Structural and functional bases for allosteric control of MMP activities: Can it pave the path for selective inhibition?

AbstractThe zinc-dependent matrix metalloproteinases (MMPs) belong to a large family of structurally homologous enzymes. These enzymes are involved in a wide variety of biological processes ranging from physiological cell proliferation and differentiation to pathological states associated with tumor metastasis, inflammation, tissue degeneration, and cell death. Controlling the enzymatic activity of specific individual MMPs by antagonist molecules is highly desirable, first, for studying their individual roles, and second as potential therapeutic agents. However, blocking the enzymatic activity with synthetic small inhibitors appears to be an extremely difficult task. Thus, this is an unmet need presumably due to the high structural homology between MMP catalytic domains. Recent reports have recognized a potential role for exosite or allosteric protein regions, distinct from the extended catalytic pocket, in mediating MMP activation and substrate hydrolysis. This raises the possibility that MMP enzymatic and non-enzymatic activities may be modified via antagonist molecules targeted to such allosteric sites or to alternative enzyme domains. In this review, we discuss the structural and functional bases for potential allosteric control of MMPs and highlight potential alternative enzyme domains as targets for designing highly selective MMP inhibitors

Building of the Tetraspanin Web: Distinct Structural Domains of CD81 Function in Different Cellular Compartments

The tetraspanin web is composed of a network of tetraspanins and their partner proteins that facilitate cellular interactions and fusion events by an unknown mechanism. Our aim was to unravel the web partnership between the tetraspanin CD81 and CD19, a cell surface signaling molecule in B lymphocytes. We found that CD81 plays multiple roles in the processing, intracellular trafficking, and membrane functions of CD19. Surprisingly, these different roles are embodied in distinct CD81 domains, which function in the different cellular compartments: the N-terminal tail of CD81 has an effect on the glycosylation of CD19; the first transmembrane domain of CD81 is sufficient to support the exit of CD19 from the endoplasmic reticulum, although the large extracellular loop (LEL) of CD81 associates physically with CD19 early during biosynthesis; and finally, the TM2 and TM3 domains of CD81 play a role in the transmission of signals initiated upon engagement of the LEL. The participation of distinct CD81 domains in varied functions may explain the pleiotropic effects of CD81 within the tetraspanin web

Development of Allogeneic NK Cell Adoptive Transfer Therapy in Metastatic Melanoma Patients: In Vitro Preclinical Optimization Studies

<div><p>Natural killer (NK) cells have long been considered as potential agents for adoptive cell therapy for solid cancer patients. Until today most studies utilized autologous NK cells and yielded disappointing results. Here we analyze various modular strategies to employ allogeneic NK cells for adoptive cell transfer, including donor-recipient HLA-C mismatching, selective activation and induction of melanoma-recognizing lysis receptors, and co-administration of antibodies to elicit antibody-dependent cell cytotoxicity (ADCC). We show that NK cell activation and induction of the relevant lysis receptors, as well as co-administration of antibodies yield substantial anti-cancer effects, which are functionally superior to HLA-C mismatching. Combination of the various strategies yielded improved effects. In addition, we developed various clinically-compatible <i>ex vivo</i> expansion protocols that were optimized according to fold expansion, purity and expression of lysis receptors. The main advantages of employing allogeneic NK cells are accessibility, the ability to use a single donor for many patients, combination with various strategies associated with the mechanism of action, e.g. antibodies and specific activation, as well as donor selection according to HLA or CD16 genotypes. This study rationalizes a clinical trial that combines adoptive transfer of highly potent allogeneic NK cells and antibody therapy.</p> </div

Combining ex-vivo NK cell activation, ADCC stimulation and HLA-C mismatching against melanoma cells.

<p>The indicated NK cultures were tested against HLA-C mismatched melanoma cells in the presence of anti-GD3 or isotype control (IC) antibodies. Y axis denotes specific killing (%). Figure shows the mean results of three independent experiments. *denotes statistical significance in <i>t-tests</i> of P = 0.01. Error bars represent SEM.</p

NK cell potency against primary melanoma cultures in relation to NK lysis receptor surface expression following <i>ex-vivo</i> expansion protocols.

<p>Cultured NK cells were analyzed at different time points: ”d1” following culturing with only IL-2 for overnight, “d14” and “d21” indicate 14 and 21 days in culture respectively. The letters “K”, “H” and “L” indicate culture condition set as delineated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057922#pone-0057922-g004" target="_blank">Figure 4</a>. (A–B) shows the results of comparing selected culturing conditions: (A) killing activity of representative NK cultures in HLA-C matched and mismatched settings. The mean results of three independent experiments is shown; (B) expression of the indicated NK lysis receptors by the various NK cultures; (C) killing activity of representative NK cultures against MHC class I negative lymphoma cells (721.221) and melanoma cells (1106mel). The mean results of three independent experiments is shown; (D–E) shows the results of comparing different time points along culture in the optimal culturing condition H: (D) expression of the indicated NK lysis receptors by the various NK cultures; (E) killing activity in HLA-C mismatched setting in the presence of IgG1 isotype control antibodies or the indicated blocking antibodies. E:T ratios were 10∶1. MFI means Median Fluorescence Intensity. Figure shows the mean of three independent experiments. *denotes statistical significance in <i>t-tests</i> of P<0.05. Error bars represent SEM.</p

HLA-C matched and mismatched NK donors and melanoma patients.

<p>The table shows the identity of each of the two alleles of HLA-A, HLA-B and HLA-C for two melanoma patients (Mel008 and Mel10) and seven healthy NK donors (depicted as “HD” followed by a serial number). The table indicates whether one of the HLA-A alleles or HLA-B alleles is a KIR-ligand (highlighted in gray). Similarly, the table indicates whether each HLA-C is of C1 or C2 subgroup, effectively classifying all patients to homozygotes of C1 or C2, or C1–C2 heterozygotes (highlighted in gray). The table is arranged to demonstrate melanoma-NK pairs that are either HLA-C matched or mismatched.</p

ADCC killing effect against melanoma.

<p>(A) A highly significant effect of pre-incubation with anti-GD3 mAbs, but not with isotype control (IC) on the potency of NK cells cultured over night with 100 IU/ml of IL-2 against melanoma; (B) GD3-targeted ADCC can be stimulated only against GD3-positive melanoma cells and is dependent on pre-incubation of the effector cells with IL-2 overnight; (C) Combination of ADCC and HLA-C mismatching. The NK cultures and their matching status towards each melanoma culture are indicated in the figure. E:T ratios were 10∶1. Y axis denotes specific killing (%). Figure shows the mean of four independent experiments. *denotes statistical significance in <i>t-tests</i> of P<0.05 and **of P<0.01. Error bars represent SEM.</p