Search

The utility of antibody therapeutics is hampered by potential cross-reactivity with healthy tissue. Over the past decade, significant advances have been made in the design of activatable antibodies, which increase, or create altogether, the therapeutic window of a parent antibody. Of these, antibody prodrugs (pro-antibodies) are masked antibodies that have advanced the most for therapeutic use. They are designed to reveal the active, parent antibody only when encountering proteases upregulated in the microenvironment of the targeted disease tissue, thereby minimizing off-target activity. However, current pro-antibody designs are relegated to fusion proteins that append masking groups restricted to the use of only canonical amino acids, offering excellent control of the site of introduction, but with no authority over where the masking group is installed other than the N -terminus of the antibody. Here, we present a palladium-based bioconjugation approach for the site-specific introduction of a masked tyrosine mimic in the complementary determining region of the FDA approved antibody therapeutic ipilimumab used as a model system. The approach enables the introduction of a protease cleavable group tethered to noncanonical polymers (polyethylene glycol (PEG)) resulting in 47-fold weaker binding to cells expressing CTLA-4, the target antigen of ipilimumab. Upon exposure to tumor-associated proteases, the masking group is cleaved, unveiling a tyrosine-mimic (dubbed hydroxyphenyl cysteine (HPC)) that restores (>90% restoration) binding affinity to its target antigen.

Advances in in vitro display and protein engineering yield therapeutics with affinities in the picomolar range. The Gyrolab ® microfluidics platform uses the kinetic exclusion assay principle to measure subnanomolar solution affinities. This work describes application of the Gyrolab solution affinity module and the new multi-curve analysis feature to determine affinity of the PD-L1 Adnectin™ positron emission tomography radioligand, which was measured as 20 pM for human PD-L1. We also report key parameters that affect assay signal-to-background ratio and data quality, such as detection reagent concentration. Gyrolab offers the necessary throughput for rapid assay development with low sample consumption, as demonstrated in this study, which also provides helpful tips for assay optimization for solution affinity measurement.

An aortoesophageal fistula (AEF) is a rare but often fatal complication associated with the placement of an aortic stent graft such as those utilized in thoracic endovascular aortic repair (TEVAR) procedures. Definitive surgical repair of AEF is the treatment of choice, as conservative management is nearly always fatal. We present the case of an AEF in a 74-year-old male managed by a unique treatment method; an esophageal stent was deployed to cover the fistula as a temporizing measure prior to definitive surgical correction, thus allowing time for resuscitation and hemodynamic optimization. The use of esophageal stents in the setting of AEF following TEVAR has been previously reported in the literature as a palliative measure for patients deemed incapable of tolerating open repair surgery. Our case demonstrates a new and innovative approach to the management of AEF following TEVAR in which the use of esophageal stenting is expanded beyond the role of palliative care and should be considered as a means to optimize at-risk patients prior to definitive corrective surgery in the hopes of improving outcomes., Competing Interests: Declaration of conflicting interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Signaling through the heterotrimeric G protein, G12, via Rho induces a striking increase in breast cancer cell invasion. In this study, evidence is provided that the c-Jun NH(2)-terminal kinase (JNK) is a key downstream effector of G12 on this pathway. Expression of constitutively-active Gα12 or activation of G12 signaling by thrombin leads to increased JNK and c-Jun phosphorylation. Pharmacologic inhibition of JNK or knockdown of JNK expression by siRNA significantly decreases G12-induced JNK activation as well as the ability of breast cancer cells to invade a reconstituted basement membrane. Furthermore, expression of dominant-negative Rho or treatment of cells with an inhibitor of the Rho kinase, ROCK, reduces G12-induced JNK and c-Jun activation, and ROCK inhibitor treatment also inhibits G12-induced cellular invasion. JNK knockdown or ROCK inhibitor treatment has no effect on activation of Rho by G12. Taken together, our data indicate that JNK activation is required for G12-induced invasion of breast cancer cells and that JNK is downstream of Rho and ROCK on this pathway. This study implicates a G12-stimulated mitogen-activated protein kinase cascade in cancer cell invasion, and supports a role for JNK in cancer progression.

The G12 subfamily of heterotrimeric guanine nucleotide-binding proteins consists of two alpha subunits, G alpha12 and G alpha13. These proteins mediate signalling via G protein-coupled receptors and have been implicated in various physiological and pathophysiological processes. A number of direct and indirect effectors of G alpha12 and G alpha13 have been identified that mediate, or have been proposed to mediate, the diverse cellular responses accompanying activation of G12 proteins. This review describes the signalling pathways and cellular events stimulated by G12 proteins, with a particular emphasis on processes that are important in regulating cell migration and invasion, and could potentially be involved in the pathophysiology of cancer metastasis. Experimental findings directly implicating G12 proteins in the spread of metastatic disease are also summarized, indicating the importance of targeted inhibition of G12 signalling as a potential therapeutic option for locally advanced and metastatic disease.

Although the prognosis for patients with early-stage breast cancer has improved, the therapeutic options for patients with locally advanced and metastatic disease are limited. To improve the treatment of these patients, the molecular mechanisms underlying breast cancer invasion and metastasis must be understood. In this study, we report that signaling through the G12 family of heterotrimeric G proteins (Galpha12 and Galpha13) promotes breast cancer cell invasion. Moreover, we demonstrate that inhibition of G12 signaling reduces the metastatic dissemination of breast cancer cells in vivo. Finally, we demonstrate that the expression of Galpha12 is significantly up-regulated in the earliest stages of breast cancer, implying that amplification of G12 signaling may be an early event in breast cancer progression. Taken together, these observations identify the G12 family proteins as important regulators of breast cancer invasion and suggest that these proteins may be targeted to limit invasion- and metastasis-induced patient morbidity and mortality.

The heterotrimeric G protein G(12) has been implicated in such cellular regulatory processes as cytoskeletal rearrangement, cell-cell adhesion, and oncogenic transformation. Although the activated alpha-subunit of G(12) has been shown to interact directly with a number of protein effectors, the roles of many of these protein-protein interactions in G(12)-mediated cell physiology are poorly understood. To begin dissecting the specific cellular pathways engaged upon G(12) activation, we produced a series of substitution mutants in the regions of Galpha(12) predicted to play a role in effector binding. Here we report the identification and characterization of an altered form of Galpha(12) that is functionally uncoupled from signaling through the monomeric G protein Rho, a protein known to propagate several Galpha(12)-mediated signals. This mutant of Galpha(12) fails to bind the Rho-specific guanine nucleotide exchange factors p115RhoGEF and LARG (leukemia-associated RhoGEF), fails to stimulate Rho-dependent transcriptional activation, and fails to trigger activation of RhoA and the Rho-mediated cellular responses of cell rounding and c-jun N-terminal kinase activation. Importantly, this mutant of Galpha(12) retains coupling to the effector protein E-cadherin, as evidenced by its ability both to bind E-cadherin in vitro and to disrupt E-cadherin-mediated cell-cell adhesion. Furthermore, this mutant retains the ability to trigger beta-catenin release from the cytoplasmic domain of cadherin. This identification of a variant of Galpha(12) that is selectively uncoupled from one signaling pathway while retaining signaling capacity through a separate pathway will facilitate investigations into the mechanisms through which G(12) proteins mediate diverse biological responses.

The refolding of barstar from its urea-unfolded state has been studied extensively using various spectroscopic probes and real-time NMR, which provide global and residue-specific information, respectively, about the folding process. Here, a preliminary structural characterization by NMR of barstar in 8 M urea has been carried out at pH 6.5 and 25 degrees C. Complete backbone resonance assignments of the urea-unfolded protein were obtained using the recently developed three-dimensional NMR techniques of HNN and HN(C)N. The conformational propensities of the polypeptide backbone in the presence of 8 M urea have been estimated by examining deviations of secondary chemical shifts from random coil values. For some residues that belong to helices in native barstar, 13C(alpha) and 13CO secondary shifts show positive deviations in the urea-unfolded state, indicating that these residues have propensities toward helical conformations. These residues are, however, juxtaposed by residues that display negative deviations indicative of propensities toward extended conformations. Thus, segments that are helical in native barstar are unlikely to preferentially populate the helical conformation in the unfolded state. Similarly, residues belonging to beta-strands 1 and 2 of native barstar do not appear to show any conformational preferences in the unfolded state. On the other hand, residues belonging to the beta-strand 3 segment show weak nonnative helical conformational preferences in the unfolded state, indicating that this segment may possess a weak preference for populating a helical conformation in the unfolded state.

Barstar is known to form a molten globule-like A form below pH 4. This form exists as a soluble aggregate of 16 monomeric subunits, and appears to remain homogeneous in solution for at least two weeks. Here, structural characterization by NMR of the flexible regions in the A form of barstar has been carried out at pH 2.7 and 25 degrees C. Significantly, the A form appears to be a symmetrical aggregate. Using the recently described fast assignment strategy from HNN and HN(C)N spectra, along with the standard triple resonance and three-dimensional NMR experiments, the flexible segment of the aggregate has been identified to belong largely to the N-terminal end of the polypeptide chain; sequential connectivities were obtained for the first 20 residues (except two) from these experiments. This segment is free in each of the monomeric subunits, and does not form a part of the aggregated core of the A form. The secondary chemical shifts of these residues suggest propensity toward an extended structure. Their (3)J(HN,H)(alpha) coupling constants have values corresponding to those in a random coil structure. However, a few medium-range NOEs, some of them involving side chain atoms, are observed between some residues in this segment. The lowered temperature coefficients of the H(N) chemical shifts compared to random coil values indicate possibilities of some hydrogen bonding in this region. Analysis of the (15)N relaxation parameters and reduced spectral density functions, in particular the negative values of heteronuclear NOEs, indicates large-amplitude high-frequency motions in the N-terminal segments; the first three residues show more negative NOEs than the others. The (15)N transverse relaxation rates and the J(0) spectral density values for residues Ser12 and Ser69 are significantly larger than for the rest, indicating some microsecond to millisecond time scale conformational exchange contributions to the relaxation of these residues. Taken all together, the data suggest that the A form of barstar is an aggregate with a rigid core, but with the N-terminal 20 residues of each of the monomeric subunits, in a highly dynamic random coil conformation which shows transient local ordering of structure. The N-terminal segment, anchored to the aggregated core, exhibits free-flight motion.

The unfolding of ribonuclease A was studied in 5.2 M guanidine hydrochloride at pH 8 and 10 degrees C using multiple optical probes, native-state hydrogen exchange (HX), and pulse labeling by hydrogen exchange. First, native-state HX studies were used to demonstrate that the protein exists in two slowly interconverting forms under equilibrium native conditions: a predominant exchange-incompetent N form and an alternative ensemble of conformations, N(I), in which some amide hydrogens are fully exposed to exchange. Pulsed HX studies indicated that, during unfolding, the rates of exposure to exchange with solvent protons were similar for all backbone NH probe protons. It is shown that two parallel routes of unfolding are available to the predominant N conformation as soon as it encounters strong unfolding conditions. A fraction of molecules appears to rapidly form N(I) on one route. On the other route an exchange-incompetent intermediate state ensemble, I(U)(2), is formed. The kinetics of unfolding measured by far-UV circular dichroism (CD) were faster than those measured by near-UV CD and intrinsic tyrosine fluorescence of the protein. The logarithms of the rate constants of the unfolding reaction measured by all three optical probes also showed a nonlinear dependence on GdnHCl concentration. All of the data suggest that N(I) and I(U)(2) are nativelike in their secondary and tertiary structures. While N(I) unfolds directly to the fully exchange-competent unfolded state (U), I(U)(2) forms another intermediate I(U)(3) which then unfolds to U. I(U)(3) is devoid of all native alpha-helical secondary structure and has only 30% of the tertiary interactions still intact. Since the rates of global unfolding measured by near-UV CD and fluorescence agree well with the rates of exposure determined for all of the backbone NH probe protons, it appears that the rate-limiting step for the unfolding of RNase A is the dissolution of the entire native tertiary structure and penetration of water into the hydrophobic core.

The slow folding of a single tryptophan-containing mutant of barstar has been studied in the presence of 2 M urea at 10 degrees C, using steady state and time-resolved fluorescence methods and far and near-UV CD measurements. The protein folds in two major phases: a fast phase, which is lost in the dead time of measurement during which the polypeptide collapses to a compact form, is followed by a slow observable phase. During the fast phase, the rotational correlation time of Trp53 increases from 2.2 ns to 7.2 ns, and its mean fluorescence lifetime increases from 2.3 ns to 3.4 ns. The fractional changes in steady-state fluorescence, far-UV CD, and near-UV CD signals, which are associated with the fast phase are, respectively, 36 %, 46 %, and 16 %. The product of the fast phase can bind the hydrophobic dye ANS. These observations together suggest that the folding intermediate accumulated at the end of the fast phase has: (a) about 20 % of the native-state secondary structure, (b) marginally formed or disordered tertiary structure, (c) a water-intruded and mobile protein interior; and (d) solvent-accessible patches of hydrophobic groups. Measurements of the anisotropy decay of Trp53 suggest that it undergoes two types of rotational motion in the intermediate: (i) fast (tau(r) approximately 1 ns) local motion of its indole side-chain, and (ii) a slower (tau(r) approximately 7.2 ns) motion corresponding to global tumbling of the entire protein molecule. The ability of the Trp53 side-chain to undergo fast local motion in the intermediate, but not in the fully folded protein where it is completely buried in the hydrophobic core, suggests that the core of the intermediate is still poorly packed. The global tumbling time of the fully folded protein is faster at 5.6 ns, suggesting that the volume of the intermediate is 25 % more than that of the fully folded protein. The rate of folding of this intermediate to the native state, measured by steady-state fluorescence, far-UV CD, and near-UV CD, is 0.07(+/-0.01) min(-1) This rate compares to a rate of folding of 0.03(+/-0.005) min(-1), determined by double-jump experiments which monitor directly formation of native protein; and to a rate of folding of 0.05 min(-1), when determined from time-resolved anisotropy measurements of the long rotational correlation time, which relaxes from an initial value of 7.2 ns to a final value of 5. 6 ns as the protein folds. On the other hand, the amplitude of the short correlation time decreases rapidly with a rate of 0.24(+/-0.06) min(-1). These results suggest that tight packing of residues in the hydrophobic core occurs relatively early during the observable slow folding reaction, before substantial secondary and tertiary structure formation and before final compaction of the protein., (Copyright 2000 Academic Press.)