Small Molecule Peptidomimetics Research Articles

Recent Advances in Amphipathic Peptidomimetics as Antimicrobial Agents to Combat Drug Resistance.

The development of antimicrobial drugs with novel structures and clear mechanisms of action that are active against drug-resistant bacteria has become an urgent need of safeguarding human health due to the rise of bacterial drug resistance. The discovery of AMPs and the development of amphipathic peptidomimetics have lay the foundation for novel antimicrobial agents to combat drug resistance due to their overall strong antimicrobial activities and unique membrane-active mechanisms. To break the limitation of AMPs, researchers have invested in great endeavors through various approaches in the past years. This review summarized the recent advances including the development of antibacterial small molecule peptidomimetics and peptide-mimic cationic oligomers/polymers, as well as mechanism-of-action studies. As this exciting interdisciplinary field is continuously expanding and growing, we hope this review will benefit researchers in the rational design of novel antimicrobial peptidomimetics in the future.

A peptidomimetic modulator of the CaV2.2 N-type calcium channel for chronic pain.

Transmembrane Cav2.2 (N-type) voltage-gated calcium channels are genetically and pharmacologically validated, clinically relevant pain targets. Clinical block of Cav2.2 (e.g., with Prialt/Ziconotide) or indirect modulation [e.g., with gabapentinoids such as Gabapentin (GBP)] mitigates chronic pain but is encumbered by side effects and abuse liability. The cytosolic auxiliary subunit collapsin response mediator protein 2 (CRMP2) targets Cav2.2 to the sensory neuron membrane and regulates their function via an intrinsically disordered motif. A CRMP2-derived peptide (CBD3) uncouples the Cav2.2-CRMP2 interaction to inhibit calcium influx, transmitter release, and pain. We developed and applied a molecular dynamics approach to identify the A1R2 dipeptide in CBD3 as the anchoring Cav2.2 motif and designed pharmacophore models to screen 27 million compounds on the open-access server ZincPharmer. Of 200 curated hits, 77 compounds were assessed using depolarization-evoked calcium influx in rat dorsal root ganglion neurons. Nine small molecules were tested electrophysiologically, while one (CBD3063) was also evaluated biochemically and behaviorally. CBD3063 uncoupled Cav2.2 from CRMP2, reduced membrane Cav2.2 expression and Ca2+ currents, decreased neurotransmission, reduced fiber photometry-based calcium responses in response to mechanical stimulation, and reversed neuropathic and inflammatory pain across sexes in two different species without changes in sensory, sedative, depressive, and cognitive behaviors. CBD3063 is a selective, first-in-class, CRMP2-based peptidomimetic small molecule, which allosterically regulates Cav2.2 to achieve analgesia and pain relief without negative side effect profiles. In summary, CBD3063 could potentially be a more effective alternative to GBP for pain relief.

Assessment of the bound conformation of bombesin to the BB1 and BB2 receptors

Bombesin is an endogenous peptide involved in a wide spectrum of physiological activities ranging from satiety, control of circadian rhythm and thermoregulation in the central nervous system, to stimulation of gastrointestinal hormone release, activation of macrophages and effects on development in peripheral tissues. Actions of the peptide are mediated through the two high affinity G-protein coupled receptors BB1R and BB2R. Under pathophysiological conditions, these receptors are overexpressed in many different types of tumors, such as prostate cancer, breast cancer, small and non-small cell lung cancer and pancreatic cancer. This observation has been used for designing cell markers, but it has not been yet exploited for therapeutical purposes. Despite the enormous biological interest of the peptide, little is known about the stereochemical features that contribute to their activity. On the one hand, mutagenesis studies identified a few receptor residues important for high bombesin affinity and on the other, a few studies focused on the relevance of diverse residues of the peptide for receptor activation. Models of the peptide bound to BB1R and BB2R can be helpful to improve our understanding of the stereochemical features granting bombesin activity. Accordingly, the present study describes the computational process followed to construct such models by means of Steered Molecular Dynamics, using models of the peptide and its receptors. Present results provide new insights into the structure-activity relationships of bombesin and its receptors, as well as render an explanation for the differential binding affinity observed towards BB1R and BB2R. Finally, these models can be further exploited to help for designing novel small molecule peptidomimetics with improved pharmacokinetics profile.

Peptidomimetic Small-Molecule Inhibitors of 3CLPro Activity and Spike-ACE2 Interaction: Toward Dual-Action Molecules against Coronavirus Infections.

The development of molecules able to target protein–protein interactions (PPIs) is of interest for the development of novel therapeutic agents. Since a high percentage of PPIs are mediated by α-helical structure at the interacting surface, peptidomimetics that reproduce the essential conformational components of helices are useful templates for the development of PPIs inhibitors. In this work, the synthesis of a constrained dipeptide isostere and insertion in the short peptide epitope EDLFYQ of the angiotensin-converting enzyme 2 (ACE2) α1 helix domain resulted in the identification of a molecule capable of inhibiting the SARS-CoV-2 ACE2/spike interaction in the micromolar range. Moreover, inhibition of SARS-CoV-2 3CLPro main protease activity was assessed as an additional inhibitory property of the synthesized peptidomimetics, taking advantage of the C-terminal Q amino acid present in both the ACE2 epitope and the Mpro recognizing motif (APSTVxLQ), thus paving the way to the development of multitarget therapeutics toward coronavirus infections.

Structure-activity relationship studies of functionalized aromatic peptidomimetics as neurolysin activators

Modulating peptidase neurolysin (Nln) has been identified as a potential cerebroprotective target for the development of therapeutics for ischemic stroke. Continued structure–activity relationship studies on peptidomimetic small molecule activators of Nln bearing electron-donating and electron- withdrawing functionalized phenyls are explored. Incorporation of fluorine or trifluoromethyl groups produces Nln activators with enhanced A50, while methoxy substitution produces derivatives with enhanced Amax. Selected activators containing methoxy or trifluoromethyl substitution are selective for Nln over related peptidases and possess increased blood–brain barrier penetrability than initial hits.

Pharmacological targeting of Sam68 functions in colorectal cancer stem cells

SummaryCancer stem cells (CSCs) are documented to play a key role in tumorigenesis and therapy resistance. Despite significant progress in clinical oncology, CSC reservoirs remain elusive and difficult to eliminate. Reverse-turn peptidomimetics were characterized as disruptors of CBP/beta-Catenin interactions and represent a promising avenue to curb hyperactive canonical Wnt/beta-Catenin signaling in CSCs. Recent studies suggested Sam68 as a critical mediator of reverse-turn peptidomimetics response in CSC populations. Using computational and biochemical approaches we confirmed Sam68 as a primary target of reverse-turn peptidomimetics. Furthermore, we executed an in silico drug discovery pipeline to identify yet uncharacterized reverse-turn peptidomimetic structures displaying superior anti-CSC activity in transformed pluripotent and colorectal cancer cell models. Thus, we identified YB-0158 as a reverse-turn peptidomimetic small molecule with enhanced translational potential, altering key hallmarks of human colorectal CSCs in patient-derived ex vivo organoids and in vivo serial tumor transplantation.

Interim Results from the Phase 1a/1b Dose-Finding Study of CWP232291 (CWP291) in Relapsed or Refractory Myeloma (RRMM) Alone or in Combination with Lenalidomide and Dexamethasone

Background: CWP291, a novel peptidomimetic small molecule, has potent and selective inhibitory activity on a Wnt gene reporter and decreases expression of the β-catenin target genes, cyclin D1 and survivin. It has broad anti-cancer efficacy in vitro, outperforming lenalidomide and bortezomib in MM murine xenografts, and improving survival in combination vs. single agents alone in bone marrow engraftment models.Methods: Subjects with RRMM were infused CWP291 IV over at least 30 minutes twice weekly for 3 weeks out of a 4-week cycle in a 3+3 dose-escalation, Phase 1 design. In Phase 1a, CWP291 was delivered as a single agent. In Phase 1b, which started at a dose lower than a demonstrably well-tolerated dose in Phase 1a, allowing for more rapid initiation of this phase of the study (J Clin Oncol 35:2017 Suppl Abstr TPS8058), subjects received CWP291 in combination with lenalidomide and dexamethasone.Results: As of July 25, 2017, 21 subjects (13M, 8F), ages 44-79 (median 61 years) were enrolled; twelve in Phase 1a at doses from 198 - 446 mg/m2 and 8 in Phase 1b at 198 - 263 mg/m2. The median duration of treatment was 2 cycles (range 1 - 6 cycles) with 5 subjects ongoing. The most common (≥20% of subjects) drug-related adverse events (AEs) were due to GI toxicities, with diarrhea in 16 subjects (76%) and nausea in 10 subjects (48%); toxicities were grade ≤2 except grade 3 in 1 subject each. Asthenia/fatigue was seen in 10 subjects (48%) with 1 grade 3. Thrombocytopenia was reported in 6 subjects (29%), 5 of which were grade ≥3. Anemia was seen in 4 subjects (29%), and in 3 subjects were grade ≥3. Serious adverse events were recorded for 11 subjects with 18 separate events, the most frequent (9 events in 6 subjects) considered unrelated were hospitalizations for infections (pneumonia and parainfluenza virus) and only 3 events (urinary tract infection, grade 3 hypotension and grade 2 hypoxia) were considered possibly related. Three dose-limiting toxicities (DLT) occurred in Phase 1a in 2 subjects at 446 mg/m2, which were hypoxia (grade 3), neutropenia (grade 4) and thrombocytopenia (grade 4). The 335 mg/m2 cohort has now been initiated in which 1 DLT (grade 3 hypoxia) has been seen, and accrual is ongoing. One DLT (hypotension grade 3) was seen in Phase 1b at 198 mg/m2 however no additional DLTs were seen and accrual is now ongoing at 263 mg/m2. Fourteen subjects were evaluable for efficacy as per IMWG criteria. Six subjects had progressive disease (1 in Phase 1b), 4 subjects had stable disease, and responses were seen in 4 subjects (2 minor). There was also documentation of a 34.6% decrease in bone marrow plasma cells in one subject for whom a bone marrow aspirate was available in Phase 1a. Changes in targeted genes in blood and BM, as well as PK, will be provided from expanded and escalated dosing cohorts.Conclusions: CWP291, a novel and first in class molecule with significant preclinical activity, shows safety as a single agent as well as in combination with standard of care for patients with relapsed/refractory multiple myeloma Early evidence of efficacy in these subjects suggests that this is an active drug in this indication, and is continuing to be explored. DisclosuresManasanch:sanofi: Research Funding; celgene: Consultancy; adaptive biotechnologies: Consultancy; takeda: Consultancy; quest diagnostics: Research Funding; merck: Research Funding. Hauptschein:Theriac, Inc.: Employment. Choi:JW Pharmaceuticals, Inc.: Employment. Lee:JW Pharmaceuticals: Employment.

Exploiting Knowledge on Structure-Activity Relationships for Designing Peptidomimetics of Endogenous Peptides.

Endogenous peptides are important mediators in cell communication, being consequently involved in many physiological processes. Their use as therapeutic agents is limited due to their poor pharmacokinetic profile. To circumvent this drawback, alternative diverse molecules based on the stereochemical features that confer their activity can be synthesized, using them as guidance; from peptide surrogates provided with a better pharmacokinetic profile, to small molecule peptidomimetics, through cyclic peptides. The design process requires a competent use of the structure-activity results available on individual peptides. Specifically, it requires synthesis and analysis of the activity of diverse analogs, biophysical information and computational work. In the present work, we show a general framework of the process and show its application to two specific examples: the design of selective AT1 antagonists of angiotensin and the design of selective B2 antagonists of bradykinin.

Pharmacologic inhibition of RGD-binding integrins ameliorates fibrosis and improves function following kidney injury.

Fibrosis is a final common pathway for many causes of progressive chronic kidney disease (CKD). Arginine–glycine–aspartic acid (RGD)‐binding integrins are important mediators of the pro‐fibrotic response by activating latent TGF‐β at sites of injury and by providing myofibroblasts information about the composition and stiffness of the extracellular matrix. Therefore, blockade of RGD‐binding integrins may have therapeutic potential for CKD. To test this idea, we used small‐molecule peptidomimetics that potently inhibit a subset of RGD‐binding integrins in a murine model of kidney fibrosis. Acute kidney injury leading to fibrosis was induced by administration of aristolochic acid. Continuous subcutaneous administration of CWHM‐12, an RGD integrin antagonist, for 28 days improved kidney function as measured by serum creatinine. CWHM‐12 significantly reduced Collagen 1 (Col1a1) mRNA expression and scar collagen deposition in the kidney. Protein and gene expression markers of activated myofibroblasts, a major source of extracellular matrix deposition in kidney fibrosis, were diminished by treatment. RNA sequencing revealed that inhibition of RGD integrins influenced multiple pathways that determine the outcome of the response to injury and of repair processes. A second RGD integrin antagonist, CWHM‐680, administered once daily by oral gavage was also effective in ameliorating fibrosis. We conclude that targeting RGD integrins with such small‐molecule antagonists is a promising therapeutic approach in fibrotic kidney disease.

Direct Transformation of N-Protected α,β-Unsaturated γ-Amino Amides into γ-Lactams through a Base-Mediated Molecular Rearrangement.

Here, we are reporting a single-step transformation of N-protected α,β-unsaturated γ-amino amides into 5,5-disubstituted γ-lactams through a base-mediated new molecular rearrangement. In contrast to the known N- to C(O) cyclization of saturated γ-amino acids into corresponding γ-lactams, the new rearrangement involves the cyclization between N-terminal Cγ- to C-terminal amide N. The cyclization process was initiated by the migration of double bond from α,β → β,γ position. The enamine-imine tautomerization of the new β,γ-double bond and subsequent 5-exo-trig cyclization of terminal amide leads to the formation of N-protected 5,5-disubstituted γ-lactam. The structures of various γ-lactams obtained from the rearrangement were studied in single crystals. Overall, the results reported here demonstrate the facile and single-step transformation of N-protected α,β-unsaturated γ-amino amides into γ-lactams and provided an excellent opportunity to construct small-molecule peptidomimetics.

Peptide and non-peptide mimetics as potential therapeutics targeting oral bacteria and oral biofilms.

The development of the oral biofilm requires a complex series of interactions between host tissues and the colonizing bacteria as well as numerous interspecies interactions between the organisms themselves. Disruption of normal host–microbe homoeostasis in the oral cavity can lead to a dysbiotic microbial community that contributes to caries or periodontal disease. A variety of approaches have been pursued to develop novel potential therapeutics that are active against the oral biofilm and/or target specific oral bacteria. The structure and function of naturally occurring antimicrobial peptides from oral tissues and secretions as well as external sources such as frog skin secretions have been exploited to develop numerous peptide mimetics and small molecule peptidomimetics that show improved antimicrobial activity, increased stability and other desirable characteristics relative to the parent peptides. In addition, a rational and minimalist approach has been developed to design small artificial peptides with amphipathic α‐helical properties that exhibit potent antibacterial activity. Furthermore, with an increased understanding of the molecular mechanisms of beneficial and/or antagonistic interspecies interactions that contribute to the formation of the oral biofilm, new potential targets for therapeutic intervention have been identified and both peptide‐based and small molecule mimetics have been developed that target these key components. Many of these mimetics have shown promising results in in vitro and pre‐clinical testing and the initial clinical evaluation of several novel compounds has demonstrated their utility in humans.

Viral mimetic priming enhances α-synuclein-induced degeneration: Implications for Parkinson’s disease

Evidence is accumulating to suggest that viral infections and consequent viral-mediated neuroinflammation may contribute to the etiology of idiopathic Parkinson’s disease. Moreover, viruses have been shown to influence α-synuclein oligomerization as well as the autophagic clearance of abnormal intra-cellular proteins aggregations, both of which are key neuropathological events in Parkinson’s disease pathogenesis. To further investigate the interaction between viral-mediated neuroinflammation and α-synuclein aggregation in the context of Parkinson’s disease, this study sought to determine the impact of viral neuroinflammatory priming on α-synuclein aggregate-induced neuroinflammation and neurotoxicity in the rat nigrostriatal pathway. To do so, male Sprague-Dawley rats were intra-nigrally injected with a synthetic mimetic of viral dsRNA (poly I:C) followed two weeks later by a peptidomimetic small molecule which accelerates α-synuclein fibril formation (FN075). The impact of the viral priming on α-synuclein aggregation-induced neuroinflammation, neurodegeneration and motor dysfunction was assessed. We found that prior administration of the viral mimetic poly I:C significantly exacerbated or precipitated the α-synuclein aggregate induced neuropathological and behavioral effects. Specifically, sequential exposure to the two challenges caused a significant increase in nigral microgliosis (p < 0.001) and astrocytosis (p < 0.01); precipitated a significant degeneration of the nigrostriatal cell bodies (p < 0.05); and precipitated a significant impairment in forelimb kinesis (p < 0.01) and sensorimotor integration (p < 0.01). The enhanced sensitivity of the nigrostriatal neurons to pathological α-synuclein aggregation after viral neuroinflammatory priming further suggests that viral infections may contribute to the etiology and pathogenesis of Parkinson’s disease.

Designed Macrocyclic Peptides as Nanomolar Amyloid Inhibitors Based on Minimal Recognition Elements.

Amyloid self-assembly is linked to the pathogenesis of Alzheimer's disease (AD) and type 2 diabetes (T2D), but so far, no anti-amyloid compound has reached the clinic. Macrocyclic peptides belong to the most attractive drug candidates. Herein we present macrocyclic peptides (MCIPs) designed using minimal IAPP-derived recognition elements as a novel class of nanomolar amyloid inhibitors of both Aβ40(42) and IAPP or Aβ40(42) alone and show that chirality controls inhibitor selectivity. Sequence optimization led to the discovery of an Aβ40(42)-selective MCIP exhibiting high proteolytic stability in human plasma and human blood-brain barrier (BBB) crossing ability in a cell model, two highly desirable properties for anti-amyloid AD drugs. Owing to their favorable properties, MCIPs should serve as leads for macrocyclic peptide-based anti-amyloid drugs and scaffolds for the design of small-molecule peptidomimetics for targeting amyloidogenesis in AD or in both AD and T2D.

Reaction profiling of a set of acrylamide-based human tissue transglutaminase inhibitors

The major function of the enzyme human tissue transglutaminase (TG2) is the crosslinking of proteins via a transamidation between the γ-carboxamide of a glutamine and the ε-amino group of a lysine. Overexpression of TG2 can lead to undesirable outcomes and has been linked to conditions such as fibrosis, celiac disease and neurodegenerative diseases. Accordingly, TG2 is a tempting drug target. The most effective TG2 inhibitors to date are small-molecule peptidomimetics featuring electrophilic warheads that irreversibly modify the active site catalytic cysteine (CYS277). In an effort to facilitate the design of such TG2 inhibitors, we undertook a quantum mechanical reaction profiling of the Michael reaction between a set of six acrylamide-based known TG2 inhibitors and the TG2 CYS277. The inhibitors were docked into the active site and the coordinates were refined by MD simulations prior to modelling the covalent modification of the CYS277 thiolate. The results of QM/MM MD umbrella sampling applied to reaction coordinates driving the Michael reaction are presented for two approximations of the Michael reaction: a concerted reaction (simultaneous thiolate attack onto the acrylamide warhead and pronation from the adjacent HIS335) and a two-stage reaction (consecutive thiolate attack and protonation). The two-stage approximation of the Michael reaction gave the better results for the evaluation of acrylamide-based potential TG2 inhibitors in silico. Good correlations were observed between the experimental TG2 IC50 data and the calculated activation energies over the range 0.0061–6.3μM (three orders of magnitude) and we propose that this approach may be used to evaluate acrylamide-based potential TG2 inhibitors.

Novel phase 1a/1b dose-finding study design of CWP232291 (CWP291) in relapsed or refractory myeloma (MM).

TPS8058 Background: CWP291, a novel peptidomimetic small molecule, has potent, selective inhibitory activity on a Wnt gene reporter, decreasing expression of β-catenin target genes, cyclin D1 and survivin. With broad anti-cancer efficacy in vitro, it significantly outperforms lenalidomide as a single agent combination in MM bone marrow engraftment models. Methods: This Phase 1a/1b study (NCT #02426723) was designed to define a well-tolerated dose of CWP291 as a single agent in subjects with R/R MM. CWP291 was administered IV over ≥30 minutes 2x weekly for 3 weeks out of a 4-week cycle, with standard 3+3 dose escalation design. But an important objective in terms of patient benefit and further clinical development was to explore activity of a combination regimen with lenalidomide. Thus, a novel study design allowed initiation of the Phase 1b as soon as CWP291 achieved a well-tolerated dose as a single agent. Combination therapy would start at one dose level lower. Enrollment of patients onto each arm was guided by Safety Review Committee assessments, including baseline laboratory values, performance status, extent of prior therapy, or prior adverse events related to lenalidomide. Results: Initiated September 2015, the starting dose was based on a prior Phase 1 study in AML, 198 mg/m2. There were 4 sites involved, and 11 patients enrolled over 12 months. Approval of the new design by regulatory authorities and IRBs was completed by November 2016. A well-tolerated single agent dose (297 mg/m2) was identified, allowing initiation of the Phase 1b at a dose of 198 mg/m2(one dose level lower) combined with lenalidomide. Four subjects were enrolled in ~2 months to the Phase 1b. Enrollment to both arms is continuing and the status of this study will be updated at presentation. Conclusions: The ability to consider combination therapy with a novel drug is clearly a motivation for patient participation in clinical trials; especially true in MM, as multiple new therapies are available. This trial design was approved and allowed based on assessment of individual patient safety and potential benefit. Rapid enrollment in the combination therapy arm may significantly foster development of novel agents with this study design. Clinical trial information: NCT #02426723.

Ongoing Phase 1a/1b Dose-Finding Study of CWP232291 (CWP291) in Relapsed or Refractory Multiple Myeloma (MM)

Background: CWP291, a novel peptidomimetic small molecule, has potent and selective inhibitory activity on a Wnt gene reporter and decreases expression of the β-catenin target genes, cyclin D1 and survivin. It has broad anti-cancer efficacy in vitro, outperforming lenalidomide and bortezomib in multiple myeloma murine xenografts and demonstrating improved survival when used in combination with lenalidomide in bone marrow engraftment models.Methods: Subjects with relapsed or refractory MM were infused CWP291 iv over at least 30 minutes twice weekly for 3 weeks out of a 4-week cycle in a 3+3 dose-escalation, Phase 1 design.Results: Results as of July 15, 2016 over a dose range of 198 - 446 mg/m2 in 9 subjects are reported. Median duration of treatment was 3 cycles (range 1.3 - 6.5 cycles) with 3 subjects ongoing. The most common (>20% of subjects) drug-related adverse events (AEs) were due to GI toxicities, including diarrhea (77.8%), nausea (55.6%) and constipation (33.3%). Toxicity was mostly ≤grade 2, however grade 3/4 events were frequently related to myelosuppression, including neutropenia (33.3%), and anemia and thrombocytopenia (22.2% each). Serious adverse events were recorded for 5 subjects with 8 separate events: 6 events in 4 subjects considered unrelated were hospitalizations for pneumonia, and 2 events in 1 subject each considered possibly related were a urinary tract infection and grade 3 hypoxia. Dose-limiting toxicities occurred in 2 patients at 446 mg/m2, which were hypoxia (grade 3) and thrombocytopenia (grade 4). Accrual at 335 mg/m2 has now been initiated. Stable disease (≥3 months on therapy with no increase in M protein as per the IMWG) was seen in 4 subjects. There was also documentation of a 34.6% decrease in bone marrow plasma cells in one subject for whom a bone marrow aspirate was available. PK in the first 3 subjectsreceiving198 mg/m2showed plasma levels of the active metabolite had very little inter-patient variability, which were comparable to patients with AML receiving this same dose in another study. There was less than 4% excreted in the urine, and there was no evidence of accumulation from Day 1 to Day 18. Changes in targeted genes in blood and BM, as well as additional PK, will be provided from expanded and escalated dosing cohorts. Furthermore, preliminary observations in the Phase 1b part of this study, which combines the administration of CWP291 with lenalidomide and dexamethasone, will be reported.Conclusions: CWP291, a novel and first in class molecule with significant preclinical activity, shows single agent safety as well as early evidence of efficacy in subjects with MM. Accrual is ongoing to establish the optimal dose level for both single agent and combination therapy. DisclosuresHauptschein:JW Theriac Pharmaceutical: Employment. Choi:JW Pharmaceutical Corporation: Employment. Lee:JW Pharmaceutical Corporation: Employment.

Identification of a Novel Inhibitory Allosteric Site in p38α

In the present study, we report the discovery of a novel allosteric inhibitory site for p38α, a subclass of the mitogen-activated protein kinases (MAPK) family. The putative site was discovered after inspection of the crystallographic structure of the p38α-MK2 complex. MK2 (MAPK-activated protein kinase 2) is an interesting protein playing a dual role as modulator and substrate of p38α. This intriguing behavior is due to the ability of the two proteins to form distinctive heterodimers when p38α is phosphorylated or not. We hypothesized that the regulatory action of MK2 is due to its capability to keep p38α in an inactive conformation and consequently, we investigated the atomic structure of the p38α-MK2 complex to understand such regulatory behavior at the molecular level. After inspection of the complex structure, two peptides designed from the MK2 regulatory loop in contact with p38α with sequences Tyr1-Ser2-Asn3-His4-Gly5-Leu6 (peptide-1) and [Phe0]-peptide-1 (peptide-2) in their zwitterionic form were investigated for their phosphorylation inhibitory capability in vitro. Since both peptides exhibited inhibitory capability of the p38α kinase mediated phosphorylation of MEF2A, in a subsequent step we pursued the discovery of small molecule peptidomimetics. For this purpose we characterized in detail the peptide-p38α interaction using molecular dynamics simulations, leading to the definition of a pharmacophore for the peptide-protein interaction. This hypothesis was used as query for a in silico screening, leading to the discovery of a fused ring compound with micromolar inhibitory activity. Site-directed mutagenesis studies support that the compound binds to the putative novel allosteric site in p38α.

Phase 1 study of CWP232291 in relapsed/refractory acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS).

7044 Background: CWP232291, a novel peptidomimetic small molecule identified via a phenotypic drug discovery screen, shows selective inhibitory activity at submicromolar levels on a Wnt gene report...

GDC-0152 attenuates the malignant progression of osteosarcoma promoted by ANGPTL2 via PI3K/AKT but not p38MAPK signaling pathway.

Angiopoietin-like protein 2 (ANGPTL2) plays an important role in inflammatory carcinogenesis and tumor metastasis. The compound GDC-0152 is a peptidomimetic small molecule antagonist of inhibitor of apoptosis (IAP) proteins with antitumor activity. However, the interaction between ANGPTL2 and GDC-0152 has not been studied. It has been proven that ANGPTL2 promotes metastasis of osteosarcoma. Therefore, in the present study, the effect of GDC-0152 on the malignant progression of osteosarcoma promoted by ANGPTL2 was investigated. Human osteosarcoma cell line SaOS2 cells were pre-treated or non-treated with GDC-0152 and then exposed to recombinant human ANGPTL2. The viability of SaOS2 cells was determined by MTT assay, the migration of SaOS2 cells was analyzed by chamber migration assay kit, and the SaOS2 cell apoptosis was determined by fluorescence-activated cell sorting (FACS) and nuclear staining. Treatment with ANGPTL2 increased SaOS2 cell growth and migration and decreased cell apoptosis. The increased cell growth and decreased cell apoptosis were significantly attenuated in SaOS2 cells receiving GDC-0152. However, the ANGPTL2-increased SaOS2 cell migration was not inhibited by GDC-0152 treatment. Furthermore, western blot analysis showed that the activation of phosphatidyl inositol 3-kinase (PI3K) (p85), PI3K (p110), protein kinase B (Akt) (Ser473), Akt (Thr308) and p38 mitogen-activated protein kinase (p38MAPK) were upregulated by ANGPTL2. Quantitative real-time polymerase chain reaction (qTR-PCR) and gelatin zymography showed that the mRNA expression and activity of matrix metalloproteinase-9 (MMP-9) and matrix metalloproteinase-2 (MMP-2) were also increased by ANGPTL2. The upregulated activation of PI3K and Akt were significantly suppressed by the treatment of GDC-0152. In contrast, GDC-0152 did not suppress ANGPTL2-induced p38MAPK phosphorylation, MMP-9/MMP-2 mRNA expression or MMP-9/MMP-2 activity. Taken together, these data indicate that GDC-0152 attenuates the malignant progression of osteosarcoma promoted by ANGPTL2 via PI3K/AKT but not p38MAPK signaling pathway. The present study indicated a novel therapeutic strategy to inhibit tumor growth by indirectly preventing ANGPTL2 signaling.

In Vitro Phenotypic Characterization of Hepatitis C Virus NS3 Protease Variants Observed in Clinical Studies of Telaprevir

Telaprevir is a linear, peptidomimetic small molecule that inhibits hepatitis C virus (HCV) replication by specifically inhibiting the NS3·4A protease. In phase 3 clinical studies, telaprevir in combination with peginterferon and ribavirin (PR) significantly improved sustained virologic response (SVR) rates in genotype 1 chronic HCV-infected patients compared with PR alone. In patients who do not achieve SVR after treatment with telaprevir-based regimens, variants with mutations in the NS3·4A protease region have been observed. Such variants can contribute to drug resistance and limit the efficacy of treatment. To gain a better understanding of the viral resistance profile, we conducted phenotypic characterization of the variants using HCV replicons carrying site-directed mutations. The most frequently observed (significantly enriched) telaprevir-resistant variants, V36A/M, T54A/S, R155K/T, and A156S, conferred lower-level resistance (3- to 25-fold), whereas A156T and V36M+R155K conferred higher-level resistance (>25-fold) to telaprevir. Rarely observed (not significantly enriched) variants included V36I/L and I132V, which did not confer resistance to telaprevir; V36C/G, R155G/I/M/S, V36A+T54A, V36L+R155K, T54S+R155K, and R155T+D168N, which conferred lower-level resistance to telaprevir; and A156F/N/V, V36A+R155K/T, V36M+R155T, V36A/M+A156T, T54A+A156S, T54S+A156S/T, and V36M+T54S+R155K, which conferred higher-level resistance to telaprevir. All telaprevir-resistant variants remained fully sensitive to alpha interferon, ribavirin, and HCV NS5B nucleoside and nonnucleoside polymerase inhibitors. In general, the replication capacity of telaprevir-resistant variants was lower than that of the wild-type replicon.