Design and synthesis of peptide-based chimeric molecules to induce degradation of the estrogen and androgen receptors

Peptide-based inducers of estrogen receptor (ER) α and androgen receptor (AR) degradations via the ubiquitin–proteasome system (UPS) were developed. The designated inducers were composed of two biologically active scaffolds: the helical peptide PERM3, which is an LXXLL-like mimic of the coactivator SRC-1, and various small molecules (MV1, LCL161, VH032, and POM) that bind to E3 ligases (IAPs, VHL, and cereblon, respectively), to induce ubiquitylation of nuclear receptors that bind to SRC-1. All of the synthesized chimeric E3 ligand-containing molecules induced the UPS-mediated degradation of ERα and AR. The PERM3 peptide was applicable for the development of the ERα and AR degraders using these E3 ligands.     

Efficient protein knockdown of HaloTag-fused proteins using hybrid molecules consisting of IAP antagonist and HaloTag ligand

We previously reported a protein knockdown system for HaloTag-fused proteins using hybrid small molecules consisting of alkyl chloride, which binds covalently to HaloTag, linked to BE04 (2), a bestatin (3) derivative with an affinity for cellular inhibitor of apoptosis protein 1 (cIAP1, a kind of ubiquitin ligase). This system addressed several limitations of prior protein knockdown technology, and was applied to degrade two HaloTag-fused proteins. However, the degradation activity of these hybrid small molecules was not potent. Therefore, we set out to improve this system. We report here the design, synthesis and biological evaluation of novel hybrid compounds 4a and 4b consisting of alkyl chloride linked to IAP antagonist MV1 (5). Compounds 4a and 4b were confirmed to reduce the levels of HaloTag-fused tumor necrosis factor α (HaloTag-TNFα), HaloTag-fused cell division control protein 42 (HaloTag-Cdc42), and unfused HaloTag protein in living cells more potently than did BE04-linked compound 1b. Analysis of the mode of action revealed that the reduction of HaloTag-TNFα is proteasome-dependent, and is also dependent on the linker structure between MV1 (5) and alkyl chloride. These compounds appear to induce ubiquitination at the HaloTag moiety of HaloTag-fused proteins. Our results indicate that these newly synthesized MV1-type hybrid compounds, 4a and 4b, are efficient tools for protein knockdown for HaloTag-fused proteins.     

Design, synthesis and biological evaluation of nuclear receptor-degradation inducers

Compounds that regulate the function(s) of nuclear receptors (NRs) are useful for biological studies and as candidate therapeutic agents. Most such compounds are agonists or antagonists. On the other hand, we have developed specific protein degradation inducers, which we designated as SNIPERs (Specific and Nongenetic IAPs-dependent Protein ERasers), for selective degradation of target proteins. SNIPERs are hybrid molecules consisting of an appropriate ligand for the protein of interest, coupled to a ligand for inhibitor of apoptosis proteins (IAPs), which target the bound protein for polyubiquitination and proteasomal degradation. We considered that protein knockdown with SNIPERs would be a promising alternative approach for modulating NR function. In this study, we designed and synthesized degradation inducers targeting retinoic acid receptor (RAR), estrogen receptor (ER), and androgen receptor (AR). These newly synthesized RAR, ER, and AR SNIPERs, 9, 11, and 13, respectively, were confirmed to significantly reduce the levels of the corresponding NRs in live cells.     

Syntheses,structures and properties of three novel coordination polymers with a flexible asymmetrical bridging ligand

Three novel coordination polymers with different structural motifs, [Ag(pmtmb)]_n (1), [Cd(pmtmb)₂(H₂O)]_n (2) and [Cu(pmtmb)₂]_n (3), have been synthesized with a flexible asymmetrical bridging ligand, 4-(2-pyrimidylthiomethyl)benzoic acid (Hpmtmb). X-ray diffraction analyses show that 1 is a 2D layer containing unusual zigzag Ag chains based on mixed ligand-supported and ligand-unsupported Ag–Ag interactions, 2 is a necklace structure that further linked through hydrogen bonding to form a 2D sheet, and 3 is a 3D compact framework full assembled from 1D centipede-like chains via π–π stacking interactions. Interestingly, the ligand pmtmb adopts different configurations and coordination modes in the solid structures of these complexes. Furthermore, three complexes exhibit remarkable thermal stability and the complexes 1 and 2 exhibit intense green and purple luminescence, respectively.     

Double protein knockdown of cIAP1 and CRABP-II using a hybrid molecule consisting of ATRA and IAPs antagonist

Protein knockdown can be achieved by the use of a small molecule that possesses affinity for both the target protein and ubiquitin ligase. We have designed such a degradation-inducing molecule targeting cIAP1 and CRABP-II, which are involved in proliferation of several cancer cell lines and in neuroblastoma growth, respectively. As a CRABP-II-recognizing moiety, all-trans retinoic acid (ATRA, 3), a physiological ligand of CRABP, was chosen. As a cIAP1-recognizing moiety, MV1 (5), which is a cIAP1/cIAP2/XIAP pan-ligand, was chosen. Although cIAP1 itself possesses ubiquitin ligase activity, we expected that its decomposition would be efficiently mediated by related molecules, including cIAP2 and XIAP, which also possess ubiquitin ligase activity. The designed degradation inducer 6, in which ATRA (3) and MV1 (5) moieties are connected via a linker, was synthesized and confirmed to induce efficient degradation of both cIAP1 and CRABP-II. It showed potently inhibited the proliferation of IMR32 cells.     

Structure-based design,synthesis and biological evaluation of a newer series of pyrazolo[1,5-a]pyrimidine analogues as potential anti-tubercular agents

In-depth study of structure-based drug designing can provide vital leads for the development of novel, clinically active molecules. In this present study, twenty six novel pyrazolo[1,5-a]pyrimidine analogues (6a-6z) were designed using molecular docking studies. The designed molecules were synthesized in good yields. Structural elucidation of the synthesized molecules was carried out using IR, MS, ¹H NMR and ¹³C NMR spectroscopy. All the synthesized compounds were evaluated for their in-vitro anti-tubercular activity against H37Rv strain by Alamar Blue assay method. Most of the synthesized compounds displayed potent anti-tubercular activities. Amongst all the tested compounds 6p, 6g, 6n and 6h exhibited promising anti-tubercular activity. Further, these potent compounds were gauged for MDR-TB, XDR-TB and cytotoxic study. None of these compounds exhibited potent cytotoxicity. Stability of protein ligand complex was further evaluated by molecular dynamics simulation for 10 ns. All these results indicate that the synthesized compounds could be potential leads for further development of new potent anti-tubercular agents.     

Two-dimensional Cu and first Pb coordination polymers based on a flexible 1,4-cyclohexanedicarboxylate ligand displaying different conformations and coordination modes

Two novel metal-organic hybrid coordination polymers {[Cu(bpo)(chdc)(H₂O)](H₂O)_0.5}_n (1) and [Pb(chdc)(H₂O)]_n (2) have been synthesized under different conditions and structurally characterized by single-crystal X-ray diffraction technique, where H₂chdc refers to a flexible 1,4-cyclohexanedicarboxylic acid ligand and bpo is 2,5-bis(4-pyridyl)-1,3,4-oxadiazole. Complex 1 has a two-dimensional (2-D) grid-like [11.28 × 13.63 Å²] framework in which the Cu^II centers are extended via bidentate bridging ligands bpo and e,e-trans-chdc along two directions, exhibiting large porous cavities. Coordination polymer 2 represents the first Pb^II complex of H₂chdc in which the larger Pb^II centers are connected by e,a-cis-chdc anions to afford a 2-D close-knit structure.     

Synthesis, characterization and crystal structure of a monomeric and a macrocyclic copper (II) complex with a large cavity using benzylacetylacetone ligand

The bidentate ligand benzylacetylacetone was used to synthesize the Cu(II) complexes 1 and 2 without and with 4,4^′-bipyridine ligand, respectively. The complexes were characterized by analytical and spectroscopic studies. The mononuclear complex [Cu(C₁₀H₉O₂)₂] (1) has been synthesized by the reaction of copper acetate with the ligand whereas the tetranuclear complex [Cu₄(4,4^′-bpy)₄(C₁₀H₉O₂)₄(C₂H₃O₂)₄] (2) has been synthesized by the reaction of copper acetate with the ligand followed by the addition of 4,4^′-bipyridine. The X-ray analysis shows that the complex 1 has square planar geometry and the complex 2 has square pyramidal geometry around the metal centers. The thermogravimetric studies showed that the complexes undergo decomposition in multiple steps.     

Novel biotin linker with alkyne and amino groups for chemical labelling of a target protein of a bioactive small molecule

We synthesized a novel linker (1) with biotin, alkyne and amino groups for the identification of target proteins using a small molecule that contains an azide group (azide probe). The alkyne in the linker bound the azide probe via an azide-alkyne Huisgen cycloaddition. A protein cross-linker effectively bound the conjugate of the linker and an azide probe with a target protein. The covalently bound complex was detected by western blotting. Linker 1 was applied to a model system using an abscisic acid receptor, RCAR/PYR/PYL (PYL). Cross-linked complexes of linker 1, the azide probes and the target proteins were successfully visualized by western blotting. This method of target protein identification was more effective than a previously developed method that uses a second linker with biotin, alkyne, and benzophenone (linker 2) that acts to photo-crosslink target proteins. The system developed in this study is a method for identifying the target proteins of small bioactive molecules and is different from photo-affinity labelling.     

Degradation of huntingtin mediated by a hybrid molecule composed of IAP antagonist linked to phenyldiazenyl benzothiazole derivative

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by aggregation of mutant huntingtin (mHtt), and removal of mHtt is expected as a potential therapeutic option. We previously reported protein knockdown of Htt by using hybrid small molecules (Htt degraders) consisting of BE04, a ligand of ubiquitin ligase (E3), linked to probes for protein aggregates. Here, in order to examine the effect of changing the ligand, we synthesized a similar Htt degrader utilizing MV1, an antagonist of the inhibitor of apoptosis protein (IAP) family (a subgroup of ubiquitin E3 ligases), which is expected to have a higher affinity and specificity for IAP, as compared with BE04. The MV1-based hybrid successfully induced interaction between Htt aggregates and IAP, and reduced mHtt levels in living cells. Its mode of action was confirmed to be the same as that of the BE04-based hybrid. However, although the affinity of MV1 for IAP is greater than that of BE04, the efficacy of Htt degradation by the MV1-based molecule was lower, suggesting that linker length between the ligand and probe might be an important determinant of efficacy.     

Synthesis and immunoregulatory activities of conjugates of a Toll-like receptor 7 inert ligand

In the synthesis and modification of the analogs of an adenine type of Toll-like receptor (TLR) 7 agonists, we found a special compound, 9-propionyloxy-8-hydroxy-2-(2-methoxyethoxy)-adenine (6). It is a synthesized TLR7 inert ligand, which does not respond to TLR7 itself. However, it can be coupled with protein or peptide antigens via propionyloxy functional group to promote their immunogenicity significantly. The compound was covalently coupled to protein and peptide to get the conjugates. The inductivity of cytokine production by the conjugates was 872.4-fold compared with the unconjugated antigens in vitro by mouse splenocyte. These data show that the immunostimulatory activity of inert TLR7 ligand can be endowed, and the activity of antigens can be amplified by conjugation with various proteins and peptides, thus broadening the potential therapeutic application and reducing the risk of TLR7 agonists’ side effects.     

Hydrothermal synthesis and characterizations of a novel 2-D double-layers metal-organic coordination polymer involving in situ ligand synthesis

A novel 2-D metal-organic coordination polymer [Ni_0.5(apddica)(H₂O)] · H₂O 1 (Hapddica=2-(4-Amino-phenyl)-1,3-dioxo-2,3-dihydro-1H-isoindole-5-carboxylic acid) has been hydrothermally synthesized and structurally characterized by element analyses, IR and XPS spectra, TG analyses and single crystal X-ray diffraction. Crystal data: C₁₅H₁₃N₂Ni_0.5O₆, monoclinic, P2(1)/c, a=12.181(2) Å, b=7.9392(6) Å, c=14.835(3) Å, β=94.25(3) °, V=1430(75) ų, Z=4. Compound 1 exhibits a novel 2-D double-layers network based on apddica and Ni center. It is noteworthy that new ligand Hapddica was first prepared with hydrothermal technique involving in situ ligand synthesis and used for constructing coordination polymers. There are unusual parallelogram tunnels along a-axis in which the dissociative water molecules filled in. The magnetic property of compound has been studied.     

Synthesis, characterization, and protein tyrosine phosphatases inhibition activities of oxovanadium(IV) complexes with Schiff base and polypyridyl derivatives

Seven new mixed-ligand vanadyl complexes, [V^IVO(5-Br-SAA)(NN)] and [V^IVO(2-OH-NAA)(NN)] (1-7) (5-Br-SAA for 5-bromosalicylidene anthranilic acid, 2-OH-NAA for 2-hydroxy-1-naphthaldehyde anthranilic acid and NN for N,N′-donor heterocyclic base, namely, 2,2′-bipyridine (bpy, 1 and 5), 1,10-phenanthroline (phen, 2 and 6), dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq, 3 and 7), dipyrido[3,2-a:2′,3′-c]phenazine (dppz, 4)), were synthesized and characterized. X-ray crystal structure of [V^IVO(5-Br-SAA)(phen)] revealed a distorted octahedral geometry with the Schiff base ligand coordinated in a tridentate ONO-fashion and the phenanthroline ligand in a bidentate fashion. Density-functional theory (DFT) calculations suggest a similar structure and the same coordination mode for all the other oxovanadium complexes synthesized. Biochemical assays demonstrate that the mixed-ligand oxovanadium(IV) complexes are potent inhibitors of protein tyrosine phosphatase 1B (PTP1B), with IC₅₀ values approximately 41-75 nM. Kinetics assays suggest that the complexes inhibit PTP1B in a competitive manner. Notably, they had moderate selectivity of PTP1B over T-cell protein tyrosine phosphatase (TCPTP) (about 2-fold) and good selectivity over Src homology phosphatase 1 (SHP-1) (about 4∼7-fold). Thus, these mixed-ligand complexes represent a promising class of PTP1B inhibitors for future development as anti-diabetic agents.     

Novel 3,4-dihydro-4-oxoquinazoline-based acetohydrazides: Design,synthesis and evaluation of antitumor cytotoxicity and caspase activation activity

In search for novel small molecules with antitumor cytotoxicity via activating procaspase-3, we have designed and synthesized three series of novel (E)-N′-benzylidene-4-oxoquinazolin-3(4H)-yl)acetohydrazides (5a-j, 6a-h, and 7a-h). On the phenyl ring ò the benzylidene part, three different substituents, including 2-OH-4-OCH₃, 4-OCH₃, and 4-N(CH₃)₂, were introduced, respectively. Biological evaluation showed that the acetohydrazides in series 5a-j, in which the phenyl ring of the benzylidene part was substituted by 2-OH-4-OCH₃ substituent, exhibited potent cytotoxicity against three human cancer cell lines (SW620, colon; PC-3, prostate; NCI-H23, lung). Most of the compounds, in this series, especially compounds 5c, 5b and 5h, also significantly activated caspase-3 activity. Among these, compound 5c displayed 1.61-fold more potent than PAC-1 as caspase-3 activator. Cell cycle analysis showed that compounds 5b, 5c, and 5h significantly arrested the cell cycle in G1 phase. Further apoptotic studies also demonstrated compounds 5b, 5c, and 5h as strong apoptotic cell death inducers. The docking simulation studies showed that these compounds could activate procaspase via chelating Zn²⁺ ion bound to the allosteric site of the zymogen.     

Tyrosine-based asymmetric urea ligand for prostate carcinoma: Tuning biological efficacy through in silico studies

In this article, we have explored the chemical interactions of tyrosine-based asymmetric urea ligands in the binding pockets of prostate specific membrane antigen (PSMA) through in silico studies. The S1 pocket of the PSMA protein offers better scope for modifications in the urea ligands to improve the binding affinity. Accordingly, tyrosine-based (S)-2-(3-((S)-1-carboxy-2-(4-(carboxymethoxy)phenyl)ethyl)ureido)pentanedioic acid (CYUE, 3) ligand was designed, synthesized and predicted to possess inhibition constant (Ki) of 55 nM with PSMA protein. The CYUE (3) ligand was further elaborated into a fluorescent diagnostic probe for detection of PSMA⁺ cancers. In vitro studies on human malignant cell lines such as LNCaP and PC-3 were performed to show the efficacy and specificity of the newly synthesized bio-construct. The fluorescent bio-conjugate was found to be very specific to the PSMA protein with an overall binding affinity constant (K_D) of 88 nM.     

Synthesis, crystal structure, antitumor activity and DNA-binding study on the Mn(II) complex of 2H-5-hydroxy-1,2,5-oxadiazo[3,4-f]1,10-phenanthroline

The complex [Mn(L)(NO₃)₂(H₂O)₂] (1) (L=2H-5-hydroxy-1,2,5-oxadiazo[3,4-f]1,10-phenanthroline) was synthesized and characterized by elemental analysis, IR and UV. The crystal and molecular structure of 1 was determined by single-crystal X-ray diffraction; crystal data: light yellow, monoclinic, space group P2₁/n, Z=4, a=7.432(2) Å, b=9.582(3) Å, c=23.445(7) Å, β=90.519(5)°. The Mn atom in 1 is hexa-coordinated in a distorted octahedral arrangement by two N atoms of the ligand L and four O atoms of two water molecules and two nitrate anions. Biological tests in vitro showed that 1 has significant antitumor activity against HL-60, KB, Hela and BGC-823 cells. The interaction of 1 with calf thymus DNA was investigated by absorption titration, thermal denaturation and viscosity measurements. The results suggest that 1 binds with DNA by intercalating via the ligand L.     

Synthesis, spectral and X-ray structural studies of a NO donor Schiff base ligand and its Ni(II) complexes

The Schiff base ligand, 4-isopropylbenzaldehyde[N-(3-oxo-3,4-dihydro-2-quinoxalinyl)hydrazone] (Ipbh), the 1:1 condensation product of 4-isopropylbenzaldehyde and 2-hydroxy-3-hydrazinoquinoxiline, has been synthesized and characterized by X-ray crystallography. A series of complexes of Ipbh with Nickel(II), viz., [Ni(Ipbh)₂]Cl₂ (1), [Ni(Ipbh)₂]Br₂ (2), [Ni(Ipbh)₂]I₂ (3), [Ni(Ipbh)₂·(CH₃OH)₂](NO₃)₂·(CH₃OH)₂ (4) and [Ni(Ipbh)₂ClO₄]ClO₄ (5) have been synthesized. All the complexes were characterized by elemental analysis, molar conductivity, CHN analysis, spectroscopic studies, magnetic susceptibility measurements and TG/DTA methods. The solid-state structure of the complex 4 was established by single crystal X-ray crystallography. In all the complexes, Ipbh acts as a bidentate NO chelating agent, coordinated to the metal ion through the imine nitrogen and quinoxaline oxygen. In complex 4, Nickel(II) is in a distorted octahedral environment with an identical set of donor atoms, N₂O₄, coming from two imine nitrogen and two quinoxaline oxygen atoms of two Ipbh moieties as well as two oxygen atoms of the two methanol molecules. The crystal packing of Ipbh and the complex 4 exhibits 1D and 2D supramolecular networks, respectively through different intermolecular hydrogen-bonding interactions.