Phosphinic acid-based inhibitors of tubulin polyglutamylases

Tubulin is subject to a reversible post-translational modification involving polyglutamylation and deglutamylation of glutamate residues in its C-terminal tail. This process plays key roles in regulating the function of microtubule associated proteins, neuronal development, and metastatic progression. This study describes the synthesis and testing of three phosphinic acid-based inhibitors that have been designed to inhibit both the glutamylating and deglutamylating enzymes. The compounds were tested against the polyglutamylase TTLL7 using tail peptides as substrates (100 μM) and the most potent inhibitor displayed an IC₅₀ value of 150 μM. The incorporation of these compounds into tubulin C-terminal tail peptides may lead to more potent TTLL inhibitors.     

Ring-fused pyrazole derivatives as potent inhibitors of lymphocyte-specific kinase (Lck): Structure,synthesis, and SAR

We have identified a novel series of ring-fused pyrazole derivatives as lymphocyte-specific kinase (Lck) inhibitors. The most potent analogs exhibited good enzyme inhibitory activity (IC₅₀s <1 nM) as well as excellent cellular activity against mixed lymphocyte reaction (MLR) (IC₅₀s <1 nM).     

EHD1 and Eps15 interact with phosphatidylinositols via their Eps15 homology domains

The C-terminal Eps15 homology domain-containing protein, EHD1, regulates the recycling of receptors from the endocytic recycling compartment to the plasma membrane. In cells, EHD1 localizes to tubular and spherical recycling endosomes. To date, the mode by which EHD1 associates with endosomal membranes remains unknown, and it has not been determined whether this interaction is direct or via interacting proteins. Here, we provide evidence demonstrating that EHD1 has the ability to bind directly and preferentially to an array of phospholipids, preferring phosphatidylinositols with a phosphate at position 3. Previous studies have demonstrated that EH domains coordinate calcium binding and interact with proteins containing the tripeptide asparagine-proline-phenylalanine (NPF). Using two-dimensional nuclear magnetic resonance analysis, we now describe a new function for the Eps15 homology (EH) domain of EHD1 and show that it is capable of directly binding phosphatidylinositol moieties. Moreover, we have expanded our studies to include the C-terminal EH domain of EHD4 and the second of the three N-terminal EH domains of Eps15 and demonstrated that phosphatidylinositol binding may be a more general property shared by certain other EH domains. Further studies identified a positively charged lysine residue (Lys-483) localized within the third helix of the EH domain, on the opposite face of the NPF-binding pocket, as being critical for the interaction with the phosphatidylinositols.     

Chemical shift assignments of the C-terminal Eps15 homology domain-3 EH domain

The C-terminal Eps15 homology (EH) domain 3 (EHD3) belongs to a eukaryotic family of endocytic regulatory proteins and is involved in the recycling of various receptors from the early endosome to the endocytic recycling compartment or in retrograde transport from the endosomes to the Golgi. EH domains are highly conserved in the EHD family and function as protein-protein interaction units that bind to Asn-Pro-Phe (NPF) motif-containing proteins. The EH domain of EHD1 was the first C-terminal EH domain from the EHD family to be solved by NMR. The differences observed between this domain and proteins with N-terminal EH domains helped describe a mechanism for the differential binding of NPF-containing proteins. Here, structural studies were expanded to include the EHD3 EH domain. While the EHD1 and EHD3 EH domains are highly homologous, they have different protein partners. A comparison of these structures will help determine the selectivity in protein binding between the EHD family members and lead to a better understanding of their unique roles in endocytic regulation.     

Development and evaluation of 4-(pyrrolidin-3-yl)benzonitrile derivatives as inhibitors of lysine specific demethylase 1

As part of our ongoing efforts to develop reversible inhibitors of LSD1, we identified a series of 4-(pyrrolidin-3-yl)benzonitrile derivatives that act as successful scaffold-hops of the literature inhibitor GSK-690. The most active compound, 21g, demonstrated a K_d value of 22 nM and a biochemical IC₅₀ of 57 nM. In addition, this compound displayed improved selectivity over the hERG ion channel compared to GSK-690, and no activity against the related enzymes MAO-A and B. In human THP-1 acute myeloid leukaemia cells, 21g was found to increase the expression of the surrogate cellular biomarker CD86. This work further demonstrates the versatility of scaffold-hopping as a method to develop structurally diverse, potent inhibitors of LSD1.     

EHDS are serine phosphoproteins: EHD1 phosphorylation is enhanced by serum stimulation

Endocytic processes are mediated by multiple protein-protein interacting modules and regulated by phosphorylation and dephosphorylation. The Eps15 homology domain containing protein 1 (EHD1) has been implicated in regulating recycling of proteins, internalized both in clathrin-dependent and clathrin-independent endocytic pathways, from the recycling compartment to the plasma membrane. EHD1 was found in a complex with clathrin, adaptor protein complex-2 (AP-2) and insulin-like growth factor-1 receptor (IGF-1R), and was shown to interact with Rabenosyn-5, SNAP29, EHBP1 (EH domain binding protein 1) and syndapin I and II. In this study, we show that EHD1, like the other human EHDs, undergoes serine-phosphorylation. Our results also indicate that EHD1 is a serum-inducible serine-phosphoprotein and that PKC (protein kinase C) is one of its kinases. In addition, we show that inhibitors of clathrin-mediated endocytosis decrease EHD1 phosphorylation, while inhibitors of caveolinmediated endocytosis do not affect EHD1 phosphorylation. The results of experiments in which inhibitors of endocytosis were employed strongly suggest that EHD1 phosphorylation occurs between early endosomes and the endocytic recycling compartment.     

Increase of anti-HIV activity of C-peptide fusion inhibitors using a bivalent drug design approach

We reported the design of fusion inhibitors with improved activity using a multivalent inhibitor design strategy. First, we chose C29 as the template sequence, which is a 29-mer peptide derived from HIV-1 gp41 CHR domain and has anti-HIV activity of IC₅₀ 118 nM in a cell–cell fusion assay. We optimized the crosslink sites and linkers of the template peptide. We found that N-terminal crosslink caused activity improvement based on the multivalent co-operative effect. Especially, the IC₅₀ of peptide (CAcaC29)₂ was improved from 49.02 (monomeric form) to 5.71 nM. Compared with long peptides, short peptides may be more suitable to analyze the co-operative effect. So we selected a shorter peptide C22 to synthesize the bivalent inhibitors. Due its weak helicity, no co-operative effect appeared. Therefore, we chose SC22EK, which were introduced salt bridges to consolidate the helicity based on the natural sequence C22. The cross-linked (CAcaSC22EK)₂ was four times more potent than the monomer SC22EK in anti-HIV activity, with an IC₅₀ value of 4.92 nM close to the high active peptide fusion inhibitor C34. The strategy used in this study may be used to design new fusion inhibitors to interfere similar processes.     

Cyclic peptides containing tryptophan and arginine as Src kinase inhibitors

A number of cyclic and linear peptides containing various combinations of amino acids were evaluated for their Src kinase inhibitory potency. Among all the peptides, cyclic decapeptide C[RW]₅ containing alternative arginine (R) and tryptophan (W) residues was found to be the most potent Src kinase inhibitor. C[RW]₅ showed higher inhibitory activity (IC₅₀ = 2.8 μM) than C[KW]₅, L(KW)₅, C[RW]₄, and C[RW]₃ with IC₅₀ values of 46.9, 69.1, 21.5, and 25.0 μM, respectively, as determined in a fluorescence intensity-based assay. Thus, the cyclic nature, the presence of arginine, ring size, and the number of amino acids in the structure of the peptide were found to be critical in Src kinase inhibitory potency. The IC₅₀ value of C[RW]₅ was found to be 0.8 μM in a radioactive assay using [γ-³²P]-ATP and polyE₄Y as the substrate. C[RW]₅ was a noncompetitive Src kinase inhibitor, showing approximately fourfold more selectivity towards Src than Abl.     

EH domain of EHD1

EHD1 is a member of the mammalian C-terminal Eps15 homology domain (EH) containing protein family, and regulates the recycling of various receptors from the endocytic recycling compartment to the plasma membrane. The EH domain of EHD1 binds to proteins containing either an Asn-Pro-Phe or Asp-Pro-Phe motif, and plays an important role in the subcellular localization and function of EHD1. Thus far, the structures of five N-terminal EH domains from other proteins have been solved, but to date, the structure of the EH domains from the four C-terminal EHD family paralogs remains unknown. In this study, we have assigned the 133 C-terminal residues of EHD1, which includes the EH domain, and solved its solution structure. While the overall structure resembles that of the second of the three N-terminal Eps15 EH domains, potentially significant differences in surface charge and the structure of the tripeptide-binding pocket are discussed.     

Synthesis and disulfide bond connectivity–activity studies of a kalata B1-inspired cyclopeptide against dengue NS2B–NS3 protease

Kalata B1 is a plant protein with remarkable thermal, chemical and enzymatic stability. Its potential applications could be centered on the possibility of using its cyclic structure and cystine knot motif as a scaffold for the design of stable pharmaceuticals. To discover potent dengue NS2B–NS3 protease inhibitors, we have prepared various kalata B1 analogues by varying the amino acid sequence. Mass spectrometric and biochemical investigations of these analogues revealed a cyclopeptide whose two fully oxidized forms are substrate-competitive inhibitors of the dengue viral NS2B–NS3 protease. Both oxidized forms showed potent inhibition with K_i of 1.39 ± 0.35 and 3.03 ± 0.75 μM, respectively.     

Comparison of the susceptibility of porcine and human dipeptidyl-peptidase IV to inhibition by protein-derived peptides

The enzyme dipeptidyl-peptidase IV (DPP-IV) is recognized to be a promising target for the management of type 2 diabetes. Over the last decade, numerous synthetic molecules and more recently, peptides from dietary proteins, have been reported to be able to inhibit DPP-IV activity. Most studies that have investigated the in vitro effect of these inhibitors have used porcine or human DPP-IV. Although structurally alike, it is unclear whether these two species display similar inhibition patterns. Therefore, the objective of this study was to compare the effects of protein-derived peptides on the activity of porcine and recombinant human DPP-IV. The two species showed different inhibition susceptibility to 43 of the 62 peptide sequences investigated. While 37 protein-derived peptides were more effective at inhibiting the porcine DPP-IV, only six caused a stronger inhibition of the activity of the human enzyme. Although the peptides WR, IPIQY and WCKDDQNPHS were found to be among the most potent inhibitors of both species, the inhibitory effect was greater on the porcine enzyme than on human DPP-IV (αK_i or K_i = 11.5, 13.4, 13.3 μM and 31.4, 28.2, 75.0 μM for porcine and human DPP-IV, respectively). Investigation into the mode of action of the most effective inhibitory peptides revealed that both species were inhibited in a similar manner by short fragments (≤5 amino acid residues), but that some of the longer peptides acted differently on the enzymes. This study shows that porcine DPP-IV is generally inhibited with greater potency by protein-derived peptides than is the human enzyme.     

EHD proteins associate with syndapin I and II and such interactions play a crucial role in endosomal recycling

EHD proteins were shown to function in the exit of receptors and other membrane proteins from the endosomal recycling compartment. Here, we identify syndapins, accessory proteins in vesicle formation at the plasma membrane, as differential binding partners for EHD proteins. These complexes are formed by direct eps15-homology (EH) domain/asparagine proline phenylalanine (NPF) motif interactions. Heterologous and endogenous coimmunoprecipitations as well as reconstitutions of syndapin/EHD protein complexes at intracellular membranes of living cells demonstrate the in vivo relevance of the interaction. The combination of mutational analysis and coimmunoprecipitations performed under different nucleotide conditions strongly suggest that nucleotide binding by EHD proteins modulates the association with syndapins. Colocalization studies and subcellular fractionation experiments support a role for syndapin/EHD protein complexes in membrane trafficking. Specific interferences with syndapin-EHD protein interactions by either overexpression of the isolated EHD-binding interface of syndapin II or of the EHD1 EH domain inhibited the recycling of transferrin to the plasma membrane, suggesting that EH domain/NPF interactions are critical for EHD protein function in recycling. Consistently, both inhibitions were rescued by co-overexpression of the attacked protein component. Our data thus reveal that, in addition to a crucial role in endocytic internalization, syndapin protein complexes play an important role in endocytic receptor recycling.     

Structure activity relationship modelling of milk protein-derived peptides with dipeptidyl peptidase IV (DPP-IV) inhibitory activity

Quantitative structure activity type models were developed in an attempt to predict the key features of peptide sequences having dipeptidyl peptidase IV (DPP-IV) inhibitory activity. The models were then employed to help predict the potential of peptides, which are currently reported in the literature to be present in the intestinal tract of humans following milk/dairy product ingestion, to act as inhibitors of DPP-IV. Two models (z- and v-scale) for short (2–5 amino acid residues) bovine milk peptides, behaving as competitive inhibitors of DPP-IV, were developed. The z- and the v-scale models (p < 0.05, R² of 0.829 and 0.815, respectively) were then applied to 56 milk protein-derived peptides previously reported in the literature to be found in the intestinal tract of humans which possessed a structural feature of DPP-IV inhibitory peptides (P at the N₂ position). Ten of these peptides were synthetized and tested for their in vitro DPP-IV inhibitory properties. There was no agreement between the predicted and experimentally determined DPP-IV half maximal inhibitory concentrations (IC₅₀) for the competitive peptide inhibitors. However, the ranking for DPP-IV inhibitory potency of the competitive peptide inhibitors was conserved. Furthermore, potent in vitro DPP-IV inhibitory activity was observed with two peptides, LPVPQ (IC₅₀ = 43.8 ± 8.8 μM) and IPM (IC₅₀ = 69.5 ± 8.7 μM). Peptides present within the gastrointestinal tract of human may have promise for the development of natural DPP-IV inhibitors for the management of serum glucose.     

Molecular docking and structure–activity relationship studies on benzothiazole based non-peptidic BACE-1 inhibitors

A similarity search on the structural analogs of an inhibitor of BACE-1 with IC₅₀ 2.8 μM, which contained a P1 benzothiazole group together with a triazine ring linked by a secondary amine group, was described in this Letter and some more potent inhibitors against BACE-1 were identified. The most potent compound 5 (IC₅₀ = 0.12 μM) increases the inhibitory potency by 24 folds. Our results suggest that a pyrrolidinyl side group at the P3′ and P4′ of the inhibitors are favored for strong inhibition and a small aromatic group at the P4 position is also essential to the potency.     

Design and biological testing of peptidic dimerization inhibitors of human Hsp90 that target the C-terminal domain

BackgroundSmall molecules targeting the dimerization interface of the C-terminal domain of Hsp90, a validated target for cancer treatment, have yet to be identified.MethodsThree peptides were designed with the aim to inhibit the dimerization of Hsp90. Computational and biophysical methods examined the α-helical structure for the three peptides. Based on the Autodisplay technology, a novel flow cytometer dimerization assay was developed to test inhibition of Hsp90 dimerization. Microscale thermophoresis was used to determine the K_D of the peptides towards the C-terminal domain of Hsp90.ResultsMD simulations and CD spectroscopy indicated an α-helical structure for two of the three peptides. By flow cytometer analysis, IC₅₀ values of 2.08 μM for peptide H2 and 8.96 μM for peptide H3 were determined. Dimer formation of the C-terminal dimerization domain was analyzed by microscale thermophoresis, and a K_D of 1.29 nM was determined. Furthermore, microscale thermophoresis studies demonstrated a high affinity binding of H2 and H3 to the C-terminal domain, with a K_D of 1.02 μM and 1.46 μM, respectively.ConclusionsThese results revealed the first peptidic inhibitors of Hsp90 dimerization targeting the C-terminal domain. Furthermore, it has been shown that these peptides bind to the C-terminal domain with a low micromolar affinity.General significanceThese results can be used to design and screen for small molecules that inhibit the dimerization of the C-terminal domain of Hsp90, which could open a new route for cancer therapy.     

Discovery of triazole aminopyrazines as a highly potent and selective series of PI3Kδ inhibitors

A novel class of potent PI3Kδ inhibitors with >1000-fold selectivity against other class I PI3K isoforms is described. Optimization of the substituents on a triazole aminopyrazine scaffold, emerging from an in-house PI3Kα program, turned moderately selective PI3Kδ compounds into highly potent and selective PI3Kδ inhibitors. These efforts resulted in a series of aminopyrazines with PI3Kδ IC₅₀ ? 1 nM in the enzyme assay, some of the most selective PI3Kδ inhibitors published to date, with a cell potency in a JeKo-cell assay of 20–120 nM.     

Strategy for designing selective α-l-rhamnosidase inhibitors: Synthesis and biological evaluation of l-DMDP cyclic isothioureas

This study shows that the cyclization of l-DMDP thioureas to bicyclic l-DMDP isothioureas improved α-l-rhamnosidase inhibition which was further enhanced by increasing the length of the alkyl chain. The addition of a long alkyl chain, such as decyl or dodecyl, to the nitrogen led to the production of highly potent inhibitors of α-l-rhamnosidase; it also caused broad inhibition spectrum against β-glucosidase and β-galactosidase. In contrast, the corresponding N-benzyl-l-DMDP cyclic isothioureas display selective inhibition of α-l-rhamnosidase; 3′,4′-dichlorobenzyl-l-DMDP cyclic isothiourea (3r) was found to display the most potent and selective inhibition of α-l-rhamnosidase, with IC₅₀ value of 0.22 μM, about 46-fold better than the positive control 5-epi-deoxyrhamnojirimycin (5-epi-DRJ; IC₅₀ = 10 μM) and occupied the active-site of this enzyme (K_i = 0.11 μM). Bicyclic isothioureas of ido-l-DMDP did not inhibit α-l-rhamnosidase. These new mimics of l-rhamnose may affect other enzymes associated with the biochemistry of rhamnose including enzymes involved in progression of tuberculosis.     

Multiple FAS1 domains and the RGD motif of TGFBI act cooperatively to bind αvβ3 integrin,leading to anti-angiogenic and anti-tumor effects

TGFBI, a transforming growth factor β-induced extracellular matrix protein, circulates at a level of ~ 300 ng/ml in humans and modulates several integrin-mediated cellular functions. The protein contains an N-terminal EMI domain, four consecutive FAS1 domains, and the RGD motif. Each FAS1 domain and the RGD motif have been known to interact with avb3 integrin. Here, we found that the binding affinity (K_d) of TGFBI for αvβ3 integrin was approximately 3.8 × 10^? 8 M, a value ~ 2300-fold higher than that of a single FAS1 domain, and demonstrated that this greater affinity was due to the cooperative action of the four FAS1 domains and the RGD motif. Moreover, TGFBI exhibited more potent anti-angiogenic and anti-tumorigenic activities, even at a 100-fold lower molar dose than the reported effective dose of the FAS1 domain. Finally, our data showed that TGFBI specifically targeted the tumor vasculature and accumulated at the tumor site. Collectively, our results support the theory that TGFBI acts as a potent endogenous anti-tumor and anti-angiogenic molecule by targeting αvβ3 integrin, and highlights the importance of physiological circulating TGFBI levels in inhibiting tumor growth.     

Y-shaped bis-arylethenesulfonic acid esters: Potential potent and membrane permeable protein tyrosine phosphatase 1B inhibitors

Known PTP1B inhibitors with bis-anionic moieties exhibit potent inhibitory activity, good selectivity, however, they are incapable of penetrating cellular membranes. Based upon our finding of a new pharmacophoric group in inhibition of PTP1B and the structural characteristics of the binding pocket of PTP1B, a series of bis-arylethenesulfonic acid ester derivatives were designed and synthesized. These novel molecules, particularly Y-shaped bis-arylethenesulfonic acid ester derivatives, exhibited high PTP1B inhibitory activity, moderate selectivity, and great potential in penetrating cellular membranes (compound 7p, CLog P = 9.73, P_app = 9.6 × 10^-6 cm/s; IC₅₀ = 140, 1290 and 920 nM on PTP1B, TCPTP and SHP2, respectively). Docking simulations suggested that these Y-shaped inhibitors might interact with multiple secondary binding sites in addition to the catalytic site of PTP1B.     

Synthesis and biological evaluation of imidazole thioacetanilides as novel non-nucleoside HIV-1 reverse transcriptase inhibitors

A series of 2-(1-aryl-1H-imidazol-2-ylthio)acetamide [imidazole thioacetanilide (ITA)] derivatives were synthesized and evaluated as potent inhibitors of human immunodeficiency virus type-1 (HIV-1). Among them, the most potent HIV-1 inhibitors were 4a5 (EC₅₀ = 0.18 μM), and 4a2 (EC₅₀ = 0.20 μM), which were more effective than the lead compound L1 (EC₅₀ = 2.053 μM) and the reference drugs nevirapine and delavirdine. The preliminary structure–activity relationship (SAR) of the newly synthesized congeners is discussed.