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Characterization of macrocycle hits. (a) Specificity profiling of macrocycle 57. (b) Prolongation of the coagulation time of human plasma in the presence of different concentrations of macrocycle 7 and 57. Coagulation was triggered to activate the intrinsic coagulation pathway (aPTT) and the extrinsic coagulation pathway (PT)
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Macrocycles provide an attractive modality for drug development, but generating ligands for new targets is hampered by the limited availability of large macrocycle libraries. We have established a solution-phase macrocycle synthesis strategy in which three building blocks are coupled sequentially in efficient alkylation reactions that eliminate the...
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... Inspired by studies using mosquito liquid handling 20,21 or contactless acoustic droplet ejection (ADE) technology [22][23][24][25] for synthesizing and screening small molecule libraries, we recently applied ADE for miniaturizing chemical reactions and screening of macrocycles at smaller scale 26 . However, the reaction yields varied substantially due to the challenging nature of the macrocyclization reactions we applied, and the small number of commercially available linker reagents (<20) limited the diversities of these libraries 19,26,27 . While the approach allowed identification of nanomolar binders to targets with defined binding pockets such as trypsin-like serine proteases 19,27 , we struggled to generate high-affinity binders to more challenging targets such as protein-protein interactions, the best ones obtained being PPI inhibitors of p53:MDM2 PPI with rather weak potency in the low micromolar range 26 . ...
... However, the reaction yields varied substantially due to the challenging nature of the macrocyclization reactions we applied, and the small number of commercially available linker reagents (<20) limited the diversities of these libraries 19,26,27 . While the approach allowed identification of nanomolar binders to targets with defined binding pockets such as trypsin-like serine proteases 19,27 , we struggled to generate high-affinity binders to more challenging targets such as protein-protein interactions, the best ones obtained being PPI inhibitors of p53:MDM2 PPI with rather weak potency in the low micromolar range 26 . ...
Macrocycles have excellent potential as therapeutics due to their ability to bind challenging targets. However, generating macrocycles against new targets is hindered by a lack of large macrocycle libraries for high-throughput screening. To overcome this, we herein established a combinatorial approach by tethering a myriad of chemical fragments to peripheral groups of structurally diverse macrocyclic scaffolds in a combinatorial fashion, all at a picomole scale in nanoliter volumes using acoustic droplet ejection technology. In a proof-of-concept, we generate a target-tailored library of 19,968 macrocycles by conjugating 104 carboxylic-acid fragments to 192 macrocyclic scaffolds. The high reaction efficiency and small number of side products of the acylation reactions allowed direct assay without purification and thus a large throughput. In screens, we identify nanomolar inhibitors against thrombin (Ki = 44 ± 1 nM) and the MDM2:p53 protein-protein interaction (Kd MDM2 = 43 ± 18 nM). The increased efficiency of macrocycle synthesis and screening and general applicability of this approach unlocks possibilities for generating leads against any protein target.
... 12 Beyond the highly prevalent Huisgen cycloaddition, other variants of Cu-catalyzed alkyne macrocyclizations 13 and cycloadditions 9b,14 are very commonly utilized, as are a diverse array of intramolecular palladium-catalyzed C−C and C−X couplings. 15 Beyond these prevalent transformations, there are also reported examples of library generation using photocatalyzed macrocyclization, 16 reductive amination, 17 nucleophilic substitution, 18 Friedel−Crafts alkylations, 19 the intramolecular Ullmann reaction, 20 Pauson−Khand reactions, 12d rhodium-catalyzed O−H/N−H insertions, 21 condensation reactions, 22 and cyclopropanations. 23 While many other synthetic methods for macrocyclic ring closure have been utilized in target-oriented syntheses, 5a,24 these methodologies are often limited by reaction efficiency, substrate scope, and functional group tolerance, thereby presenting a key challenge for DOS. 25 Given the limited scope of transformations available for macrocyclic ring closure, we have employed a reaction screening approach to identify new C−C bond forming macrocyclizations that are tolerant of diverse substitutions, ring sizes, and functional groups. ...
... 21.3 Macrocyclizations of Alkynyl Sulfonyl Hydrazone Substrate 9 To Produce 11 and 17. 10,10-Dimethyl -3,8-diazaspiro[bicyclo[13.3.1]nonadecane-5,1′-cyclohexane]-1 (18),15 (19),16-trien-12-yne-2,7dione (11). A mixture of alkynyl tosylhydrazone 9 (5 mg, 8.85 μmol), 2-propanol (2.2 mL), NaOt-Bu (13 μL, 2 M in THF), and copper(I) trifluoromethanesulfonate toluene complex (0.46 mg, 0.89 μmol) was irradiated using a microwave at 90°C for 5 min. ...
... 135.1, 134.9, 133.6, 129.9, 129.7, 128.8, 128.8, 128.7, 127.5, 81.8, 70.9, 62.7, 48.9, 47.1, 45.6, 37.7, 34.9, 34.8, 30.0, 26.0, 25.1, 21.8, 21.7 3116,2926,2869,1641,1542,1470,1456,1368,1297,1248,1213 MHz) δ 172. 3, 167.1, 161.6, 135.3, 134.5, 130.8, 129.8, 129.1, 128.0, 81.8, 70.9, 48.9, 47.3, 45.5, 37.7, 35.0, 34.8, 30.0, 26.1, 25. (18). Colorless oil; R f = 0.33 (35% acetone in hexanes); IR (thin film): ν max 3302, 2929,1636,1528,1453,1265,1209,1036,733,701,635 3, 167.8, 141.6, 134.8, 129.9, 128.8, 126.3, 125.6, 81.9, 70.9, 64.9, 49.0, 47.1, 45.3, 37.8, 34.9, 34.8, 30. ...
... We tested the acoustic dispensing strategy using a recently developed approach for macrocycle synthesis (Figure 1 d) in which m N a -bromoacetyl-activated linear peptides were reacted with n primary amines, followed by a cyclization with o linkers to yield m n o macrocycles. [17] We had previously used this approach to affinity mature a macrocyclic thrombin inhibitor (P2, K i = 42 nm) through combinatorial synthesis of thousands of variants of it. However, we had not applied the strategy to the synthesis or screening of random macrocyclic compound libraries, nor to any target other than thrombin. ...
Macrocyclic compounds are an attractive class of therapeutic ligands against challenging targets, such as protein–protein interactions. However, the development of macrocycles as drugs is hindered by the lack of large combinatorial macrocyclic libraries, which are cumbersome, expensive, and time consuming to make, screen, and deconvolute. Here, we established a strategy for synthesizing and screening combinatorial libraries on a picomolar scale by using acoustic droplet ejection to combine building blocks at nanoliter volumes, which reduced the reaction volumes, reagent consumption, and synthesis time. As a proof‐of‐concept, we assembled a 2700‐member target‐focused macrocyclic library that we could subsequently assay in the same microtiter synthesis plates, saving the need for additional transfers and deconvolution schemes. We screened the library against the MDM2–p53 protein–protein interaction and generated micromolar and sub‐micromolar inhibitors. Our approach based on acoustic liquid transfer provides a general strategy for the development of macrocycle ligands.
... We tested the acoustic dispensing strategy using a recently developed approach for macrocycle synthesis (Figure 1 d) in which m N a -bromoacetyl-activated linear peptides were reacted with n primary amines, followed by a cyclization with o linkers to yield m n o macrocycles. [17] We had previously used this approach to affinity mature a macrocyclic thrombin inhibitor (P2, K i = 42 nm) through combinatorial synthesis of thousands of variants of it. However, we had not applied the strategy to the synthesis or screening of random macrocyclic compound libraries, nor to any target other than thrombin. ...
Macrocyclic compounds are an attractive class of therapeutic ligands against challenging targets such as protein‐protein interactions. However, the development of macrocycles as drugs is hindered by the lack of large combinatorial macrocyclic libraries, which are cumbersome, expensive, and time consuming to make, screen, and deconvolute. Here, we established a strategy for synthesizing and screening combinatorial libraries on a picomolar scale using acoustic droplet ejection to combine building blocks at nanoliter volumes, which reduced reaction volumes, reagent consumption, and synthesis time. As a proof‐of‐concept, we assembled a 2,700‐member target‐focused macrocyclic library that we could subsequently assay in the same microtiter synthesis plates, saving the need for additional transfers and deconvolution schemes. We screened the library against the MDM2‐p53 protein‐protein interaction and generated micromolar and sub‐micromolar inhibitors. Our work synthesizing combinatorial macrocycle libraries at the picomole‐scale using acoustic liquid transfer provides a general strategy towards macrocycle ligand development.
The enormous social and economic consequences of thrombotic diseases, as well as the significant dangers associated with present medications, need the development of more effective and safer anticoagulants. In silico structure‐based design was used to find new peptide‐based thrombin inhibitors and cyclic peptide‐based molecules are attracting wide attention in that regard. Here we have shown the synthesis, conformational and in silico analysis of tetrahydrofuran amino acid (TAA) based 15‐membered cyclic tetrapeptides (CTPs) containing guanidine side chain as thrombin inhibitors. Solution NMR conformational analysis suggests that the βγ fused structures of CTPs are retained in the core and there is no effect of guanidine functionality on the scaffold. Molecular docking results showed that these CTPs can inhibit thrombin protein by interacting with Asp189 residue and the core structure helps to retain the guanidine group inside the active site of protein without any alteration in the backbone H‐bonding. Overall, we have presented here the synthesis, conformational analysis of TAA containing cyclic tetrapeptides along with their docking study against thrombin protein. Further these results will help to design peptide based novel thrombin inhibitors.
Peptides can be converted to highly active compounds by introducing appropriate substituents on the suitable amino acid residue. Although modifiable residues in peptides can be systematically identified by peptide scanning methodologies, there is no practical method for optimization at the "scanned" position. With the purpose of using derivatives not only for scanning but also as a starting point for further chemical functionalization, we herein report the "scanning and direct derivatization" strategy through chemoselective acylation of embedded threonine residues by a serine/threonine ligation (STL) with the help of in situ screening chemistry. We have applied this strategy to the optimization of the polymyxin antibiotics, which were selected as a model system to highlight the power of the rapid derivatization of active scanning derivatives. Using this approach, we explored the structure-activity relationships of the polymyxins and successfully prepared derivatives with activity against polymyxin-resistant bacteria and those with Pseudomonas aeruginosa selective antibacterial activity. This strategy opens up efficient structural exploration and further optimization of peptide sequences.
The development of highly active and selective enzyme inhibitors is one of the priorities of medicinal chemistry. Typically, various high-throughput screening methods are used to find lead compounds from a large pool of synthetic compounds, and these are further elaborated and structurally refined to achieve the desired properties. In an effort to streamline this complex and laborious process, new selection strategies based on different principles have recently emerged as an alternative. Herein, we compare three such selection strategies with the aim of identifying potent and selective inhibitors of human carbonic anhydrase II. All three approaches, in situ click chemistry, phage-display libraries and synthetic peptide libraries, led to the identification of more potent inhibitors when compared to the parent compounds. In addition, one of the inhibitor-peptide conjugates identified from the phage libraries showed greater than 100-fold selectivity for the enzyme isoform used for the compound selection. In an effort to rationalize the binding properties of the conjugates, we performed detailed crystallographic and NMR structural analysis, which revealed the structural basis of the compound affinity towards the enzyme and led to the identification of a novel exosite that could be utilized in the development of isoform specific inhibitors.
Macrocyclic peptides are an important class of bioactive substances. When inserting an aromatic foldamer segment in a macrocyclic peptide, the strong folding propensity of the former may influence the conformation and alter the properties of the latter. Such an insertion is relevant because some foldamer-peptide hybrids have recently been shown to be tolerated by the ribosome, prior to forming macrocycles, and can thus be produced using an in vitro translation system. We have investigated the interplay of peptide and foldamer conformations in such hybrid macrocycles. We show that foldamer helical folding always prevails and stands as a viable means to stretch, i.e. unfold, peptides in a solvent dependent manner. Conversely, the peptide systematically has a reciprocal influence and gives rise to strong foldamer helix handedness bias as well as foldamer helix stabilisation. The hybrid macrocycles also show resistance towards proteolytic degradation.
Combining different compound classes gives molecular hybrids that can offer access to novel chemical space and unique properties. Peptides provide ideal starting points for such molecular hybrids, which can be easily modified with a variety of molecular entities. The addition of small molecules can improve the potency, stability and cell permeability of therapeutically relevant peptides. Furthermore, they are often applied to create peptide-based tools in chemical biology. In this review, we discuss general methods that allow the discovery of this compound class and highlight key examples of peptide–small molecule hybrids categorised by the application and function of the small molecule entity.
