Bifunctional ligands that target cells displaying the alpha v beta3 integrin

Strategies to eliminate tumor cells have long been sought. We envisioned that a small molecule could be used to decorate the offending cells with immunogenic carbohydrates and evoke an immune response. To this end, we describe the synthesis of bifunctional ligands possessing two functional motifs: one binds a cell-surface protein and the other binds a naturally occurring human antibody. Our conjugates combine an RGD-based peptidomimetic, to target cells displaying the alpha v beta3 integrin, with the carbohydrate antigen galactosyl-alpha(1-3)galactose [Galalpha(1-3)Gal or alpha-Gal]. To generate such bifunctional ligands, we designed and synthesized RGD mimetics 1 b and 2 c, which possess a free amino group for modification. These compounds were used to generate bifunctional derivatives 1 c and 2 d, with dimethyl squarate serving as the linchpin; thus, our synthetic approach is modular. To evaluate the binding of our peptidomimetics to the target alpha v beta3-displaying cells, we implemented a cell-adhesion assay. Results from this assay indicate that the designed, small-molecule ligands inhibit alpha v beta3-dependent cell adhesion. Additionally, our most effective bifunctional ligand exhibits a high degree of selectivity (4000-fold) for alpha v beta3 over the related alpha v beta5 integrin, a result that augurs its utility in specific cell targeting. Finally, we demonstrate that the bifunctional ligands can bind to alpha v beta3-positive cells and recruit human anti-Gal antibodies. These results indicate that both the integrin-binding and the anti-Gal-binding moieties can act simultaneously. Bifunctional conjugates of this type can facilitate the development of new methods for targeting cancer cells by exploiting endogenous antibodies. We anticipate that our modifiable alpha v beta3-binding ligands will be valuable in a variety of applications, including drug delivery and tumor targeting.     

Self-assembling multimeric integrin alpha5beta1 ligands for cell attachment and spreading

Substrates utilising clustered arginine-glycine-aspartic acid (RGD) ligand displays support greater cell adhesion over random displays. However, cell adhesion to integrin alpha5beta1 requires the synergy site on the 9th type III fibronectin domain (FIII) in addition to RGD on the 10th FIII domain. Here, we have designed and expressed soluble protein chimeras consisting of an N-terminal 9th-10th FIII domain pair, IgG-derived hinge and leucine zipper-derived helix; the latter mutated to yield di-, tri- and tetrameric coiled coils and thus self-assembling, multimeric integrin alpha5beta1 ligands. A unique C-terminal cysteine was appended to the helix to facilitate 'anchoring' of the chimeras with a defined orientation on a surface. Size-exclusion chromatography and circular dichroism demonstrated that the chimeras self-assembled as multimers in solution with defined secondary structures predicted from theoretical calculations. Biotinylation via a thioether bond was used to selectively bind the chimeras to streptavidin-coated surfaces, each of which was then shown to bind integrin alpha5beta1 by surface plasmon resonance. Spreading of fibroblasts to surfaces derivatised with the chimeras was found to proceed in the order: tetramer > trimer > dimer > monomer. Thus, we describe novel polyvalent integrin alpha5beta1 ligands for facile derivatisation of substrates to improve cell adhesion in vitro.     

Synthesis and biological investigation of novel tricyclic benzodiazepinedione-based RGD analogues

Integrins, a widely expressed family of heterodimeric cell surface adhesion proteins, are expressed in a variety of cell types. They play a decisive role in cell-cell adhesion or cell to extracellular matrix adhesion events. Antagonists of alpha(v)beta(3) or alpha(IIb)beta(3) integrin may have a potential use in suppression of pathological processes. We present the synthesis of novel tricyclic benzodiazepinedione-based RGD analogues, which were subsequently tested in a solid-phase receptor assay in order to investigate their binding affinities towards alpha(v)beta(3) and alpha(IIb)beta(3) integrin.     

A potent integrin antagonist from a small library of cyclic RGD pentapeptide mimics including benzyl-substituted azabicycloalkane amino acids

A small library of cyclic RGD pentapeptide mimics, including benzyl-substituted azabicycloalkane amino acids, was synthesized with the aim of developing active and selective integrin antagonists. In vitro binding assays established one particular compound with affinity for both the alpha(v)beta(3) and the alpha(v)beta(5) integrins. The synthesis in solution and the in vitro screening of these RGD derivatives, as well as the determination of the conformational properties of the integrin ligands by spectroscopic and computational methods are described.     

Identification of a Suitable Peptidic Molecular Platform for the Development of NPY(Y1)R-Specific Imaging Agents

NPY(Y₁)R (neuropeptide Y receptor subtype 1) is an important target structure for tumor-specific imaging and therapy as this receptor subtype is overexpressed in very high density and incidence especially in human breast cancer. Targeting this receptor with radiolabeled truncated analogues of the endogenous ligand NPY (neuropeptide Y) has, however, not yet resulted in satisfactory imaging results when using positron emission tomography (PET). This can be attributed to the limited stability of these PET imaging agents caused by their fast proteolytic degradation. Although highly promising NPY analogues were developed, their stability has only been investigated in very few cases. In this systematical work, we comparatively determined the stability of the five most promising truncated analogues of NPY that were developed over the last years, showing the highest receptor affinities and subtype selectivities. The stability of the peptides was assessed in human serum as well as in a human liver microsomal stability assay; these gave complementary results, thus demonstrating the necessity to perform both assays and not just conventional serum stability testing. Of the tested peptides, only [Lys(lauroyl)²⁷,Pro³⁰,Lys(DOTA)³¹,Bip³²,Leu³⁴]NPY_27-36 showed high stability against peptidase degradation; thus this is the best-suited truncated NPY analogue for the development of NPY(Y₁)R-specific imaging agents.     

Biological Relevance of RGD-Integrin Subtype-Specific Ligands in Cancer

Integrins are heterodimeric transmembrane proteins able to connect cells with the micro-environment. They represent a family of receptors involved in almost all the hallmarks of cancer. Integrins recognizing the Arg-Gly-Asp (RGD) peptide in their natural extracellular matrix ligands have been particularly investigated as tumoral therapeutic targets. In the last 30 years, intense research has been dedicated to designing specific RGD-like ligands able to discriminate selectively the different RGD-recognizing integrins. Chemists′ efforts have led to the proposition of modified peptide or peptidomimetic libraries to be used for tumor targeting and/or tumor imaging. Here we review, from the biological point of view, the rationale underlying the need to clearly delineate each RGD-integrin subtype by selective tools. We describe the complex roles of RGD-integrins (mainly the most studied αvβ3 and α5β1 integrins) in tumors, the steps towards selective ligands and the current usefulness of such ligands. Although the impact of integrins in cancer is well acknowledged, the biological characteristics of each integrin subtype in a specific tumor are far from being completely resolved. Selective ligands might help us to reconsider integrins as therapeutic targets in specific clinical settings.     

A “Three-in-One” Multifunctional Probe for Bcl-2/Mcl-1 Profiling and Visualizing in Situ

Bcl-2 and Mcl-1, the two arms of the anti-apoptotic Bcl-2 family proteins, have been identified as key regulators of apoptosis and effective therapeutic targets of cancer. However, no small-molecular probe is capable of profiling and visualizing both Bcl-2 and Mcl-1 simultaneously in situ. Herein, we report a multifunctional molecular probe (BnN₃-OPD-Alk) by a “three-in-one” molecular designing strategy, which integrated the Bcl-2/Mcl-1 binding ligand, fluorescent reporter group and photoreactive group azido into the same scaffold. BnN₃-OPD-Alk exhibited sub-micromolar affinities to Bcl-2/Mcl-1 and bright green self-fluorescence. It was then successfully applied for Bcl-2/Mcl-1 labeling, capturing, enriching, and bioimaging both in vitro and in cells. This strategy could facilitate the precise early diagnosis and effective therapy of dual Bcl-2/Mcl-1-related diseases.     

Development of novel 1,2,3,4-tetrahydroisoquinoline derivatives and closely related compounds as potent and selective dopamine D3 receptor ligands

Based on N-alkylated 1,2,3,4-tetrahydroisoquinoline derivatives, which are structurally related to the partial agonist BP 897, a series of novel, selective dopamine D3 receptor antagonists has been synthesised. Derivatisation included changes in the arylamide moiety and the tetrahydroisoquinoline substructure leading to compounds with markedly improved selectivities and affinities in the low nanomolar concentration range. From the 55 structures presented here, (E)-3-(4-iodophenyl)-N-(4-(1,2,3,4-tetrahydroisoquinolin-2-yl)butyl)acrylamide (51) has high affinity (Ki(hD3)=12 nM) and a 123-fold preference for the D3 receptor relative to the D2 receptor subtype. Its pharmacological profile offers the prospect of a novel radioligand as a tool for various dopamine D3-receptor-related in vitro and in vivo investigations.     

A tale of two targets: differential RNA selectivity of nucleobase-aminoglycoside conjugates

Aminoglycoside antibiotics are RNA-binding polyamines that can bind with similar affinities to structurally diverse RNA targets. To design new semisynthetic aminoglycosides with improved target selectivity, it is important to understand the energetic and structural basis by which diverse RNA targets recognize similar ligands. It is also imperative to discover how novel aminoglycosides could be rationally designed to have enhanced selectivity for a given target. Two RNA drug targets, the prokaryotic ribosomal A-site and the HIV-1 TAR, provide an excellent model system in which to dissect the issue of target selectivity, in that they each have distinctive interactions with aminoglycosides. We report herein the design, synthesis, and binding activity of novel nucleobase-aminoglycoside conjugates that were engineered to be more selective for the A-site binding pocket. Contrary to the structural design, the conjugates bind the A-site more weakly than does the parent compound and bind the TAR more tightly than the parent compound. This result implies that the two RNA targets differ in their ability to adapt to structurally diverse ligands and thus have inherently different selectivities. This work emphasizes the importance of considering the inherent selectivity traits of the RNA target when engineering new ligands.     

Construction of an artificial receptor protein ("anticalin") based on the human apolipoprotein D

Human apolipoprotein D (ApoD) is a prominent member of the lipocalin family of proteins and transports arachidonic acid and progesterone in various body fluids. Lipocalins share a structurally conserved beta barrel as their central folding unit, which supports a set of four hypervariable loops that form the entrance to the ligand pocket. Based on this structural pattern ApoD was employed as a scaffold for the combinatorial design of artificial receptor proteins termed anticalins. After randomization of 24 amino acids located within the loop region, several ApoD variants were selected against hemoglobin, a biochemically well-characterized model target, by using bacterial phagemid display and colony screening. One variant, dubbed HbgA, was further investigated by surface plasmon resonance interaction analysis and found to complex hemoglobin specifically and with a dissociation constant of about 2 microM. While our previous work on the structurally related insect bilin-binding protein was focused on the generation of binding activity towards low-molecular-weight ligands, this study demonstrates for the first time that a lipocalin can also be tailored to recognize a protein target. The fact that even a human member of this protein family has now been successfully recruited for anticalin construction opens the possibility for future application of such engineered lipocalins as target-recognition vehicles in medical therapy.     

Development of a structural model for the cytoplasmic domain of an integrin

The cytoplasmic tails of integrin heterodimers play central roles in controlling the activation states of integrins and in transmitting intracellular signals. Despite their short length, no structure of any integrin cytoplasmic domain has been determined. Therefore, molecular models for the cytoplasmic domain of alpha(IIb)beta3, the major platelet integrin, were generated, including models for the individual cytoplasmic tails, the binary alphaIIb-calcium complex, and the ternary alphaIIb-beta3-calcium complex. Structural analysis of circular dichroism spectra were compiled with data obtained from short homologous sequences within crystallized proteins, and with secondary structural predictions to develop starting models for each subunit. These models were subjected to a series of energy minimization and molecular dynamic simulations to generate final models. AlphaIIb was predicted to be ordered at its N-terminus and its C-terminus could accommodate a cation in a multicoordinated complex. The structure of beta3 was dominated by a beta-turn at its NPXY motif (beta3 744-747). In docking of alphaIIb to different sites within beta3, the conformation of the beta3 juxta-transmembrane (beta3 716-721) was greatly altered. This region was confirmed to be a conformational 'hot- spot' by circular dichroism. The conformational flexibility of this juxta-transmembrane region, which is highly conserved amongst integrins, is ideally located to regulate signaling.      

Synthesis of Aminoethyl-Substituted Piperidine Derivatives as σ1 Receptor Ligands with Antiproliferative Properties

A series of novel σ₁ receptor ligands with a 4-(2-aminoethyl)piperidine scaffold was prepared and biologically evaluated. The underlying concept of our project was the improvement of the lipophilic ligand efficiency of previously synthesized potent σ₁ ligands. The key steps of the synthesis comprise the conjugate addition of phenylboronic acid at dihydropyridin-4(1H)-ones 7 , homologation of the ketones 8 and introduction of diverse amino moieties and piperidine N-substituents. 1-Methylpiperidines showed particular high σ₁ receptor affinity and selectivity over the σ₂ subtype, whilst piperidines with a proton, a tosyl moiety or an ethyl moiety exhibited considerably lower σ₁ affinity. Molecular dynamics simulations with per-residue binding free energy deconvolution demonstrated that different interactions of the basic piperidine-N-atom and its substituents (or the cyclohexane ring) with the lipophilic binding pocket consisting of Leu105, Thr181, Leu182, Ala185, Leu186, Thr202 and Tyr206 are responsible for the different σ₁ receptor affinities. Recorded logD_7.4 and calculated clogP values of 4a and 18a indicate low lipophilicity and thus high lipophilic ligand efficiency. Piperidine 4a inhibited the growth of human non-small cell lung cancer cells A427 to a similar extent as the σ₁ antagonist haloperidol. 1-Methylpiperidines 20a , 21a and 22a showed stronger antiproliferative effects on androgen negative human prostate cancer cells DU145 than the σ₁ ligands NE100 and S1RA.     

GPCR antitarget modeling: pharmacophore models for biogenic amine binding GPCRs to avoid GPCR-mediated side effects

G protein-coupled receptors (GPCRs) form a large protein family that plays an important role in many physiological and pathophysiological processes. However, the central role that the biogenic amine binding GPCRs and their ligands play in cell signaling poses a risk in new drug candidates that reveal side affinities towards these receptor sites. These candidates have the potential to interfere with the physiological signaling processes and to cause undesired effects in preclinical or clinical studies. Here, we present 3D cross-chemotype pharmacophore models for three biogenic amine antitargets: the alpha(1A) adrenergic, the 5-HT(2A) serotonin, and the D2 dopamine receptors. These pharmacophores describe the key chemical features present within these biogenic amine antagonists and rationalize the biogenic amine side affinities found for numerous new drug candidates. First applications of the alpha(1A) adrenergic receptor model reveal that these in silico tools can be used to guide the chemical optimization towards development candidates with fewer alpha(1A)-mediated side effects (for example, orthostatic hypotension) and, thus, with an improved clinical safety profile.     

CCL4 Stimulates Cell Migration in Human Osteosarcoma via the mir-3927-3p/Integrin αvβ3 Axis

Osteosarcoma is the most common type of primary malignant bone cancer, and it is associated with high rates of pulmonary metastasis. Integrin αvβ3 is critical for osteosarcoma cell migratory and invasive abilities. Chemokine (C-C motif) ligand 4 (CCL4) has diverse effects on different cancer cells through its interaction with its specific receptor, C-C chemokine receptor type 5 (CCR5). Analysis of mRNA expression in human osteosarcoma tissue identified upregulated levels of CCL4, integrin αv and β3 expression. Similarly, an analysis of records from the Gene Expression Omnibus (GEO) dataset showed that CCL4 was upregulated in human osteosarcoma tissue. Importantly, the expression of both CCL4 and integrin αvβ3 correlated positively with osteosarcoma clinical stages and lung metastasis. Analysis of osteosarcoma cell lines identified that CCL4 promotes integrin αvβ3 expression and cell migration by activating the focal adhesion kinase (FAK), protein kinase B (AKT), and hypoxia inducible factor 1 subunit alpha (HIF-1α) signaling pathways, which can downregulate microRNA-3927-3p expression. Pharmacological inhibition of CCR5 by maraviroc (MVC) prevented increases in integrin αvβ3 expression and cell migration. This study is the first to implicate CCL4 as a potential target in the treatment of metastatic osteosarcoma.     

Synthesis and Pharmacological Evaluation of σ2 Receptor Ligands Based on a 3-Alkoxyisoxazole Scaffold: Potential Antitumor Effects against Osteosarcoma

Since its initial discovery as the basis for nicotinic acetylcholine receptor ligands, the 3-alkoxyisoxazole scaffold has been shown to be a versatile platform for the development of potent σ1 and σ2 receptor ligands. Herein we report a further SAR exploration of the 3-alkoxyisoxazole scaffold with the aim of obtaining potent σ2 receptor ligands. Various substitutions on the benzene ring and at the basic amino regions resulted in a total of 21 compounds that were tested for their binding affinities for the σ2 receptor. In particular, compound 51 [(2S)-1-(4-ammoniobutyl)-2-(((5-((3,4-dichlorophenoxy)methyl)isoxazol-3-yl)oxy)methyl)pyrrolidin-1-ium chloride] was identified as one of the most potent σ2 ligands within the series, with a K_i value of 7.9 nM. It demonstrated potent antiproliferative effects on both osteosarcoma cell lines 143B and MOS−J (IC₅₀ values of 0.89 and 0.71 μM, respectively), relative to siramesine (IC₅₀ values of 1.81 and 2.01 μM). Moreover, compound 51 inhibited clonal formation of osteosarcoma 143B cells at 1 μM, corresponding to half the dose required of siramesine for similar effects. The general cytotoxicity profile of compound 51 was assessed in a number of normal cell lines, including HaCaT, HAF, and LO2 cells. Furthermore, FACS analysis showed that compound 51 likely inhibits osteosarcoma cell growth by disruption of the cell cycle and promotion of apoptosis.     

18F-Labeled FAUC 346 and BP 897 derivatives as subtype-selective potential PET radioligands for the dopamine D3 receptor

Disturbances of neutrotransmission at the dopamine D3 receptor are related to several neuropsychiatric diseases and in particular to drug addiction. Herein, we report the computer-assisted prediction of D3 selectivities of new fluoroalkoxy-substituted receptor ligands by means of 3D-QSAR analysis. As close analogues of the D3-selective lead compound FAUC 346 and BP 879, the (19)F-substituted test compounds 4 a-d were synthesized and evaluated. In vitro investigation of their binding characteristics in transfected Chinese Hamster Ovary (CHO) cells led to excellent K(i) values between 0.12 and 0.69 nM at the dopamine D3 subtype. The benzothiophene-substituted carboxamide 4 a (K(i)=0.12 nM) displayed 133 and 283-fold selectivity over the structurally related D2(Long) and D4 subtypes, respectively. Mitogenesis assays showed the behavior of partial agonists. Based on these data, we synthesized the [(18)F]fluoroethoxy-substituted radioligands [(18)F]4 a-d. The N-[4-[4-(2-hydroxyphenyl)piperazin-1-yl]butyl]-2-carboxamides 3 a-d were prepared and labeled with 2-[(18)F]fluoroethyltosylate in a two-step procedure. Optimization of the (18)F-labeling conditions led to radiochemical yields between 24 and 65 %.     

Disaccharide mimetics of the aminoglycoside antibiotic neamine

A highly convergent approach has been employed for the facile synthesis of a library of 24 disaccharides that are alpha(1-3), beta(1-3), alpha(1-4), or beta(1-4) linked and contain 2-4 amino groups. Fourier-transformation ion cyclotron resonance mass spectrometry (FT-ICR MS) has been used to determine dissociation constant (Kd) values for the binding of the disaccharides to a prototypical fragment of 16S ribosomal RNA. Several derivatives bound with affinities similar to that of neamine. Structure-activity relationships have revealed the substitution pattern that is important for high-affinity binding. The compounds described here are unique lead compounds for the design of novel aminoglycoside antibiotics.     

Escaping Antiangiogenic Therapy: Strategies Employed by Cancer Cells

Tumor angiogenesis is widely recognized as one of the “hallmarks of cancer”. Consequently, during the last decades the development and testing of commercial angiogenic inhibitors has been a central focus for both basic and clinical cancer research. While antiangiogenic drugs are now incorporated into standard clinical practice, as with all cancer therapies, tumors can eventually become resistant by employing a variety of strategies to receive nutrients and oxygen in the event of therapeutic assault. Herein, we concentrate and review in detail three of the principal mechanisms of antiangiogenic therapy escape: (1) upregulation of compensatory/alternative pathways for angiogenesis; (2) vasculogenic mimicry; and (3) vessel co-option. We suggest that an understanding of how a cancer cell adapts to antiangiogenic therapy may also parallel the mechanisms employed in the bourgeoning tumor and isolated metastatic cells delivering responsible for residual disease. Finally, we speculate on strategies to adapt antiangiogenic therapy for future clinical uses.     

Synthesis and Pharmacological Evaluation of SNC80 Analogues with a Bridged Piperazine Ring

To discover novel δ‐opioid receptor ligands derived from SNC80 ( 1 ), a series of 6,8‐diazabicyclo[3.2.2]nonane derivatives bearing two aromatic moieties was prepared, and the affinity toward δ, μ, and κ receptors, as well as σ receptors, was investigated. After removal of the 4‐methoxybenzyl and 2,4‐dimethoxybenzyl protecting groups, the pharmacophoric N,N‐diethylcarbamoylbenzyl residue was attached to the 6,8‐diazabicyclo[3.2.2]nonane framework to yield the designed δ receptor ligands. In a first series of compounds the benzhydryl moiety of SNC80 was dissected, and one phenyl ring was attached to the bicyclic framework. In a second series of δ ligands the complete benzhydryl moiety was introduced into the bicyclic scaffold. The determined δ receptor affinities show that compounds based on an (R)‐glutamate‐derived bicyclic scaffold possess higher δ receptor affinity than their (S)‐glutamate‐derived counterparts. Furthermore, an intact benzhydryl moiety leads to δ receptor ligands that are more potent than compounds with two separated aromatic moieties. Compound 24 , with the same spatial arrangement of substituents around the benzhydryl stereocenter as SNC80, shows the highest δ receptor affinity of this series: K_i=24 nM . Whereas the highly potent δ ligands reveal good selectivity against μ and κ receptors, the σ₁ and/or σ₂ affinities of some compounds are almost in the same range as their δ receptor affinities, such as compound 25 (σ₂: K_i=83 nM ; δ: K_i=75 nM ). In [³⁵S]GTPγS assays the most potent δ ligands 24 and 25 showed almost the same intrinsic activity as the full agonist SNC80, proving the agonistic activity of 24 and 25 . The enantiomeric 4‐benzylidene derivatives 15 and ent‐ 15 showed selective cytotoxicity toward the 5637 (bladder) and A‐427 (small‐cell lung) human tumor cell lines.     

Molecular Docking and Molecular Dynamics Simulations Discover Curcumin Analogue as a Plausible Dual Inhibitor for SARS-CoV-2

Recently, the world has been witnessing a global pandemic with no effective therapeutics yet, while cancer continues to be a major disease claiming many lives. The natural compound curcumin is bestowed with multiple medicinal applications in addition to demonstrating antiviral and anticancer activities. In order to elucidate the impact of curcumin on COVID-19 and cancer, the current investigation has adapted several computational techniques to unfold its possible inhibitory activity. Accordingly, curcumin and similar compounds and analogues were retrieved and assessed for their binding affinities at the binding pocket of SARS-CoV-2 main protease and DDX3. The best binding pose was escalated to molecular dynamics simulation (MDS) studies to assess the time dependent stability. Our findings have rendered one compound that has demonstrated good molecular dock score complemented by key residue interactions and have shown stable MDS results inferred by root mean square deviation (RMSD), radius of gyration (Rg), binding mode, hydrogen bond interactions, and interaction energy. Essential dynamics results have shown that the systemadapts minimum energy conformation to attain a stable state. The discovered compound (curA) could act as plausible inhibitor against SARS-CoV-2 and DDX3. Furthermore, curA could serve as a chemical scaffold for designing and developing new compounds.