Query-guided protein–protein interaction inhibitor discovery

Protein–protein interactions (PPIs) are central to biological mechanisms, and can serve as compelling targets for drug discovery. Yet, the discovery of small molecule inhibitors of PPIs remains challenging given the large and typically shallow topography of the interacting protein surfaces. Here, we describe a general approach to the discovery of orthosteric PPI inhibitors that mimic specific secondary protein structures. Initially, hot residues at protein–protein interfaces are identified in silico or from experimental data, and incorporated into secondary structure-based queries. Virtual libraries of small molecules are then shape-matched against the queries, and promising ligands docked to target proteins. The approach is exemplified experimentally using two unrelated PPIs that are mediated by an α-helix (p53/hDM2) and a β-strand (GKAP/SHANK1-PDZ). In each case, selective PPI inhibitors are discovered with low μM activity as determined by a combination of fluorescence anisotropy and ¹H–¹⁵N HSQC experiments. In addition, hit expansion yields a series of PPI inhibitors with defined structure–activity relationships. It is envisaged that the generality of the approach will enable discovery of inhibitors of a wide range of unrelated secondary structure-mediated PPIs.

Small-molecule protein–protein interaction inhibitors were prioritised on the basis of shape similarity to secondary structure-based queries incorporating hot-spot residues.     

Different phosphorylation and farnesylation patterns tune Rnd3–14-3-3 interaction in distinct mechanisms

Protein posttranslational modifications (PTMs) are often involved in the mediation or inhibition of protein–protein interactions (PPIs) within many cellular signaling pathways. Uncovering the molecular mechanism of PTM-induced multivalent PPIs is vital to understand the regulatory factors to promote inhibitor development. Herein, Rnd3 peptides with different PTM patterns as the binding epitopes and 14-3-3ζ protein were used as models to elucidate the influences of phosphorylation and farnesylation on binding thermodynamics and kinetics and their molecular mechanism. The quantitative thermodynamic results indicate that phosphorylated residues S210 and S218 (pS210 and pS218) and farnesylated C241 (fC241) enhance Rnd3–14-3-3ζ interactions in the presence of the essential pS240. However, distinct PTM patterns greatly affect the binding process. Initial association of pS240 with the phosphate-binding pocket of one monomer of the 14-3-3ζ dimer triggers the binding of pS210 or pS218 to another monomer, whereas the binding of fC241 to the hydrophobic groove on one 14-3-3ζ monomer induces the subsequent binding of pS240 to the adjacent pocket on the same monomer. Based on the experimental and molecular simulation results, we estimate that pS210/pS218 and pS240 mediate the multivalent interaction through an additive mechanism, whereas fC241 and pS240 follow an induced fit mechanism, in which the cooperativity of these two adjacent PTMs is reflected by the index ε described in our established thermodynamic binding model. Besides, these proposed binding models have been further used for describing the interaction between 14-3-3ζ and other substrates containing adjacent phosphorylation and lipidation groups, indicating their potential in general applications. These mechanistic insights are significant for understanding the regulatory factors and the design of PPI modulators.

Different protein posttranslational modifications (PTMs) patterns affect the binding thermodynamics and kinetics and their molecular mechanism of multivalent protein–protein interaction (PPIs).     

Identification of β-strand mediated protein–protein interaction inhibitors using ligand-directed fragment ligation

β-Strand mediated protein–protein interactions (PPIs) represent underexploited targets for chemical probe development despite representing a significant proportion of known and therapeutically relevant PPI targets. β-Strand mimicry is challenging given that both amino acid side-chains and backbone hydrogen-bonds are typically required for molecular recognition, yet these are oriented along perpendicular vectors. This paper describes an alternative approach, using GKAP/SHANK1 PDZ as a model and dynamic ligation screening to identify small-molecule replacements for tranches of peptide sequence. A peptide truncation of GKAP functionalized at the N- and C-termini with acylhydrazone groups was used as an anchor. Reversible acylhydrazone bond exchange with a library of aldehyde fragments in the presence of the protein as template and in situ screening using a fluorescence anisotropy (FA) assay identified peptide hybrid hits with comparable affinity to the GKAP peptide binding sequence. Identified hits were validated using FA, ITC, NMR and X-ray crystallography to confirm selective inhibition of the target PDZ-mediated PPI and mode of binding. These analyses together with molecular dynamics simulations demonstrated the ligands make transient interactions with an unoccupied basic patch through electrostatic interactions, establishing proof-of-concept that this unbiased approach to ligand discovery represents a powerful addition to the armory of tools that can be used to identify PPI modulators.

Dynamic ligation screening is used to identify acylhydrazone-linked peptide-fragment hybrids which bind to the SHANK1 PDZ domain with comparable affinity to the native GKAP peptide as shown by biophysical and structural analyses.     

Unravelling the effect of N(ε)-(carboxyethyl)lysine on the conformation,dynamics and aggregation propensity of α-synuclein

α-Synuclein (αS) aggregation is a hallmark in several neurodegenerative diseases. Among them, Parkinson''s disease is highlighted, characterized by the intraneuronal deposition of Lewy bodies (LBs) which causes the loss of dopaminergic neurons. αS is the main component of LBs and in them, it usually contains post-translational modifications. One of them is the formation of advanced glycation end-products (mainly CEL and MOLD) arising from its reaction with methylglyoxal. Despite its biological relevance, there are no data available proving the effect of glycation on the conformation of αS, nor on its aggregation mechanism. This has been hampered by the formation of a heterogeneous set of compounds that precluded conformational studies. To overcome this issue, we have here produced αS homogeneously glycated with CEL. Its use, together with different biophysical techniques and molecular dynamics simulations, allowed us to study for the first time the effect of glycation on the conformation of a protein. CEL extended the conformation of the N-terminal domain as a result of the loss of transient N-/C-terminal long-range contacts while increasing the heterogeneity of the conformational population. CEL also inhibited the αS aggregation, but it was not able to disassemble preexisting amyloid fibrils, thus proving that CEL found on LBs must be formed in a later event after aggregation.

We study the effect of an advanced glycation end product (N(ε)-(carboxyethyl)lysine), found on the Lewy bodies of people suffering from Parkinson’s disease, on the conformational and aggregation features of alpha-synuclein.     

Discovering protein–protein interaction stabilisers by native mass spectrometry

Protein–protein interactions (PPIs) are key therapeutic targets. Most PPI-targeting drugs in the clinic inhibit these important interactions; however, stabilising PPIs is an attractive alternative in cases where a PPI is disrupted in a disease state. The discovery of novel PPI stabilisers has been hindered due to the lack of tools available to monitor PPI stabilisation. Moreover, for PPI stabilisation to be detected, both the stoichiometry of binding and the shift this has on the binding equilibria need to be monitored simultaneously. Here, we show the power of native mass spectrometry (MS) in the rapid search for PPI stabilisers. To demonstrate its capability, we focussed on three PPIs between the eukaryotic regulatory protein 14-3-3σ and its binding partners estrogen receptor ERα, the tumour suppressor p53, and the kinase LRRK2, whose interactions upon the addition of a small molecule, fusicoccin A, are differentially stabilised. Within a single measurement the stoichiometry and binding equilibria between 14-3-3 and each of its binding partners was evident. Upon addition of the fusicoccin A stabiliser, a dramatic shift in binding equilibria was observed with the 14-3-3:ERα complex compared with the 14-3-3:p53 and 14-3-3:LRRK2 complexes. Our results highlight how native MS can not only distinguish the ability of stabilisers to modulate PPIs, but also give important insights into the dynamics of ternary complex formation. Finally, we show how native MS can be used as a screening tool to search for PPI stabilisers, highlighting its potential role as a primary screening technology in the hunt for novel therapeutic PPI stabilisers.

Stabilising protein–protein interactions is challenging, yet therapeutically important. Native mass spectrometry can be used to monitor binding equilibria, allowing identification and measurement of novel protein–protein interaction stabilisers.     

3D-visualization of amyloid-β oligomer interactions with lipid membranes by cryo-electron tomography

Amyloid-β (Aβ) assemblies have been shown to bind to lipid bilayers. This can disrupt membrane integrity and cause a loss of cellular homeostasis, that triggers a cascade of events leading to Alzheimer''s disease. However, molecular mechanisms of Aβ cytotoxicity and how the different assembly forms interact with the membrane remain enigmatic. Here we use cryo-electron tomography (cryoET) to obtain three-dimensional nano-scale images of various Aβ assembly types and their interaction with liposomes. Aβ oligomers and curvilinear protofibrils bind extensively to the lipid vesicles, inserting and carpeting the upper-leaflet of the bilayer. Aβ oligomers concentrate at the interface of vesicles and form a network of Aβ-linked liposomes, while crucially, monomeric and fibrillar Aβ have relatively little impact on the membrane. Changes to lipid membrane composition highlight a significant role for GM1-ganglioside in promoting Aβ-membrane interactions. The different effects of Aβ assembly forms observed align with the highlighted cytotoxicity reported for Aβ oligomers. The wide-scale incorporation of Aβ oligomers and curvilinear protofibrils into the lipid bilayer suggests a mechanism by which membrane integrity is lost.

Cryo-electron tomography 3D imaging of amyloid-β oligomers carpeting the surface of lipid bilayers in near native conditions.     

Scalable synthesis and coupling of quaternary α-arylated amino acids: α-aryl substituents are tolerated in α-helical peptides

Quaternary amino acids are important tools for the modification and stabilisation of peptide secondary structures. Here we describe a practical and scalable synthesis applicable to quaternary alpha-arylated amino acids (Q4As), and the development of solid-phase synthesis conditions for their incorporation into peptides. Monomeric and dimeric α-helical peptides are synthesised with varying degrees of Q4A substitution and their structures examined using biophysical methods. Both enantiomers of the Q4As are tolerated in folded monomeric and oligomeric α-helical peptides, with the (R)-enantiomer slightly more so than the (S).

Both R and S enantiomers of Fmoc-protected amino acids bearing α-aryl substituents may be made on gram scale. Solid-phase synthesis leads to helical peptides unperturbed by the presence of these additional α-aryl groups.     

Single-molecule analysis of interaction between p53TAD and MDM2 using aerolysin nanopores

Protein–protein interactions (PPIs) are regarded as important, but undruggable targets. Intrinsically disordered p53 transactivation domain (p53TAD) mediates PPI with mouse double minute 2 (MDM2), which is an attractive anticancer target for therapeutic intervention. Here, using aerolysin nanopores, we probed the p53TAD peptide/MDM2 interaction and its modulation by small-molecule PPI inhibitors or p53TAD phosphorylation. Although the p53TAD peptide showed short-lived (<100 ms) translocation, the protein complex induced the characteristic extraordinarily long-lived (0.1 s ∼ tens of min) current blockage, indicating that the MDM2 recruitment by p53TAD peptide almost fully occludes the pore. Simultaneously, the protein complex formation substantially reduced the event frequency of short-lived peptide translocation. Notably, the addition of small-molecule PPI inhibitors, Nutlin-3 and AMG232, or Thr18 phosphorylation of p53TAD peptide, were able to diminish the extraordinarily long-lived events and restore the short-lived translocation of the peptide rescued from the complex. Taken together, our results elucidate a novel mechanism of single-molecule sensing for analyzing PPIs and their inhibitors using aerolysin nanopores. This novel methodology may contribute to remarkable improvements in drug discovery targeted against undruggable PPIs.

Using aerolysin nanopores, we probed protein–protein interaction (PPI) between p53TAD and MDM2 and its modulation by small-molecule PPI inhibitors and p53TAD phosphorylation.     

Catalytic asymmetric synthesis of quaternary trifluoromethyl α- to ε-amino acid derivatives via umpolung allylation/2-aza-Cope rearrangement

In this study, we developed an efficient Ir-catalyzed cascade umpolung allylation/2-aza-Cope rearrangement of tertiary α-trifluoromethyl α-amino acid derivatives for the preparation of a variety of quaternary α-trifluoromethyl α-amino acids in high yields with excellent enantioselectivities. The umpolung reactivity empowered by the activation of the key isatin-ketoimine moiety obviates the intractable enantioselectivity control in Pd-catalyzed asymmetric linear α-allylation. In combination with quasi parallel kinetic resolution or kinetic resolution, the generality of this method is further demonstrated by the first preparation of enantioenriched quaternary trifluoromethyl β-, γ-, δ- and ε-amino acid derivatives.

In this study, we developed an efficient Ir-catalyzed cascade umpolung allylation/2-aza-Cope rearrangement for the preparation of a variety of quaternary trifluoromethyl α-ε-amino acids in high yields with excellent enantioselectivities.     

Selective α,δ-hydrocarboxylation of conjugated dienes utilizing CO2 and electrosynthesis

To date the majority of diene carboxylation processes afford the α,δ-dicarboxylated product, the selective mono-carboxylation of dienes is a significant challenge and the major product reported under transition metal catalysis arises from carboxylation at the α-carbon. Herein we report a new electrosynthetic approach, that does not rely on a sacrificial electrode, the reported method allows unprecedented direct access to carboxylic acids derived from dienes at the δ-position. In addition, the α,δ-dicarboxylic acid or the α,δ-reduced alkene can be easily accessed by simple modification of the reaction conditions.

Selective electrosynthetic α,δ-hydrocarboxylation of 1,3-dienes is reported, utilising non-sacrificial electrodes that provide access to the previously challenging δ-carboxylated regioisomer.     

Asymmetric synthesis of γ-chiral borylalkanes via sequential reduction/hydroboration using a single copper catalyst

The synthesis of γ-chiral borylalkanes through copper-catalyzed enantioselective S_N2′-reduction of γ,γ-disubstituted allylic substrates and subsequent hydroboration was reported. A copper–DTBM-Segphos catalyst produced a range of γ-chiral alkylboronates from easily accessible allylic acetate or benzoate with high enantioselectivities up to 99% ee. Furthermore, selective organic transformations of the resulting γ-chiral alkylboronates generated the corresponding γ-chiral alcohol, arene and amine compounds.

Copper-catalyzed reductive hydroboration of γ,γ-disubstituted allylic substrates enables preparation of γ-chiral alkylboron compounds in a one-pot cascade manner.     

Nickel-catalyzed asymmetric reductive arylation of α-chlorosulfones with aryl halides

We report an asymmetric Ni-catalyzed reductive cross-coupling of aryl/heteroaryl halides with racemic α-chlorosulfones to afford enantioenriched sulfones. The reaction tolerates a variety of functional groups under mild reaction conditions, which complements the current methods. The utility of this work was demonstrated by facile late-stage functionalization of commercial drugs.

In this work Ni-Catalyzed reductive cross-coupling between (hetero)aryl halides and racemic α-chlorosulfones to prepare enantioenriched α,α-disubstituted sulfones was demonstrated, allowing facile structural derivatization of drug precursors.     

An explicitly designed paratope of amyloid-β prevents neuronal apoptosis in vitro and hippocampal damage in rat brain

Synthetic antibodies hold great promise in combating diseases, diagnosis, and a wide range of biomedical applications. However, designing a therapeutically amenable, synthetic antibody that can arrest the aggregation of amyloid-β (Aβ) remains challenging. Here, we report a flexible, hairpin-like synthetic paratope (SP1, ∼2 kDa), which prevents the aggregation of Aβ monomers and reverses the preformed amyloid fibril to a non-toxic species. Structural and biophysical studies further allowed dissecting the mode and affinity of molecular recognition events between SP1 and Aβ. Subsequently, SP1 reduces Aβ-induced neurotoxicity, neuronal apoptosis, and ROS-mediated oxidative damage in human neuroblastoma cells (SH-SY5Y). The non-toxic nature of SP1 and its ability to ameliorate hippocampal neurodegeneration in a rat model of AD demonstrate its therapeutic potential. This paratope engineering module could readily implement discoveries of cost-effective molecular probes to nurture the basic principles of protein misfolding, thus combating related diseases.

Herein, the therapeutic potentials of an explicitly designed peptide probe are systematically illuminated in vitro and in vivo against Aβ aggregation. The probe demonstrates remarkable potency for attenuating neurotoxicity and hippocampal damage.     

Cooperative stabilisation of 14-3-3σ protein–protein interactions via covalent protein modification

14-3-3 proteins are an important family of hub proteins that play important roles in many cellular processes via a large network of interactions with partner proteins. Many of these protein–protein interactions (PPI) are implicated in human diseases such as cancer and neurodegeneration. The stabilisation of selected 14-3-3 PPIs using drug-like ‘molecular glues’ is a novel therapeutic strategy with high potential. However, the examples reported to date have a number of drawbacks in terms of selectivity and potency. Here, we report that WR-1065, the active species of the approved drug amifostine, covalently modifies 14-3-3σ at an isoform-unique cysteine residue, Cys38. This modification leads to isoform-specific stabilisation of two 14-3-3σ PPIs in a manner that is cooperative with a well characterised molecular glue, fusicoccin A. Our findings reveal a novel stabilisation mechanism for 14-3-3σ, an isoform with particular involvement in cancer pathways. This mechanism can be exploited to harness the enhanced potency conveyed by covalent drug molecules and dual ligand cooperativity. This is demonstrated in two cancer cell lines whereby the cooperative behaviour of fusicoccin A and WR-1065 leads to enhanced efficacy for inducing cell death and attenuating cell growth.

The aminothiol WR-1065 covalently modifies 14-3-3σ to stabilse its interactions with p53 and ERα. It enhances the effect of fusicoccin A via a cooperative mechanism that leads to 14-3-3 partner-protein specific activty against cancer cells.      

A step-economic and one-pot access to chiral Cα-tetrasubstituted α-amino acid derivatives via a bicyclic imidazole-catalyzed direct enantioselective C-acylation

C^α-Tetrasubstituted α-amino acids are ubiquitous and unique structural units in bioactive natural products and pharmaceutical compounds. The asymmetric synthesis of these molecules has attracted a lot of attention, but a more efficient method is still greatly desired. Here we describe the first sequential four-step acylation reaction for the efficient synthesis of chiral C^α-tetrasubstituted α-amino acid derivatives from simple N-acylated amino acids via an auto-tandem catalysis using a single nucleophilic catalyst. The synthetic efficiency is improved via a direct enantioselective C-acylation; the methodology affords the corresponding C^α-tetrasubstituted α-amino acid derivatives with excellent enantioselectivities (up to 99% ee). This step-economic, one-pot, and auto-tandem strategy provides facile access to important chiral building blocks, such as peptides, serines, and oxazolines, which are often used in medicinal and synthetic chemistry.

The first four-step sequential reaction for the synthesis of C^α-tetrasubstituted chiral α-amino acid derivatives via auto-tandem catalysis has been developed.     

Acrylic boronate: a multifunctional C3 building block for catalytic synthesis of rare organoborons and chemoselective heterobifunctional ligations

A novel C3 acylboron building block; acrylic boronate was successfully prepared and its versatility for catalytic synthesis of several previously inaccessible organoborons is described. Cross-metathesis and Pd-catalyzed coupling of acrylic boronate enabled two complementary routes to highly functionalized α,β-unsaturated acylborons and two new types of conjugated borylated products: α,β,γ,δ-unsaturated and bis-α,β unsaturated acylborons. The synthetic application of α,β-unsaturated acylborons was demonstrated for the first time, thereby providing a general and highly regioselective route to medicinally important 3-boryl pyrazoles. Acrylic boronate also provided a unique bis-electrophilic platform for rapid and chemoselective labeling of cysteines with acylboron tags which are potentially useful for site-selective functionalization and orthogonal ligation of proteins.

A novel C3 acylboron building block; acrylic boronate has been developed and its versatility for synthesis of several previously inaccessible organoborons and heterobifunctional ligations is described.     

Deoxygenative α-alkylation and α-arylation of 1,2-dicarbonyls

Construction of C–C bonds at the α-carbon is a challenging but synthetically indispensable approach to α-branched carbonyl motifs that are widely represented among drugs, natural products, and synthetic intermediates. Here, we describe a simple approach to generation of boron enolates in the absence of strong bases that allows for introduction of both α-alkyl and α-aryl groups in a reaction of readily accessible 1,2-dicarbonyls and organoboranes. Obviation of unselective, strongly basic and nucleophilic reagents permits carrying out the reaction in the presence of electrophiles that intercept the intermediate boron enolates, resulting in two new α-C–C bonds in a tricomponent process.

α-Branched carboxylic acids and other carbonyls are readily accessed by a metal- and base-free deoxygenative α-alkylation and α-arylation of 1,2-dicarbonyls via boron enolates, resulting in a tricomponent coupling with unconventional electrophiles.     

Pyrazolone ligation-mediated versatile sequential bioconjugations

Bioconjugation chemistries are critical tools in biotherapeutics discovery. The past efforts have been exclusively focused on two-segment conjugations. However, emerging research directions, such as polypharmacy biotherapeutics, desire multiple-component bioconjugations where more than two pharmacologically related biomolecules can be assembled into a single construct in high efficiency. We present here a set of sequential bioconjugation chemistries centered on a pyrazolone structural motif. It starts with a clickable “pyrazolone ligation” between a hydrazine group and a β-ketoester moiety followed by the conjugation between the newly formed pyrazolone core and an aldehyde-bearing biomolecule through a Knoevenagel reaction forming a Michael addition acceptor that can effectively capture a thiol-bearing biomolecule. When utilized intermolecularly, it quickly assembles four segments together forming a quadruple functional construct. When applied intramolecularly, it offers a set of highly diverse biomolecule scaffolds including stapled peptides and poly-macrocyclic peptides. We envision broad utilities of such sequential ligation chemistries.

A multiple component sequential bioconjugation chemistry establishes upon the joined force of hydrazine, β-keto ester, thiol and aldehyde.     

Templating S100A9 amyloids on Aβ fibrillar surfaces revealed by charge detection mass spectrometry,microscopy, kinetic and microfluidic analyses

The mechanism of amyloid co-aggregation and its nucleation process are not fully understood in spite of extensive studies. Deciphering the interactions between proinflammatory S100A9 protein and Aβ₄₂ peptide in Alzheimer''s disease is fundamental since inflammation plays a central role in the disease onset. Here we use innovative charge detection mass spectrometry (CDMS) together with biophysical techniques to provide mechanistic insight into the co-aggregation process and differentiate amyloid complexes at a single particle level. Combination of mass and charge distributions of amyloids together with reconstruction of the differences between them and detailed microscopy reveals that co-aggregation involves templating of S100A9 fibrils on the surface of Aβ₄₂ amyloids. Kinetic analysis further corroborates that the surfaces available for the Aβ₄₂ secondary nucleation are diminished due to the coating by S100A9 amyloids, while the binding of S100A9 to Aβ₄₂ fibrils is validated by a microfluidic assay. We demonstrate that synergy between CDMS, microscopy, kinetic and microfluidic analyses opens new directions in interdisciplinary research.

Templating mechanism of S100A9 amyloids on Aβ fibrillar surfaces during amyloid co-aggregation process was revealed by synergy of biophysical methods including charge detection mass spectrometry, microscopy, kinetic and microfluidic analyses.