating that chitosan may contribute to increased hydrophilic properties. The in vitro degradation test revealed that the higher chitosan content corresponded to a lower degradation rate in PGC membranes within 7 days. All PGC membranes exhibited similar cell proliferation; however, cell proliferation was lower than tissue culture polystyrene (P < 0.05). To compare antiadhesion ability, the adhesion between the cecum and abdominal wall was created in a rat model. Assessment after implantation of electrospun membranes revealed that PGCs with higher chitosan content (PGC2) had better antiadhesion effects, as evaluated by an adhesion score at day 14 postsurgery. Thus, PGC2 was effective in reducing the formation of tissue adhesion. Therefore, PGC electrospun membrane may provide a potential peritoneal antiadhesion barrier for clinical use.

Characterization of Protoporphyrin IX Species in Vitro Using Fluorescence Spectroscopy and Polar Plot Analysis.

Protoporphyrin IX (PPIX) is a photodynamic therapy (PDT) agent for the treatment of various types of cancer. The effectiveness of PDT is believed to be associated with aggregation of PPIX in cells. However, the aggregation equilibrium of PPIX in the cellular environment and in solution is still poorly understood. This is attributed by the lack of a method that allows for controllable generation of PPIX aggregates and robust analysis technique for measuring their photophysical properties. In this study, the dynamics of PPIX aggregation were investigated under high pressure and different solvent conditions using time-resolved fluorescence spectroscopy. The data were analyzed on a polar plot, a model-free analysis method that has become increasingly popular for fluorescence lifetime studies. We discovered that increasing hydrostatic pressure enhanced the formation of J-type aggregates based on measured absorbance, spectra, and lifetime features. Formation of large aggregates, which have a subnanosecond lifetime in the excited state, was observed under the increasing concentration of divalent cations as well as under a solvent of around neutral pH. PPIX monomerizes from the aggregate as pH becomes more basic, not dimerization as proposed by previous studies. Here, we demonstrate that the combination of time-resolved measurement and polar plot analysis is very robust for monitoring the presence of different types of PPIX aggregates formed in various chemical environments.

Labeling proteins inside living cells using external fluorophores for microscopy.

Site-specific fluorescent labeling of proteins inside live mammalian cells has been achieved by employing Streptolysin O, a bacterial enzyme which forms temporary pores in the membrane and allows delivery of virtually any fluorescent probes, ranging from labeled IgG's to small ligands, with high efficiency (>85% of cells). The whole process, including recovery, takes 30 min, and the cell is ready to be imaged immediately. A variety of cell viability tests were performed after treatment with SLO to ensure that the cells have intact membranes, are able to divide, respond normally to signaling molecules, and maintains healthy organelle morphology. When combined with Oxyrase, a cell-friendly photostabilizer, a ~20x improvement in fluorescence photostability is achieved. By adding in glutathione, fluorophores are made to blink, enabling super-resolution fluorescence with 20-30 nm resolution over a long time (~30 min) under continuous illumination. Example applications in conventional and super-resolution imaging of native and transfected cells include p65 signal transduction activation, single molecule tracking of kinesin, and specific labeling of a series of nuclear and cytoplasmic protein complexes.

Multidentate Polymer Coatings for Compact and Homogeneous Quantum Dots with Efficient Bioconjugation.

Quantum dots are fluorescent nanoparticles used to detect and image proteins and nucleic acids. Compared with organic dyes and fluorescent proteins, these nanocrystals have enhanced brightness, photostability, and wavelength tunability, but their larger size limits their use. Recently, multidentate polymer coatings have yielded stable quantum dots with small hydrodynamic dimensions (≤10 nm) due to high-affinity, compact wrapping around the nanocrystal. However, this coating technology has not been widely adopted because the resulting particles are frequently heterogeneous and clustered, and conjugation to biological molecules is difficult to control. In this article we develop new polymeric ligands and optimize coating and bioconjugation methodologies for core/shell CdSe/Cd(x)Zn(1-x)S quantum dots to generate homogeneous and compact products. We demonstrate that "ligand stripping" to rapidly displace nonpolar ligands with hydroxide ions allows homogeneous assembly with multidentate polymers at high temperature. The resulting aqueous nanocrystals are 7-12 nm in hydrodynamic diameter, have quantum yields similar to those in organic solvents, and strongly resist nonspecific interactions due to short oligoethylene glycol surfaces. Compared with a host of other methods, this technique is superior for eliminating small aggregates identified through chromatographic and single-molecule analysis. We also demonstrate high-efficiency bioconjugation through azide-alkyne click chemistry and self-assembly with hexa-histidine-tagged proteins that eliminate the need for product purification. The conjugates retain specificity of the attached biomolecules and are exceptional probes for immunofluorescence and single-molecule dynamic imaging. These results are expected to enable broad utilization of compact, biofunctional quantum dots for studying crowded macromolecular environments such as the neuronal synapse and cellular cytoplasm.