The Screening of Combinatorial Peptide Libraries for Targeting Key Molecules or Protein-Protein Interactions in the NF-κB Pathway.

Peptides are emerging as an increasingly dependable class of therapeutics in the treatment of cancer and metabolic and cardiovascular diseases, which are all areas of high interest to the pharmaceutical industry. The global market for peptide therapeutics was valued at about 25 billion USD in 2018 and is estimated to reach 57.2 billion USD by the end of 2027. Here, we describe a method for the screening and deconvolution of combinatorial peptide libraries to discover compounds that target discrete signaling components of the NF-κB pathway. Recently, we used this approach to specifically disrupt the interaction between the JNK-activating kinase, MKK7, and the NF-κB-regulated antiapoptotic factor, GADD45ß, in multiple myeloma (MM). We showed that the GADD45ß/MKK7 complex is a functionally critical survival module downstream of NF-κB in MM cells and as such provides an attractive therapeutic target to selectively inhibit NF-κB antiapoptotic signaling in cancer cells. By integrating the library screening and deconvolution methods described here with a rational chemical optimization strategy, we developed the first-in-class GADD45ß/MKK7 inhibitor, DTP3 (a D-tripeptide), which is now being trialed in MM and diffuse large B-cell lymphoma (DLBCL) patients. The same drug discovery approach may be generally applied to therapeutically target other key components of the NF-κB pathway in cancers beyond MM and DLBCL, as well as in non-malignant NF-κB-driven diseases.

Enhanced triacylglycerol catabolism by carboxylesterase 1 promotes aggressive colorectal carcinoma.

The ability to adapt to low-nutrient microenvironments is essential for tumor cell survival and progression in solid cancers, such as colorectal carcinoma (CRC). Signaling by the NF-κB transcription factor pathway associates with advanced disease stages and shorter survival in patients with CRC. NF-κB has been shown to drive tumor-promoting inflammation, cancer cell survival, and intestinal epithelial cell (IEC) dedifferentiation in mouse models of CRC. However, whether NF-κB affects the metabolic adaptations that fuel aggressive disease in patients with CRC is unknown. Here, we identified carboxylesterase 1 (CES1) as an essential NF-κB-regulated lipase linking obesity-associated inflammation with fat metabolism and adaptation to energy stress in aggressive CRC. CES1 promoted CRC cell survival via cell-autonomous mechanisms that fuel fatty acid oxidation (FAO) and prevent the toxic build-up of triacylglycerols. We found that elevated CES1 expression correlated with worse outcomes in overweight patients with CRC. Accordingly, NF-κB drove CES1 expression in CRC consensus molecular subtype 4 (CMS4), which is associated with obesity, stemness, and inflammation. CES1 was also upregulated by gene amplifications of its transcriptional regulator HNF4A in CMS2 tumors, reinforcing its clinical relevance as a driver of CRC. This subtype-based distribution and unfavorable prognostic correlation distinguished CES1 from other intracellular triacylglycerol lipases and suggest CES1 could provide a route to treat aggressive CRC.

Effects of combining manual lymphatic drainage and Kinesiotaping on pain, edema, and range of motion in patients with total knee replacement: a randomized clinical trial.

Total knee replacement (TKR) is continuously increasing with significantly faster recovery times. Soft tissue pain and edema of operated limbs play an important role in early functional recovery. The present study aims to evaluate the effectiveness of the combination of Kinesiotaping and Lymphatic drainage for the containment of pain and edema as well as the improvement of the range of motion of the knee as integration with standard postoperative rehabilitation. Ninety-nine TKR patients were included in the randomized clinical trial and divided into three groups: Kinesiotaping and Lymphatic drainage Group, Lymphatic drainage Group, and Kinesiotaping Group. The assessment was carried out on days 2-4-6 postoperation. All the patients had also standard reeducation sessions. It was observed that both Kinesiotaping and Lymphatic drainage was useful in reducing pain and edema. A significantly higher improvement was observed in the group in which Lymphatic drainage was associated with Kinesiotaping with respect to the individual treatments, for pain and leg circumference over and under the knee, and at the ankle. Range of motion of the knee did not show any difference since the flexion degree was similar in all the three groups. No difference was found between Kinesiotaping and Lymphatic drainage. In conclusion, the treatment with a combination Kinesiotaping and Lymphatic drainage provided better results on pain and edema observed as early as the first days after the intervention, thus may be considered a valid support for standard rehabilitation and pharmacological intervention.

Insights into the Interaction Mechanism of DTP3 with MKK7 by Using STD-NMR and Computational Approaches.

GADD45ß/MKK7 complex is a non-redundant, cancer cell-restricted survival module downstream of the NF-kB survival pathway, and it has a pathogenically critical role in multiple myeloma, an incurable malignancy of plasma cells. The first-in-class GADD45ß/MKK7 inhibitor DTP3 effectively kills MM cells expressing its molecular target, both in vitro and in vivo, by inducing MKK7/JNK-dependent apoptosis with no apparent toxicity to normal cells. DTP3 combines favorable drug-like properties, with on-target-specific pharmacology, resulting in a safe and cancer-selective therapeutic effect; however, its mode of action is only partially understood. In this work, we have investigated the molecular determinants underlying the MKK7 interaction with DTP3 by combining computational, NMR, and spectroscopic methods. Data gathered by fluorescence quenching and computational approaches consistently indicate that the N-terminal region of MKK7 is the optimal binding site explored by DTP3. These findings further the understanding of the selective mode of action of GADD45ß/MKK7 inhibitors and inform potential mechanisms of drug resistance. Notably, upon validation of the safety and efficacy of DTP3 in human trials, our results could also facilitate the development of novel DTP3-like therapeutics with improved bioavailability or the capacity to bypass drug resistance.

Preclinical toxicology and safety pharmacology of the first-in-class GADD45ß/MKK7 inhibitor and clinical candidate, DTP3.

Aberrant NF-κB activity drives oncogenesis and cell survival in multiple myeloma (MM) and many other cancers. However, despite an aggressive effort by the pharmaceutical industry over the past 30 years, no specific IκBα kinase (IKK)ß/NF-κB inhibitor has been clinically approved, due to the multiple dose-limiting toxicities of conventional NF-κB-targeting drugs. To overcome this barrier to therapeutic NF-κB inhibition, we developed the first-in-class growth arrest and DNA-damage-inducible (GADD45)ß/mitogen-activated protein kinase kinase (MKK)7 inhibitor, DTP3, which targets an essential, cancer-selective cell-survival module downstream of the NF-κB pathway. As a result, DTP3 specifically kills MM cells, ex vivo and in vivo, ablating MM xenografts in mice, with no apparent adverse effects, nor evident toxicity to healthy cells. Here, we report the results from the preclinical regulatory pharmacodynamic (PD), safety pharmacology, pharmacokinetic (PK), and toxicology programmes of DTP3, leading to the approval for clinical trials in oncology. These results demonstrate that DTP3 combines on-target-selective pharmacology, therapeutic anticancer efficacy, favourable drug-like properties, long plasma half-life and good bioavailability, with no target-organs of toxicity and no adverse effects preclusive of its clinical development in oncology, upon daily repeat-dose administration in both rodent and non-rodent species. Our study underscores the clinical potential of DTP3 as a conceptually novel candidate therapeutic selectively blocking NF-κB survival signalling in MM and potentially other NF-κB-driven cancers.

Probing the interaction interface of the GADD45ß/MKK7 and MKK7/DTP3 complexes by chemical cross-linking mass spectrometry.

GADD45ß is selectively and constitutively expressed in Multiple Myeloma cells, and this expression correlates with an unfavourable clinical outcome. GADD45ß physically interacts with the JNK kinase, MKK7, inhibiting its activity to enable the survival of cancer cells. DTP3 is a small peptide inhibitor of the GADD45ß/MKK7 complex and is able to restore MKK7/JNK activation, thereby promoting selective cell death of GADD45ß-overexpressing cancer cells. Enzymatic MS foot-printing and diazirine-based chemical cross-linking MS (CX-MS) strategies were applied to study the interactions between GADD45ß and MKK7 kinase domain (MKK7_KD) and between DTP3 and MKK7_KD. Our data show that the binding between GADD45ß and MKK7 largely occurs between GADD45ß loop 2 (region 103-117) and the kinase enzymatic pocket. We also show that DTP3 interferes with this GADD45ß/MKK7 interaction by contacting the MKK7 peptides, 113-136 and 259-274. Accordingly, an MKK7_KD Δ(101-136) variant lacking Trp135 did not produce a fluorescence quenching effect upon the binding of DTP3. The assessment of the interaction between GADD45ß and MKK7 and the elucidation of the recognition surfaces between DTP3 and MKK7 significantly advance the understanding of the mechanism underlying the inhibition of the GADD45ß/MKK7 interaction by DTP3 and pave the way to the design of small-molecule DTP3 analogues.

NF-κB in the crosshairs: Rethinking an old riddle.

Constitutive NF-κB signalling has been implicated in the pathogenesis of most human malignancies and virtually all non-malignant pathologies. Accordingly, the NF-κB pathway has been aggressively pursued as an attractive therapeutic target for drug discovery. However, the severe on-target toxicities associated with systemic NF-κB inhibition have thus far precluded the development of a clinically useful, NF-κB-targeting medicine as a way to treat patients with either oncological or non-oncological diseases. This minireview discusses some of the more promising approaches currently being developed to circumvent the preclusive safety liabilities of global NF-κB blockade by selectively targeting pathogenic NF-κB signalling in cancer, while preserving the multiple physiological functions of NF-κB in host defence responses and tissue homeostasis.

GADD45ß Loss Ablates Innate Immunosuppression in Cancer.

T-cell exclusion from the tumor microenvironment (TME) is a major barrier to overcoming immune escape. Here, we identify a myeloid-intrinsic mechanism governed by the NF-κB effector molecule GADD45ß that restricts tumor-associated inflammation and T-cell trafficking into tumors. In various models of solid cancers refractory to immunotherapies, including hepatocellular carcinoma and ovarian adenocarcinoma, Gadd45b inhibition in myeloid cells restored activation of proinflammatory tumor-associated macrophages (TAM) and intratumoral immune infiltration, thereby diminishing oncogenesis. Our results provide a basis to interpret clinical evidence that elevated expression of GADD45B confers poor clinical outcomes in most human cancers. Furthermore, they suggest a therapeutic target in GADD45ß for reprogramming TAM to overcome immunosuppression and T-cell exclusion from the TME.Significance: These findings define a myeloid-based immune checkpoint that restricts T-cell trafficking into tumors, with potentially important therapeutic implications to generally improve the efficacy of cancer immunotherapy. Cancer Res; 78(5); 1275-92. ©2017 AACR.

Unlocking the NF-κB Conundrum: Embracing Complexity to Achieve Specificity.

Transcription factors of the nuclear factor κB (NF-κB) family are central coordinating regulators of the host defence responses to stress, injury and infection. Aberrant NF-κB activation also contributes to the pathogenesis of some of the most common current threats to global human health, including chronic inflammatory diseases, autoimmune disorders, diabetes, vascular diseases and the majority of cancers. Accordingly, the NF-κB pathway is widely considered an attractive therapeutic target in a broad range of malignant and non-malignant diseases. Yet, despite the aggressive efforts by the pharmaceutical industry to develop a specific NF-κB inhibitor, none has been clinically approved, due to the dose-limiting toxicities associated with the global suppression of NF-κB. In this review, we summarise the main strategies historically adopted to therapeutically target the NF-κB pathway with an emphasis on oncology, and some of the emerging strategies and newer agents being developed to pharmacologically inhibit this pathway.

Cancer-selective targeting of the NF-κB survival pathway with GADD45ß/MKK7 inhibitors.

Constitutive NF-κB signaling promotes survival in multiple myeloma (MM) and other cancers; however, current NF-κB-targeting strategies lack cancer cell specificity. Here, we identify the interaction between the NF-κB-regulated antiapoptotic factor GADD45ß and the JNK kinase MKK7 as a therapeutic target in MM. Using a drug-discovery strategy, we developed DTP3, a D-tripeptide, which disrupts the GADD45ß/MKK7 complex, kills MM cells effectively, and, importantly, lacks toxicity to normal cells. DTP3 has similar anticancer potency to the clinical standard, bortezomib, but more than 100-fold higher cancer cell specificity in vitro. Notably, DTP3 ablates myeloma xenografts in mice with no apparent side effects at the effective doses. Hence, cancer-selective targeting of the NF-κB pathway is possible and, at least for myeloma patients, promises a profound benefit.

The nuclear factor kappa B signaling pathway: integrating metabolism with inflammation.

Nuclear factor kappa B (NF-κB) transcription factors are evolutionarily conserved, coordinating regulators of immune and inflammatory responses. They also play a pivotal role in oncogenesis and metabolic disorders. Several studies during the past two decades have highlighted the key role of the IKK/NF-κB pathway in the induction and maintenance of the state of inflammation that underlies metabolic diseases such as obesity and type 2 diabetes. Recent reports, however, reveal an even more intimate connection between NF-κB and metabolism. These studies demonstrate that NF-κB regulates energy homeostasis via direct engagement of the cellular networks governing glycolysis and respiration, with profound implications beyond metabolic diseases, including cancer, ageing and anticancer therapy. In this review, we discuss these emerging bioenergetic functions of NF-κB and their significance to oncogenesis.

Cancer: NF-κB regulates energy metabolism.

NF-κB transcription factors are evolutionarily conserved, central coordinators of immune and inflammatory responses. They also play a pivotal role in oncogenesis. NF-κB exerts these functions by regulating the transcription of genes encoding many immunoregulators, inflammatory mediators and inhibitors of apoptosis. Several studies during the past few years have also underscored the key role of the IKK/NF-κB pathway in the induction and maintenance of the state of inflammation that underlies metabolic pathologies such as obesity, insulin resistance and type-2 diabetes, reflecting the co-evolution and integration of nutrient- and pathogen-sensing systems. Recent reports, however, are revealing an even more intimate, direct connection between NF-κB and metabolism. These studies demonstrate that NF-κB regulates energy homeostasis via direct engagement of the cellular networks governing glycolysis and respiration, with profound implications that extend beyond metabolic pathologies, to cellular physiology, cancer, and anti-cancer therapy. In this review article, we discuss these emerging metabolic functions of NF-κB and their significance to oncogenesis and cancer treatment.

NF-κB controls energy homeostasis and metabolic adaptation by upregulating mitochondrial respiration.

Cell proliferation is a metabolically demanding process. It requires active reprogramming of cellular bioenergetic pathways towards glucose metabolism to support anabolic growth. NF-κB/Rel transcription factors coordinate many of the signals that drive proliferation during immunity, inflammation and oncogenesis, but whether NF-κB regulates the metabolic reprogramming required for cell division during these processes is unknown. Here, we report that NF-κB organizes energy metabolism networks by controlling the balance between the utilization of glycolysis and mitochondrial respiration. NF-κB inhibition causes cellular reprogramming to aerobic glycolysis under basal conditions and induces necrosis on glucose starvation. The metabolic reorganization that results from NF-κB inhibition overcomes the requirement for tumour suppressor mutation in oncogenic transformation and impairs metabolic adaptation in cancer in vivo. This NF-κB-dependent metabolic pathway involves stimulation of oxidative phosphorylation through upregulation of mitochondrial synthesis of cytochrome c oxidase 2 (SCO2; ref. ). Our findings identify NF-κB as a physiological regulator of mitochondrial respiration and establish a role for NF-κB in metabolic adaptation in normal cells and cancer.

Protein-protein interactions: a simple strategy to identify binding sites and peptide antagonists.

Secondary structure motifs and small protein domains can act as building blocks that are isolated and investigated to gain insights into protein global structure but can also modulate interactions with external partners. Most progress has been made in this field using synthetic peptides. Fragmentation of folded proteins by proteolytic enzymes that act preferentially on exposed and less structured sites can help to isolate shorter polypeptides with preserved secondary and tertiary structures that mimic the original protein architecture. Such molecules can be used as probes for structural studies and as tools for in vitro assays to select active fragments useful as agonists or antagonists of the original protein or as scaffolds for the design of more potent and selective ligands. This simple but effective proteolytic methodology has been successfully applied to determine antagonists of protein-protein interactions, allowing the identification of inhibitors with high efficacy and specificity. Here, we present several studies including the complex between phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes and phospholipase 1, believed to play a relevant role in the insulin resistance mechanism in phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes-overexpressing tissues, the self-association of BCL10 caspase recruitment domain that mediates a protein oligomerization process responsible for NF-kappaB activation and the self-association of growth arrest and DNA damage-inducible factor 45 beta, a major player of the endogenous NF-kappaB-mediated resistance to apoptosis.

Gadd45 beta forms a homodimeric complex that binds tightly to MKK7.

Gadd45 alpha, beta, and gamma proteins, also known as growth arrest and DNA damage-inducible factors, have a number of cellular functions, including cell-cycle regulation and propagation of signals produced by a variety of cellular stimuli, maintaining genomic stability and apoptosis. Furthermore, Gadd45 beta has been indicated as a major player in the endogenous NF-kappaB-mediated resistance to apoptosis in a variety of cell lines. In fibroblasts this mechanism involves the inactivation of MKK7, the upstream activator of JNK, by direct binding within the kinase ATP pocket. On the basis of a number of experimental data, the structures of Gadd45 beta and the Gadd45 beta-MKK7 complex have been predicted recently and data show that interactions are mediated by acidic loops 1 and 2, and helices 3 and 4 of Gadd45 beta. Here, we provide further evidence that Gadd45 beta is a prevailingly alpha-helical protein and that in solution it is able to form non covalent dimers but not higher-order oligomers, in contrast to what has been reported for the homologous Gadd45 alpha. We show that the contact region between the two monomers is comprised of the predicted helix 1 (residues Q17-Q33) and helix 5 (residues K131-R146) of the protein, which appear to be antiparallel and to form a large dimerisation surface not involved in MKK7 recognition. The results suggest the occurrence of a large complex containing at least an MKK7-Gadd45 beta:Gadd45 beta-MKK7 tetrameric unit whose complexity could be further increased by the dimeric nature of the isolated MKK7.