Small molecule- and peptide-drug conjugates addressing integrins: A story of targeted cancer treatment.

Targeted cancer treatment should avoid side effects and damage to healthy cells commonly encountered during traditional chemotherapy. By combining small molecule or peptidic ligands as homing devices with cytotoxic drugs connected by a cleavable or non-cleavable linker in peptide-drug conjugates (PDCs) or small molecule-drug conjugates (SMDCs), cancer cells and tumours can be selectively targeted. The development of highly affine, selective peptides and small molecules in recent years has allowed PDCs and SMDCs to increasingly compete with antibody-drug conjugates (ADCs). Integrins represent an excellent target for conjugates because they are overexpressed by most cancer cells and because of the broad knowledge about native binding partners as well as the multitude of small-molecule and peptidic ligands that have been developed over the last 30 years. In particular, integrin αVß3 has been addressed using a variety of different PDCs and SMDCs over the last two decades, following various strategies. This review summarises and describes integrin-addressing PDCs and SMDCs while highlighting points of great interest.

Environment-Responsive Peptide Dimers Bind and Stabilize Double-Stranded RNA.

Double-stranded RNAs (dsRNA) possess immense potential for biomedical applications. However, their therapeutic utility is limited by low stability and poor cellular uptake. Different strategies have been explored to enhance the stability of dsRNA, including the incorporation of modified nucleotides, and the use of diverse carrier systems. Nevertheless, these have not resulted in a broadly applicable approach thereby preventing the wide-spread application of dsRNA for therapeutic purposes. Herein, we report the design of dimeric stapled peptides based on the RNA-binding protein TAV2b. These dimers are obtained via disulfide formation and mimic the natural TAV2b assembly. They bind and stabilize dsRNA in the presence of serum, protecting it from degradation. In addition, peptide binding also promotes cellular uptake of dsRNA. Importantly, peptide dimers monomerize under reducing conditions which results in a loss of RNA binding. These findings highlight the potential of peptide-based RNA binders for the stabilization and protection of dsRNA, representing an appealing strategy towards the environment-triggered release of RNA. This can broaden the applicability of dsRNA, such as short interfering RNAs (siRNA), for therapeutic applications.

RGD Peptidomimetic MMAE-Conjugate Addressing Integrin αVß3-Expressing Cells with High Targeting Index.

Small molecule-drug conjugates (SMDCs) mimicking the RGD sequence (-Arg-Gly-Asp-) with a non-peptide moiety require a pharmacophore-independent attachment site. A library of 36 sulfonamide-modified RGD mimetics with nM to pM affinity for integrin αV ß3 was synthesized and analysed via DAD mapping. The best structure of the conjugable RGD mimetic was used and a linker was attached to an aromatic ring by Negishi cross-coupling. The product retained high affinity and selectivity for integrin αV ß3 . The conjugable RGD mimetic was then attached to an enzymatically cleavable GKGEVA linker equipped with a self-immolative PABC and the antimitotic drug monomethyl auristatin E (MMAE). The resulting SMDC preferred binding to integrin αV ß3 over α5 ß1 in a ratio of 1 : 4519 (ELISA) and showed selectivity for αV ß3 -positive WM115 cells over αV ß3 -negative M21-L cells in the in vitro cell adhesion assay as well as in cell viability assays with a targeting index of 134 (M21-L/WM115).

Recombinant l-Amino Acid Oxidase with Broad Substrate Spectrum for Co-substrate Recycling in (S)-Selective Transaminase-Catalyzed Kinetic Resolutions.

Chiral and enantiopure amines can be produced by enantioselective transaminases via kinetic resolution of amine racemates. This transamination reaction requires stoichiometric amounts of co-substrate. A dual-enzyme recycling system overcomes this limitation: l-amino acid oxidases (LAAO) recycle the accumulating co-product of (S)-selective transaminases in the kinetic resolution of racemic amines to produce pure (R)-amines. However, availability of suitable LAAOs is limited. Here we use the heterologously produced, highly active fungal hcLAAO4 with broad substrate spectrum. H2 O2 as byproduct of hcLAAO4 is detoxified by a catalase. The final system allows using sub-stoichiometric amounts of 1 mol% of the transaminase co-substrate as well as the initial application of l-amino acids instead of α-keto acids. With an optimized protocol, the synthetic potential of this kinetic resolution cascade was proven at the preparative scale (>90 mg) by the synthesis of highly enantiomerically pure (R)-methylbenzylamine (>99 %ee) at complete conversion (50 %).

Synthesis and Evaluation of a Non-Peptide Small-Molecule Drug Conjugate Targeting Integrin αVß3.

An integrin αVß3-targeting linear RGD mimetic containing a small-molecule drug conjugate (SMDC) was synthesized by combining the antimitotic agent monomethyl auristatin E (MMAE), an enzymatically cleavable Val-Ala-PABC linker with a linear conjugable RGD mimetic. The structure proposal for the conjugable RGD mimetic was suggested upon the DAD mapping analysis of a previously synthesized small-molecule RGD mimetic array based on a tyrosine scaffold. Therefore, a diversifying strategy was developed as well as a novel method for the partial hydrogenation of pyrimidines in the presence of the hydrogenolytically cleavable Cbz group. The small-molecule RGD mimetics were evaluated in an ELISA-like assay, and the structural relationships were analyzed by DAD mapping revealing activity differences induced by structural changes as visualized in dependence on special structural motifs. This provided a lead structure for generation of an SMDC containing the antimitotic drug MMAE. The resulting SMDC containing a linear RGD mimetic was tested in a cell adhesion and an in vitro cell viability assay in comparison to reference SMDCs containing cRGDfK or cRADfK as the homing device. The linear RGD SMDC and the cRGDfK SMDC inhibited adhesion of αVß3-positive WM115 cells to vitronectin with IC50 values in the low µM range, while no effect was observed for the αVß3-negative M21-L cell line. The cRADfK SMDC used as a negative control was about 30-fold less active in the cell adhesion assay than the cRGDfK SMDC. Conversely, both the linear RGD SMDC and the cRGDfK SMDC are about 55-fold less cytotoxic than MMAE against the αVß3-positive WM115 cell line with IC50 values in the nM range, while the cRADfK SMDC is 150-fold less cytotoxic than MMAE. Hence, integrin binding also influences the antiproliferative activity giving a targeting index of 2.8.

Physiological Response of Corynebacterium glutamicum to Indole.

The aromatic heterocyclic compound indole is widely spread in nature. Due to its floral odor indole finds application in dairy, flavor, and fragrance products. Indole is an inter- and intracellular signaling molecule influencing cell division, sporulation, or virulence in some bacteria that synthesize it from tryptophan by tryptophanase. Corynebacterium glutamicum that is used for the industrial production of amino acids including tryptophan lacks tryptophanase. To test if indole is metabolized by C. glutamicum or has a regulatory role, the physiological response to indole by this bacterium was studied. As shown by RNAseq analysis, indole, which inhibited growth at low concentrations, increased expression of genes involved in the metabolism of iron, copper, and aromatic compounds. In part, this may be due to iron reduction as indole was shown to reduce Fe3+ to Fe2+ in the culture medium. Mutants with improved tolerance to indole were selected by adaptive laboratory evolution. Among the mutations identified by genome sequencing, mutations in three transcriptional regulator genes were demonstrated to be causal for increased indole tolerance. These code for the regulator of iron homeostasis DtxR, the regulator of oxidative stress response RosR, and the hitherto uncharacterized Cg3388. Gel mobility shift analysis revealed that Cg3388 binds to the intergenic region between its own gene and the iolT2-rhcM2D2 operon encoding inositol uptake system IolT2, maleylacetate reductase, and catechol 1,2-dioxygenase. Increased RNA levels of rhcM2 in a cg3388 deletion strain indicated that Cg3388 acts as repressor. Indole, hydroquinone, and 1,2,4-trihydroxybenzene may function as inducers of the iolT2-rhcM2D2 operon in vivo as they interfered with DNA binding of Cg3388 at physiological concentrations in vitro. Cg3388 was named IhtR.

Photocontrolled DNA minor groove interactions of imidazole/pyrrole polyamides.

Azobenzenes are photoswitchable molecules capable of generating significant structural changes upon E-to-Z photoisomerization in peptides or small molecules, thereby controlling geometry and functionality. E-to-Z photoisomerization usually is achieved upon irradiation at 350 nm (π-π* transition), while the Z-to-E isomerization proceeds photochemically upon irradiation at >400 nm (n-π* transition) or thermally. Photoswitchable compounds have frequently been employed as modules, e.g., to control protein-DNA interactions. However, their use in conjunction with minor groove-binding imidazole/pyrrole (Im/Py) polyamides is yet unprecedented. Dervan-type Im/Py polyamides were equipped with an azobenzene unit, i.e., 3-(3-(aminomethyl)phenyl)azophenylacetic acid, as the linker between two Im/Py polyamide strands. Only the (Z)-azobenzene-containing polyamides bound to the minor groove of double-stranded DNA hairpins. Photoisomerization was exemplarily evaluated by 1H NMR experiments, while minor groove binding of the (Z)-azobenzene derivatives was proven by CD titration experiments. The resulting induced circular dichroism (ICD) bands of the bound ligands, together with the photometric determination of the dsDNA melting temperature, revealed a significant stabilization of the DNA upon association with the ligand. The (Z)-azobenzene acted as a building block inducing a reverse turn, which favored hydrogen bonds between the pyrrole/imidazole amide and the DNA bases. In contrast, the E-configured polyamides did not induce any ICD characteristic for minor groove binding. The incorporation of the photoswitchable azobenzene unit is a promising strategy to obtain photoswitchable Im/Py hairpin polyamides capable of interacting with the dsDNA minor groove only in the Z-configuration.

Intramolecular π-π Interactions in Flexibly Linked Partially Fluorinated Bisarenes in the Gas Phase.

Three compounds with phenyl and pentafluorophenyl rings bridged by (CH2 )3 and (CH2 )2 SiMe2 units were synthesized by hydrosilylation and C-C coupling reactions. Their solid-state structures are dominated by intermolecular π stacking interactions, primarily leading to dimeric or chain-type aggregates. Analysis of free molecules in the gas phase by electron diffraction revealed the most abundant conformer to be significantly stabilized by intramolecular π-π interactions. For the silicon compounds, structures characterized by σ-π interactions between methyl and pentafluorophenyl groups are second lowest in energy and cannot be excluded completely by the gas electron diffraction experiments. C6 H5 (CH2 )3 C6 F5 , in contrast, is present as a single conformer. The gas-phase structures served as a reference for the evaluation of a series of (dispersion-corrected) quantum-chemical calculations.