Large Libraries of Structurally Diverse Macrocycles Suitable for Membrane Permeation.

Macrocycles offer an attractive format for drug development due to their good binding properties and potential to cross cell membranes. To efficiently identify macrocyclic ligands for new targets, methods for the synthesis and screening of large combinatorial libraries of small cyclic peptides were developed, many of them using thiol groups for efficient peptide macrocyclization. However, a weakness of these libraries is that invariant thiol-containing building blocks such as cysteine are used, resulting in a region that does not contribute to library diversity but increases molecule size. Herein, we synthesized a series of structurally diverse thiol-containing elements and used them for the combinatorial synthesis of a 2,688-member library of small, structurally diverse peptidic macrocycles with unprecedented skeletal complexity. We then used this library to discover potent thrombin and plasma kallikrein inhibitors, some also demonstrating favorable membrane permeability. X-ray structure analysis of macrocycle-target complexes showed that the size and shape of the newly developed thiol elements are key for binding. The strategy and library format presented in this work significantly enhance structural diversity by allowing combinatorial modifications to a previously invariant region of peptide macrocycles, which may be broadly applied in the development of membrane permeable therapeutics.

Folding of Class IIa HDAC Derived Peptides into α-helices Upon Binding to Myocyte Enhancer Factor-2 in Complex with DNA.

Interaction of transcription factor myocyte enhancer factor-2 (MEF2) family members with class IIa histone deacetylases (HDACs) has been implicated in a wide variety of diseases. Though considerable knowledge on this topic has been accumulated over the years, a high resolution and detailed analysis of the binding mode of multiple class IIa HDAC derived peptides with MEF2D is still lacking. To fulfil this gap, we report here the crystal structure of MEF2D in complex with double strand DNA and four different class IIa HDAC derived peptides, namely HDAC4, HDAC5, HDAC7 and HDAC9. All class IIa HDAC derived peptides form extended amphipathic α-helix structures that fit snugly in the hydrophobic groove of MEF2D domain. Binding mode of class IIa HDAC derived peptides to MEF2D is very similar and occur primarily through nonpolar interactions mediated by highly conserved branched hydrophobic amino acids. Further studies revealed that class IIa HDAC derived peptides are unstructured in solution and appear to adopt a folded α-helix structure only upon binding to MEF2D. Comparison of our peptide-protein complexes with previously characterized structures of MEF2 bound to different co-activators and co-repressors, highlighted both differences and similarities, and revealed the adaptability of MEF2 in protein-protein interactions. The elucidation of the three-dimensional structure of MEF2D in complex with multiple class IIa HDAC derived peptides provide not only a better understanding of the molecular basis of their interactions but also have implications for the development of novel antagonist.

Crystal structure of human serum albumin in complex with megabody reveals unique human and murine cross-reactive binding site.

The pharmacokinetic properties of small biotherapeutics can be enhanced via conjugation to cross-reactive albumin-binding ligands in a process that improves their safety and accelerates testing through multiple pre-clinical animal models. In this context, the small and stable heavy-chain-only nanobody NbAlb1, capable of binding both human and murine albumin, has recently been successfully applied to improve the stability and prolong the in vivo plasma residence time of multiple small therapeutic candidates. Despite its clinical efficacy, the mechanism of cross-reactivity of NbAlb1 between human and murine serum albumins has not yet been investigated. To unveil the molecular basis of such an interaction, we solved the crystal structure of human serum albumin (hSA) in complex with NbAlb1. The structure was obtained by harnessing the unique features of a megabody chimeric protein, comprising NbAlb1 grafted onto a modified version of the circularly permutated and bacterial-derived protein HopQ. This structure showed that NbAlb1 contacts a yet unexplored binding site located in the peripheral region of domain II that is conserved in both human and mouse serum albumin proteins. Furthermore, we show that the binding of NbAlb1 to both serum albumin proteins is retained even at acidic pH levels, thus explaining its extended in vivo half-life. The elucidation of the molecular basis of NbAlb1 cross-reactivity to human and murine albumins might guide the design of novel nanobodies with broader reactivity toward a larger panel of serum albumins, thus facilitating the pre-clinical and clinical phases in humans.

In vitro binding analysis of legacy-linear and new generation-cyclic perfluoro-alkyl substances on sex hormone binding globulin and albumin, suggests low impact on serum hormone kinetics of testosterone.

In humans, serum testosterone (T) is largely bound to the sex hormone binding globulin (SHBG) and human serum albumin (hSA), resulting in a 2-3 % of unbound or "free" active quote (FT). Endocrine-disrupting chemicals, including perfluoro-alkyl substances (PFAS), are recognized to interfere with the hormonal axes, but the possible impact on the FT quote has not been addressed so far. Here we investigated the possible competition of two acknowledged PFAS molecules on T binding to SHBG and hSA. In particular, perfluoro-octanoic acid (PFOA) and acetic acid, 2,2-difluoro-2-((2,2,4,5-tetrafluoro-5(trifluoromethoxy)-1,3-dioxolan-4-yl)oxy)-ammonium salt (1:1) (C6O4) were used as, respectively, legacy-linear and new-generation-cyclic PFASs. Human recombinant SHBG 30-234 domain (SHBG30-234), produced in HEK293-F cells, and delipidated recombinant hSA were used as in vitro protein models. Isothermal Titration Calorimetry (ITC) and tryptophan fluorescence quencing (TFQ) were used to evaluate the binding modes of T and PFAS to SHBG30-234 and hSA. ITC revealed the binding of T to SHBG30-234 with a Kd of 44 ± 2 nM whilst both PFOA and C6O4 showed no binding activity. Results were confirmed by TFQ, since only T modified the fluorescence profile of SHBG30-234. In hSA, TFQ confirmed the binding of T on FA6 site of the protein. A similar binding mode was observed for PFOA but not for C6O4, as further verified by displacement experiments with T. Although both PFASs were previously shown to bind hSA, only PFOA is predicted to possibly compete with T for the binding to hSA. However, on the base of the binding stoichiometry and affinity of PFOA for hSA, this appears unlikely at the blood concentrations of the chemical documented to date.

A novel genetically-encoded bicyclic peptide inhibitor of human urokinase-type plasminogen activator with better cross-reactivity toward the murine orthologue.

The inhibition of human urokinase-type plasminogen activator (huPA), a serine protease that plays an important role in pericellular proteolysis, is a promising strategy to decrease the invasive and metastatic activity of tumour cells. However, the generation of selective small molecule huPA inhibitors has proven to be challenging due to the high structural similarity of huPA to other paralogue serine proteases. Efforts to generate more specific therapies have led to the development of cyclic peptide-based inhibitors with much higher selectivity against huPA. While this latter property is desired, the sparing of the orthologue murine poses difficulties for the testing of the inhibitor in preclinical mouse model. In this work, we have applied a Darwinian evolution-based approach to identify phage-encoded bicyclic peptide inhibitors of huPA with better cross-reactivity towards murine uPA (muPA). The best selected bicyclic peptide (UK132) inhibited huPA and muPA with Ki values of 0.33 and 12.58 µM, respectively. The inhibition appears to be specific for uPA, as UK132 only weakly inhibits a panel of structurally similar serine proteases. Removal or substitution of the second loop with one not evolved in vitro led to monocyclic and bicyclic peptide analogues with lower potency than UK132. Moreover, swapping of 1,3,5-tris-(bromomethyl)-benzene with different small molecules not used in the phage selection, resulted in an 80-fold reduction of potency, revealing the important structural role of the branched cyclization linker. Further substitution of an arginine in UK132 to a lysine resulted in a bicyclic peptide UK140 with enhanced inhibitory potency against both huPA (Ki = 0.20 µM) and murine orthologue (Ki = 2.79 µM). By combining good specificity, nanomolar affinity and a low molecular mass, the bicyclic peptide inhibitor developed in this work may provide a novel human and murine cross-reactive lead for the development of a potent and selective anti-metastatic therapy.

Antiviral mechanisms of two broad-spectrum monoclonal antibodies for rabies prophylaxis and therapy.

Rabies is an acute and lethal encephalomyelitis caused by lyssaviruses, among which rabies virus (RABV) is the most prevalent and important for public health. Although preventable through the post-exposure administration of rabies vaccine and immunoglobulins (RIGs), the disease is almost invariably fatal since the onset of clinical signs. Two human neutralizing monoclonal antibodies (mAbs), RVC20 and RVC58, have been shown to be effective in treating symptomatic rabies. To better understand how these mAbs work, we conducted structural modeling and in vitro assays to analyze their mechanisms of action, including their ability to mediate Fc-dependent effector functions. Our results indicate that both RVC20 and RVC58 recognize and lock the RABV-G protein in its pre-fusion conformation. RVC58 was shown to neutralize more potently the extra-cellular virus, while RVC20 mainly acts by reducing viral spreading from infected cells. Importantly, RVC20 was more effective in promoting effector functions compared to RVC58 and 17C7-RAB1 mAbs, the latter of which is approved for human rabies post-exposure treatment. These results provide valuable insights into the multiple mechanisms of action of RVC20 and RVC58 mAbs, offering relevant information for the development of these mAbs as treatment for human rabies.

Development and characterization of a latex turbidimetric immunoassay using rabbit anti-CRP single-chain Fv antibodies.

In this study, we developed and demonstrated a latex turbidimetric immunoassay (LTIA) using latex beads immobilized with rabbit monoclonal single-chain variable fragments (scFvs) selected from an scFv-displayed phage library. Sixty-five different anti-c-reactive protein (anti-CRP) scFv clones were identified after biopanning selection using antigen-coupled multi-lamellar vesicles. By ranking antigen-binding clones using the apparent dissociation rate constant (appkoff) as a sorting index, scFv clones with a dissociation constant (KD free) ranging from 4.07 × 10-9 M to 1.21 × 10-11 M were isolated. Among them, three candidates (R2-6, R2-45, and R3-2) were produced in the culture supernatant at concentrations of 50 mg/L or higher in flask culture and maintained at considerably high antigen-binding activity in immobilized state on the CM5 sensor chip surface. All the scFv-immobilized latexes (scFv-Ltxs) prepared were well-dispersed in 50 mM MOPS at pH 7.0, without additives for dispersion, and their antigen-dependent aggregation was sufficiently detectable. The reactivity of scFv-Ltx to antigen differed among the scFv clones, in particular, R2-45 scFv-Ltx detected the CRP with the highest signal. Furthermore, the reactivity of scFv-Ltx varied significantly with salt concentration, scFv immobilization density, and the type of blocking protein. Particularly, antigen-dependent latex aggregation improved significantly in all rabbit scFv clones when scFv-Ltx was blocked with horse muscle myoglobin compared with conventional bovine serum albumin; while their baseline signals in the absence of antigen were fully stable. Under optimal conditions, R2-45 scFv-Ltx exhibited greater aggregation signals with antigen concentrations higher than those produced by conventional polyclonal antibody-immobilized latex for CRP detection in LTIA. The methodology for rabbit scFv isolation, immobilization, and antigen-dependent latex aggregation demonstrated in the present study can be applicable to scFv-based LTIA for various target antigens.

Amphiphilic Poly(vinyl alcohol) Membranes Leaving Out Chemical Cross-Linkers: Design, Synthesis, and Function of Tailor-Made Poly(vinyl alcohol)-b-poly(styrene) Copolymers.

Tailor-made poly(vinyl alcohol)-b-poly(styrene) copolymers (PVA-b-PS) for separation membranes are synthesized by the combination of reversible-deactivation radical polymerization techniques. The special features of these di-block copolymers are the high molecular weight (>70 kDa), the high PVA content (>80 wt%), and the good film-forming property. They are soluble only in hot dimethyl sulfoxide, but by the "solvent-switch" technique, they self-assemble in aqueous media to form micelles. When the self-assembled micelles are cast on a porous substrate, thin-film membranes with higher water permeance than that of PVA homopolymer are obtained. Thus, by using these tailor-made PVA-b-PS copolymers, it is demonstrated that chemical cross-linkers and acid catalysts can no longer be needed to produce PVA membranes, since the PS nanodomains within the PVA matrix act as cross-linking points. Lastly, subsequent thermal annealing of the thin film enhances the membrane selectivity due to the improved microphase separation.

Nanobodies targeting LexA autocleavage disclose a novel suppression strategy of SOS-response pathway.

Antimicrobial resistance threatens the eradication of infectious diseases and impairs the efficacy of available therapeutics. The bacterial SOS pathway is a conserved response triggered by genotoxic stresses and represents one of the principal mechanisms that lead to resistance. The RecA recombinase acts as a DNA-damage sensor inducing the autoproteolysis of the transcriptional repressor LexA, thereby derepressing SOS genes that mediate DNA repair, survival to chemotherapy, and hypermutation. The inhibition of such pathway represents a promising strategy for delaying the evolution of antimicrobial resistance. We report the identification, via llama immunization and phage display, of nanobodies that bind LexA with sub-micromolar affinity and block autoproteolysis, repressing SOS response in Escherichia coli. Biophysical characterization of nanobody-LexA complexes revealed that they act by trapping LexA in an inactive conformation and interfering with RecA engagement. Our studies pave the way to the development of new-generation antibiotic adjuvants for the treatment of bacterial infections.

Investigation of the Interaction between Human Serum Albumin and Branched Short-Chain Perfluoroalkyl Compounds.

The current trend dealing with the production of per- and polyfluoroalkyl substances (PFASs) involves the shifting toward branched short-chain fluorinated compounds known as new-generation PFASs. A key aspect to be clarified, to address the adverse health effects associated with the exposure to PFASs, is their binding mode to human serum albumin (hSA), the most abundant protein in plasma. In this study, we investigated the interaction between hSA and two representative branched short-chain PFASs, namely, HPFO-DA and C6O4. In-solution studies revealed that both compounds bind hSA with affinities and stoichiometries lower than that of the legacy long-chain perfluoroalkyl compound PFOA. Competition experiments using hSA-binding drugs with known site-selectivity revealed that both HPFO-DA and C6O4 bound to pockets located in subdomain IIIA. The crystal structure of hSA in complex with HPFO-DA unveiled the presence of two binding sites. The characterization and direct comparison of hSA interactions with new-generation PFASs may be key elements for the understanding of the toxicological impact of these compounds.

Synthesis and direct assay of large macrocycle diversities by combinatorial late-stage modification at picomole scale.

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.

Molecular analysis and therapeutic applications of human serum albumin-fatty acid interactions.

Human serum albumin (hSA) is the major carrier protein for fatty acids (FAs) in plasma. Its ability to bind multiple FA moieties with moderate to high affinity has inspired the use of FA conjugation as a safe and natural platform to generate long-lasting therapeutics with enhanced pharmacokinetic properties and superior efficacy. In this frame, the choice of the FA is crucial and a comprehensive elucidation of the molecular interactions of FAs with hSA cannot be left out of consideration. To this intent, we report here a comparative analysis of the binding mode of different FA moieties with hSA. The choice among different albumin-binding FAs and how this influence the pharmacokinetics properties of a broad spectrum of therapeutic molecules will be discussed including a critical description of some clinically relevant FA conjugated therapeutics.

Bicyclic peptide-based assay for uPA cancer biomarker.

The use of synthetic bioreceptors to develop biosensing platforms has been recently gaining momentum. This case study compares the performance of a biosensing platform for the human biomarker urokinase-type plasminogen activator (h-uPA) when using two bicyclic peptides (P1 and P2) with different affinities for the target protein. The bioreceptors P1 and P2 were immobilized on magnetic microbeads and tested within a sandwich-type affinity electrochemical assay. Apart from enabling h-uPA quantification at nanomolar levels (105.8 ng/mL for P1 and 32.5 ng/mL for P2), this case study showed the potential of synthetic bicyclic peptides applicability and how bioreceptor affinity can influence the performance of the final sensing platform.

Guidelines, Strategies, and Principles for the Directed Evolution of Cross-Reactive Antibodies Using Yeast Surface Display Technology.

The ability of cross-reactive antibodies to bind multiple related or unrelated targets derived from different species provides not only superior therapeutic efficacy but also a better assessment of treatment toxicity, thereby facilitating the transition from preclinical models to human clinical studies. This chapter provides some guidelines for the directed evolution of cross-reactive antibodies using yeast surface display technology. Cross-reactive antibodies are initially isolated from a naïve library by combining highly avid magnetic bead separations followed by multiple cycles of flow cytometry sorting. Once initial cross-reactive clones are identified, sequential rounds of mutagenesis and two-pressure selection strategies are applied to engineer cross-reactive antibodies with improved affinity and yet retained or superior cross-reactivity.

Transcriptomic and genomic studies classify NKL54 as a histone deacetylase inhibitor with indirect influence on MEF2-dependent transcription.

In leiomyosarcoma class IIa HDACs (histone deacetylases) bind MEF2 and convert these transcription factors into repressors to sustain proliferation. Disruption of this complex with small molecules should antagonize cancer growth. NKL54, a PAOA (pimeloylanilide o-aminoanilide) derivative, binds a hydrophobic groove of MEF2, which is used as a docking site by class IIa HDACs. However, NKL54 could also act as HDAC inhibitor (HDACI). Therefore, it is unclear which activity is predominant. Here, we show that NKL54 and similar derivatives are unable to release MEF2 from binding to class IIa HDACs. Comparative transcriptomic analysis classifies these molecules as HDACIs strongly related to SAHA/vorinostat. Low expressed genes are upregulated by HDACIs, while abundant genes are repressed. This transcriptional resetting correlates with a reorganization of H3K27 acetylation around the transcription start site (TSS). Among the upregulated genes there are several BH3-only family members, thus explaining the induction of apoptosis. Moreover, NKL54 triggers the upregulation of MEF2 and the downregulation of class IIa HDACs. NKL54 also increases the binding of MEF2D to promoters of genes that are upregulated after treatment. In summary, although NKL54 cannot outcompete MEF2 from binding to class IIa HDACs, it supports MEF2-dependent transcription through several actions, including potentiation of chromatin binding.

Structural Analysis of Human Serum Albumin in Complex with the Fibrate Drug Gemfibrozil.

Gemfibrozil (GEM) is an orally administered lipid-regulating fibrate derivative drug sold under the brand name Lopid®, among others. Since its approval in the early 80s, GEM has been largely applied to treat hypertriglyceridemia and other disorders of lipid metabolism. Though generally well tolerated, GEM can alter the distribution and the free, active concentration of some co-administered drugs, leading to adverse effects. Most of them appear to be related to the ability of GEM to bind with high affinity human serum albumin (HSA), the major drug-carrier protein in blood plasma. Here, we report the crystal structure of HSA in complex with GEM. Two binding sites have been identified, namely Sudlow's binding sites I (FA7) and II (FA3-FA4). A comparison of the crystal structure of HSA in complex with GEM with those of other previously described HSA-drug complexes enabled us to appreciate the analogies and differences in their respective binding modes. The elucidation of the molecular interaction between GEM and HSA might offer the basis for the development of novel GEM derivatives that can be safely and synergistically co-administered with other drugs, enabling augmented therapeutic efficacies.

Peptide-Based Inhibitors of ADAM and ADAMTS Metalloproteinases.

ADAM and ADAMTS are two large metalloproteinase families involved in numerous physiological processes, such as shedding of cell-surface protein ectodomains and extra-cellular matrix remodelling. Aberrant expression or dysregulation of ADAMs and ADAMTSs activity has been linked to several pathologies including cancer, inflammatory, neurodegenerative and cardiovascular diseases. Inhibition of ADAM and ADAMTS metalloproteinases have been attempted using various small molecules and protein-based therapeutics, each with their advantages and disadvantages. While most of these molecular formats have already been described in detail elsewhere, this mini review focuses solely on peptide-based inhibitors, an emerging class of therapeutic molecules recently applied against some ADAM and ADAMTS members. We describe both linear and cyclic peptide-based inhibitors which have been developed using different approaches ranging from traditional medicinal chemistry and rational design strategies to novel combinatorial peptide-display technologies.

Native mass spectrometry for the design and selection of protein bioreceptors for perfluorinated compounds.

Biosensing platforms are answering the increasing demand for analytical tools for environmental monitoring of small molecules, such as per- and polyfluoroalkyl substances (PFAS). By transferring toxicological findings in bioreceptor design we can develop innovative pathways for biosensor design. Indeed, toxicological studies provide fundamental information about PFAS-biomolecule complexes that can help evaluate the applicability of the latter as bioreceptors. The toolbox of native mass spectrometry (MS) can support this evaluation, as shown by the two case studies reported in this work. The analysis of model proteins' (i.e. albumin, haemoglobin, cytochrome c and neuroglobin) interactions with well-known PFAS, such as perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS), demonstrated the potential of this native MS screening approach. In the first case study, untreated albumin and delipidated albumin were compared in the presence and absence of PFOA confirming that the delipidation step increases albumin affinity for PFOA without affecting protein stability. In the second case study, the applicability of our methodology to identify potential bioreceptors for PFOS/PFOA was extended to other proteins. Structurally related haemoglobin and neuroglobin revealed a 1 : 1 complex, whereas no binding was observed for cytochrome c. These studies have value as a proof-of-concept for a general application of native MS to identify bioreceptors for toxic compounds.

Unveiling the binding mode of perfluorooctanoic acid to human serum albumin.

Perfluorooctanoic acid (PFOA) is a toxic compound that is absorbed and distributed throughout the body by noncovalent binding to serum proteins such as human serum albumin (hSA). Though the interaction between PFOA and hSA has been already assessed using various analytical techniques, a high resolution and detailed analysis of the binding mode is still lacking. We report here the crystal structure of hSA in complex with PFOA and a medium-chain saturated fatty acid (FA). A total of eight distinct binding sites, four occupied by PFOAs and four by FAs, have been identified. In solution binding studies confirmed the 4:1 PFOA-hSA stoichiometry and revealed the presence of one high and three low affinity binding sites. Competition experiments with known hSA-binding drugs allowed locating the high affinity binding site in sub-domain IIIA. The elucidation of the molecular basis of the interaction between PFOA and hSA might provide not only a better assessment of the absorption and elimination mechanisms of these compounds in vivo but also have implications for the development of novel molecular receptors for diagnostic and biotechnological applications.

Covalent immobilization of delipidated human serum albumin on poly(pyrrole-2-carboxylic) acid film for the impedimetric detection of perfluorooctanoic acid.

The immobilization of biomolecules at screen printed electrodes for biosensing applications is still an open challenge. To enrich the toolbox of bioelectrochemists, graphite screen printed electrodes (G-SPE) were modified with an electropolymerized film of pyrrole-2-carboxilic acid (Py-2-COOH), a pyrrole derivative rich in carboxylic acid functional groups. These functionalities are suitable for the covalent immobilization of biomolecular recognition layers. The electropolymerization was first optimized to obtain stable and conductive polymeric films, comparing two different electrolytes: sodium dodecyl sulphate (SDS) and sodium perchlorate. The G-SPE modified with Py-2-COOH in 0.1 M SDS solution showed the required properties and were further tested. A proof-of-concept study for the development of an impedimetric sensor for perfluorooctanoic acid (PFOA) was carried out using the delipidated human serum albumin (hSA) as bioreceptor. The data interpretation was supported by size exclusion chromatography and small-angle X-ray scattering (SEC-SAXS) analysis of the bioreceptor-target complex and the preliminary results suggest the possibility to further develop this biosensing strategy for toxicological and analytical studies.