A mating mechanism to generate diversity for the Darwinian selection of DNA-encoded synthetic molecules.

DNA-encoded library technologies enable the screening of synthetic molecules but have thus far not tapped into the power of Darwinian selection with iterative cycles of selection, amplification and diversification. Here we report a simple strategy to rapidly assemble libraries of conformationally constrained peptides that are paired in a combinatorial fashion (suprabodies). We demonstrate that the pairing can be shuffled after each amplification cycle in a process similar to DNA shuffling or mating to regenerate diversity. Using simulations, we show the benefits of this recombination in yielding a more accurate correlation of selection fitness with affinity after multiple rounds of selection, particularly if the starting library is heterogeneous in the concentration of its members. The method was validated with selections against streptavidin and applied to the discovery of PD-L1 binders. We further demonstrate that the binding of self-assembled suprabodies can be recapitulated by smaller (∼7 kDa) synthetic products that maintain the conformational constraint of the peptides.

SARS-CoV-2 infection as a trigger of humoral response against apolipoprotein A-1.

BACKGROUND: Unravelling autoimmune targets triggered by SARS-CoV-2 infection may provide crucial insights into the physiopathology of the disease and foster the development of potential therapeutic candidate targets and prognostic tools. We aimed at determining (a) the association between anti-SARS-CoV-2 and anti-apoA-1 humoral response and (b) the degree of linear homology between SARS-CoV-2, apoA-1 and Toll-like receptor 2 (TLR2) epitopes. DESIGN: Bioinformatics modelling coupled with mimic peptides engineering and competition experiments were used to assess epitopes sequence homologies. Anti-SARS-CoV-2 and anti-apoA-1 IgG as well as cytokines were assessed by immunoassays on a case-control (n = 101), an intensive care unit (ICU; n = 126) and a general population cohort (n = 663) with available samples in the pre and post-pandemic period. RESULTS: Using bioinformatics modelling, linear sequence homologies between apoA-1, TLR2 and Spike epitopes were identified but without experimental evidence of cross-reactivity. Overall, anti-apoA-1 IgG levels were higher in COVID-19 patients or anti-SARS-CoV-2 seropositive individuals than in healthy donors or anti-SARS-CoV-2 seronegative individuals (P < .0001). Significant and similar associations were noted between anti-apoA-1, anti-SARS-CoV-2 IgG, cytokines and lipid profile. In ICU patients, anti-SARS-CoV-2 and anti-apoA-1 seroconversion rates displayed similar 7-day kinetics, reaching 82% for anti-apoA-1 seropositivity. In the general population, SARS-CoV-2-exposed individuals displayed higher anti-apoA-1 IgG seropositivity rates than nonexposed ones (34% vs 16.8%; P = .004). CONCLUSION: COVID-19 induces a marked humoral response against the major protein of high-density lipoproteins. As a correlate of poorer prognosis in other clinical settings, such autoimmunity signatures may relate to long-term COVID-19 prognosis assessment and warrant further scrutiny in the current COVID-19 pandemic.

Dual Bcl-XL /Bcl-2 inhibitors discovered from DNA-encoded libraries using a fragment pairing strategy.

A dual Bcl-XL / Bcl-2 inhibitor was discovered from DNA-encoded libraries using a two steps process. In the first step, DNA was used to pair PNA-encoded fragments exploring > 250 000 combinations. In the second step, a focused library combining the selected fragments with linkers of different lengths and geometries led to the identification of tight binding adducts that were further investigated for their selective target engagement in pull-down assays, for their affinity by SPR, and their selectivity in a cytotoxicity assay. The best compound showed comparable cellular activity to venetoclax, the first-in-class therapeutic targeting Bcl-2.

Experimental Identification of Immuno- dominant B-cell Epitopes from SARS-CoV-2.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the current public health crisis with devastating consequences to our societies. This COVID-19 pandemic has become the most serious threat to global public health in recent history. Given the unprecedented economic and social impact that it is causing, identification of immunodominant epitopes from SARS-CoV-2 is of great interest, not only to gain better insight into the adaptive immune response, but also for the development of vaccines, treatments and diagnostic tools. In this review, we summarize the already published or preprinted reports on the experimental identification of B-cell linear epitopes of SARS-CoV-2 proteins. Six different epitopes leading to neutralizing antibodies have been identified. Moreover, a summary of peptide candidates to be used for diagnostic tools is also included.

Identification of immunodominant linear epitopes from SARS-CoV-2 patient plasma.

A novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) is the source of a current pandemic (COVID-19) with devastating consequences in public health and economic stability. Using a peptide array to map the antibody response of plasma from healing patients (12) and heathy patients (6), we identified three immunodominant linear epitopes, two of which correspond to key proteolytic sites on the spike protein (S1/S2 and S2') known to be critical for cellular entry. We show biochemical evidence that plasma positive for the epitope adjacent to the S1/S2 cleavage site inhibits furin-mediated proteolysis of spike.

PNA-Based Dynamic Combinatorial Libraries (PDCL) and screening of lectins.

Selections from dynamic combinatorial libraries (DCL) benefit from the dynamic nature of the library that can change constitution upon addition of a selection pressure, such as ligands binding to a protein. This technology has been predominantly used with small molecules interacting with each other through reversible covalent interaction. However, application of this technology in biomedical research and drug discovery has been limited by the reversibility of covalent exchange and the analytical deconvolution of small molecule fragments. Here we report a supramolecular approach based on the use of a constant short PNA tag to direct the combinatorial pairing of fragment. This PNA tag yields fast exchange kinetics, while still delivering the benefits of cooperativity, and provides favourable properties for analytical deconvolution by MALDI. A selection from >6,000 assemblies of glycans (mono-, di-, tri-saccharides) targeting AFL, a lectin from pathogenic fungus, yielded a 95 nM assembly, nearly three orders of magnitude better in affinity than the corresponding glycan alone (41 µM).

Small-Molecule Modulators of the ATPase VCP/p97 Affect Specific p97 Cellular Functions.

VCP/p97 belongs to the AAA+ ATPase family and has an essential role in several cellular processes ranging from cell division to protein homeostasis. Compounds targeting p97 inhibit the main ATPase domain and cause cell death. Here, using PNA-encoded chemical libraries, we have identified two small molecules that target the regulatory domain of p97, comprising the N-terminal and the D1 ATPase domains, and do not cause cell death. One molecule, NW1028, inhibits the degradation of a p97-dependent reporter, whereas the other, NW1030, increases it. ATPase assays show that NW1028 and NW1030 do not affect the main catalytic domain of p97. Mapping of the binding site using a photoaffinity conjugate points to a cleft at the interface of the N-terminal and the D1 ATPase domains. We have therefore discovered two new compounds that bind to the regulatory domain of p97 and modulate specific p97 cellular functions. Using these compounds, we have revealed a role for p97 in the regulation of mitotic spindle orientation in HeLa cells.

Novel PTP1B inhibitors identified by DNA display of fragment pairs.

DNA display of PNA-encoded libraries was used to pair fragments containing different phosphotyrosine surrogates with diverse triazoles. Microarray-based screening of the combinatorially paired fragment sets (62,500 combinations) against a prototypical phosphatase, PTP1B, was used to identify the fittest fragments. A focused library (10,000 members) covalently pairing identified fragments with linkers of different length and geometry was synthesized. Screening of the focused library against PTP1B and closely related TCPTP revealed orthogonal inhibitors. The selectivity of the identified inhibitors for PTP1B versus TCPT was confirmed by enzymatic inhibition assay.

Identification of Covalent Bromodomain Binders through DNA Display of Small Molecules.

The regulation of transcriptional programs by epigenetic readers (bromodomains) has been linked to the development of several pathologies. Notably, it has been implicated in the regulation of cellular growth and evasion of apoptosis, in cancer as well as in inflammation. The discovery of small-molecule probes to dissect the role of bromodomains is thus important. We demonstrate that specific cysteine residues conserved across the bromodomains can be harnessed for covalent trapping. We report the discovery of two small molecules that form a covalent bond with cysteine residues conserved across the bromodomain family, analyze the subset of bromodomains that can be addressed through covalent binding, and show proteomic analyses enabled by the enrichment of bromodomains from native lysates.

Assembly of PNA-tagged small molecules, peptides, and carbohydrates onto DNA templates: programming the combinatorial pairing and inter-ligand distance.

The biochemical stability and desirable hybridization properties of peptide nucleic acids (PNA) coupled to the robustness of the peptidic chemistry involved in their oligomerization make them an attractive nucleic acid tag to encode molecules and program their assembly into higher order oligomers. The ability to program the dimerization of ligands with controlled distance between the ligands has important applications in emulating multimeric interactions. Additionally, the ability to program different permutations of ligand assemblies in a combinatorial fashion provides access to a broad diversity and offers a rapid screening method for fragment based approaches to drug discovery. Herein, we describe protocols to covalently link diverse carbohydrates, peptides, or small molecules to PNA and combinatorially assemble them in solution onto libraries of DNA templates or onto DNA microarrays using a commercial platform without recourse to specialized equipment or heavy upfront investment.

Discovery of an entropically-driven small molecule streptavidin binder from nucleic acid-encoded libraries.

Dehydrocholic acid was identified as a selective streptavidin binder from a PNA-tagged library. Isothermal calorimetry titration measurements showed this interaction to be entropically driven. Peptides tagged with dehydrocholic acid can be captured on a streptavidin resin and released under thermal conditions.

Selection of a synthetic glycan oligomer from a library of DNA-templated fragments against DC-SIGN and inhibition of HIV gp120 binding to dendritic cells.

We report the synthesis of a nucleic acid-encoded carbohydrate library, its combinatorial self-assembly into 37,485 pairs and a screen against DC-SIGN leading to the identification of consensus ligand motifs. A prototypical example from the selected pairs was shown to have enhanced binding. A dendrimer incorporating the selected motifs inhibited gp120's binding to dendritic cells with higher efficiency than mannan.

Autogenous modulation of the Bacillus subtilis sacB-levB-yveA levansucrase operon by the levB transcript.

Silencing of levB, the second structural gene of the tricistronic levansucrase operon encoding the endolevanase LevB, decreases the level of levansucrase expression in Bacillus subtilis. Conversely, independent expression of levB greatly stimulates operon expression. This autogenous effect is mediated by the levB transcript, which carries an internal sequence (5'-AAAGCAGGCAA-3') involved in the enhancing effect. In vitro, the levB transcript displays an affinity for the N-terminal fragment of SacY (K(D) 0.2 microM), the regulatory protein that prevents transcription termination of the levansucrase operon. This positive-feedback loop leads to an increase in the operon expression when B. subtilis is growing in the presence of high sucrose concentrations. Under these conditions, extracellular levan synthesized by the fructosyl polymerase activity of levansucrase can be degraded mainly into levanbiose by the action of LevB. Levanbiose is neither taken up nor metabolized by the bacteria. This work modifies the present view of the status of levansucrase in B. subtilis physiology.

Transcripts of the genes sacB, amyE, sacC and csn expressed in Bacillus subtilis under the control of the 5' untranslated sacR region display different stabilities that can be modulated.

When Bacillus subtilis levanase (SacC), alpha-amylase (AmyE) and chitosanase (Csn) structural genes were expressed under the regulated control of sacR, the inducible levansucrase (SacB) leader region in a degU32(Hy) mutant, it was observed that the production yields of the various extracellular proteins were quite different. This is mainly due to differences in the stabilities of their corresponding mRNAs which lead to discrepancies between the steady-state level of mRNA of sacB and csn on the one hand and amyE and sacC on the other. In contrast to levansucrase mRNA, the decay curves of alpha-amylase and levanase mRNAs obtained by Northern blotting analysis did not match the decay curves of their functional mRNA. This suggested that only a part of the population of the amyE and sacC transcripts was fully translated, while the others were possibly poorly bound to ribosomes and thus were only partially translated or not at all and consequently submitted to rapid endonuclease degradation. This hypothesis was substantiated by the finding that the introduction of a Shine-Dalgarno sequence upstream from the ribosome-binding site in the sacC transcript resulted in a fourfold increase in both the half-life of this transcript and the production of levanase. An additional cause of low-level levanase production is the premature release of mRNA by the polymerase. It was attempted to correlate this event with internal secondary structures of sacC mRNA.