Many peptide drugs rely on nonproteinogenic amino acids and chemical modifications for improved activity and proteolytic stability. However, these features also make drug production expensive and challenging to scale. Here, we engineered small, linear, proteinogenic peptides that bind human programmed death-ligand 1 (hPD-L1) with high affinity and stability using mRNA display affinity maturation. The resulting peptides, SPAM2 and SPAM3, have antibody-like affinities for hPD-L1 (dissociation constants between ~250 and 300 pM) and are selective for hPD-L1. Both SPAM2 and SPAM3 compete with hPD-L1 ligands known to interact with the programmed cell death protein 1 site and are stable in human serum. SPAM3 bound human glioma D423 cells with high affinity in flow cytometry experiments comparable to that of a clinical therapeutic antibody. These results support the use of affinity maturation selections to dramatically enhance the biophysical properties of linear, proteinogenic peptides for translational applications.

The binding affinity of antibodies to specific antigens stems from a remarkably broad repertoire of hypervariable loops known as complementarity-determining regions (CDRs). While recognizing the pivotal role of the heavy-chain 3 CDRs (CDR-H3s) in maximizing antibody-antigen affinity and specificity, the key structural determinants responsible for their adaptability to diverse loop sequences, lengths, and noncanonical structures are hitherto unknown. To address this question, we achieved a de novo synthesis of bulged CDR-H3 mimics excised from their full antibody context. CD and NMR data revealed that these stable standalone β-hairpin scaffolds are well-folded and retain many of the native bulge CDR-H3 features in water. In particular, the tryptophan residue, highly conserved across CDR-H3 sequences, was found to extend the kinked base of these β-bulges through a combination of stabilizing intramolecular hydrogen bond and CH/π interaction. The structural ensemble consistent with our NMR observations exposed the dynamic nature of residues at the base of the loop, suggesting that β-bulges act as molecular hinges connecting the rigid stem to the more flexible loops of CDR-H3s. We anticipate that this deeper structural understanding of CDR-H3s will lay the foundation to inform the design of antibody drugs broadly and engineer novel CDR-H3 peptide scaffolds as therapeutics.

Aberrant and dysregulated protein-protein interactions (PPIs) drive a significant number of human diseases, which is why they represent a major class of targets in drug discovery. Although a number of high-affinity antibody-based drugs have emerged in this therapeutic space, the discovery of smaller PPI inhibitors is lagging far behind, underscoring the need for novel scaffold modalities. To bridge this gap, we introduce a biomimetic platform technology - adaptive design of antibody paratopes into therapeutics (ADAPT) - that enables the paratope-forming binding loops of antibodies to be crafted into large β-hairpin scaffolds (ADAPTins). In this study, we describe a novel strategy for engineering native CDR-H3 "hot loops" with varying sequences, lengths, and rigidity into ADAPTins, ultimately transforming these compounds into irreversible covalent inhibitors. A proof-of-concept was established by creating a series of ADAPTin blockers of the PD1:PDL1 immune checkpoint PPI (blocking activity EC50 < 0.3 μM) which were subsequently modified into potent covalent PD1 inhibitors. The compelling rate of stable and folded ADAPTins above physiological temperature (21 out of 29) obtained across six different scaffolds suggests that the platform technology could provide a novel opportunity for high-quality peptide display and biological screening.

A biomimetic semisynthesis of the diterpenoid (+)-providencin (2) and the unexpected novel C14 regioisomer 3 was achieved by photoirradiation of the proposed biosynthetic cembranoid precursor (-)-bipinnatin E (1). The absolute configuration assignments of 1 and 2 by correlation were established by X-ray analysis. A combination of NOESY data and photochemical reaction results revealed that both C2 and C14 positions of the macrocycle (-)-1 are suitable for hydrogen abstraction, thus affording an explanation to the mixture of cyclobutane photoproduct isomers obtained by a Norrish-Yang cyclization. These results also support the proposed biosynthetic hypothesis describing the genuine photochemical transformation of (-)-1 into (+)-2, without refuting that both regioisomer products 2/3 might be artifacts of isolation.

The tryptophan zipper (Trpzip) is an iconic folding motif of β-hairpin peptides capitalizing on two pairs of cross-strand tryptophans, each stabilized by an aromatic–aromatic stacking in an edge-to-face (EtF) geometry. Yet, the origins and the contribution of this EtF packing to the unique Trpzip stability remain poorly understood. To address this question of structure–stability relationship, a library of Trpzip hairpins was developed by incorporating readily accessible nonproteinogenic tryptophans of varying electron densities. We found that each EtF geometry was, in fact, stabilized by an intricate combination of XH/π interactions. By tuning the π-electron density of Trp face rings, CH/π interactions are strengthened to gain additional stability. On the contrary, our DFT calculations support the notion that Trp edge modulations are challenging due to their simultaneous paradoxical engagement as H-bond donors in CH/π and acceptors in NH/π interactions.

The recent shift toward increasingly larger drug modalities has created a significant demand for novel classes of compounds with high membrane permeability that can inhibit intracellular protein-protein interactions (PPIs). While major advances have been made in the design of cell-permeable helices, stapled β-sheets, and cyclic peptides, the development of large acyclic β-hairpins lags far behind. Therefore, we investigated a series of 26 β-hairpins (MW > 1.6 kDa) belonging to a chemical space far beyond the Lipinski "rule of five" (fbRo5) and showed that, in addition to their innate plasticity, the lipophilicity of these peptides (log D 7.4 ≈ 0 ± 0.7) can be tuned to drastically improve the balance between aqueous solubility and passive membrane permeability.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

Immune check point inhibitor(s) have become a main stay of treatment for melanoma regardless of its mutational status. However, not all patients respond to treatment. In fact, only 40-60% respond to treatment. Currently, only PD-L1/PD-1 expression and mutational burden can predict response to treatment. Published data have shown that exosomal PD-L1 is an important factor contributing to response/failure to checkpoint inhibitor treatment. Exosome is known to carry several types of cargoes and is one of the important delivery systems in the body. We have begun to examine the role of exosomal PD-L1, PD-1 in predicting response, and as well as how to overcome drug resistance. We initiated an institutional study in metastatic melanoma patients who underwent immunotherapy treatment (anti-PD-1 + anti-CTLA 4 drugs). Blood samples were obtained at baseline and at subsequent treatment. Both serum and plasma exosomes were isolated. Eighteen patients were entered in the study. We have found that 1) Patients with PD-L1 negative tumor can have high exosomal PD-L1 expression, 2) all patients had exosomal PD-L1, and 3) all patients with decreased exosomal PD-L1 expression after treatment showed antitumor response [partial response (PR) or complete response (CR)]. In contrast, patients who had consistently high exosomal PD-L1 expression post-treatment showed no response, and this can be seen after the third cycle of treatment. Patients who achieved CR showed low exosomal PD-L1 and no exosomal PD-1 post-treatment. Importantly, we have also found that exosomes carry antiapoptotic protein and proapoptotic protein, as well as ATG family protein, which is related to autophagy. Whether these proteins also play a role in determining the cell fate in the metastatic sites is not known and will need future investigation. Interestingly, the two patients who showed no response also had high p-AKT. Since traditional anti PD-1 and anti-PD-L1 drugs do not have effect on exosomal PD-L1 and PD-1, we formed a collaboration with Dr. Stephane Roche at Florida Atlantic University, who has designed novel peptides by exploiting the innate plasticity of the PD-1 receptor (pembrolizumab H3 loop mimics). Thus far, we have screened many compounds and have selected 2 compounds for further testing. Together, we have also designed new PD-L1 peptides that can inhibit both PD-L1 and PI3K/AKT pathways simultaneously. Hence one can overcome checkpoint inhibitor resistance due to exosomal PD-L1/PD-1. Supported by grant from Sylvester Comprehensive Cancer Center and R21GM132754. Citation Format: Pablo Eduardo Puente, Dan Nguyen, Niramol Savaraj, Chunjing Wu, Medhi Wangpaichitr, Stephane Roche, Guangkuan Zhao, Alexis Richaud, Jose Lutzky, Leonel Hernandez Aya, Mecker Moller, Jessica Crystal, Neha Goel, Lynn Feun. Can exosomal cargoes predict checkpoint inhibitor response in melanoma, and can we overcome drug resistance. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4114.

Checkpoint blockade of the Programmed cell Death-1 (PD-1)immunoreceptor with its ligand 1 (PD-L1) by the monoclonal antibody pembrolizumabprovided compelling clinical results among various cancer types, yet themolecular mechanism by which this drug blocks the PD-1:PD-L1 binding interface andreactivates exhausted T cells remains unclear. To address this question, weexamined the conformational motion of PD-1 associated with the binding ofpembrolizumab. The largely overlooked innate plasticity of both PD-1 C’D and FGloops appears crucial to closing in the receptor edges on the drug. Herein, wedescribe how PD-1 bends to initiate the formation of a deep binding groove (371Å3) across several epitopes while engaging pembrolizumab. Our analysisultimately provided a rational design for mimicking the pembrolizumab H3 loop [RDYRFDMGFD]as a PD-1 inhibitor. A series of H3 loop mimics were synthesized and theirfolding characterized by CD and NMR spectroscopy. As a result, a first-in-classb-hairpin peptide inhibitorof the PD-1/PD-L1 interface was identified (IC50 of 0.6 ± 0.2 μM). Overall, this studydemonstrates that the dynamic groove formed between the C’D and FG loops ofPD-1 is an attractive target for the development of peptide-based PD-1inhibitors.

Despite their pivotal role in protein function and antibody binding affinity, β-hairpins bearing long non-canonical loops are a challenge to modern synthesis because of the large entropic penalty associated with their folding. Little is known about the contribution and impact of stabilizing motifs on the folding of β-hairpins of variable length and plasticity. Here we report a direct comparison between these b-straps thermodynamics and their thermal stability behavior using several local spectroscopic probes of the folding/unfolding landscape. The judicious cooperative interactions crafted in β-Strap RW(VW)•••(WV/H)WE (strap = strand + cap) greatly stabilized hairpins with up to 10-residue loops lacking an innate nucleating-turn locus (Tm up to 52 oC; 88 ± 1% folded at 291 K). The present design of novel β-straps aims to provide the foundation to study new classes of long hairpins and ultimately offer an attractive alternative to macrocyclic peptides for the mimicry of functional loops from proteins and antibodies.

Macrocyclic furanobutenolide-derived cembranoids (FBCs)are the biosynthetic precursors to a wide variety of highly congested andoxygenated polycyclic (nor)diterpenes (e.g.plumarellide, verrillin or bielschowskysin). These architecturally complexmetabolites are thought to originate from site-selective oxidation of themacrocycles’ backbone and a series of intricate transannular reactions. Yet thedevelopment of a common biomimetic route has been hampered by a lack of syntheticmethods for the pivotal furan dearomatization in a regio- and stereoselectivemanner. To address these shortcomings, a concise strategy of chemo- and stereoselectiveepoxidation followed by a kinetically-controlled furan dearomatization is reported. Thesurprising switch of facial a:b-discrimination observed in the epoxidations of the most strained E-acerosolideversus E-deoxypukalideand E-bipinnatinJ derived macrocycles has been rationalized by the 3D-conformationalpreferences of the macrocyclic scaffolds. The downstreamfunctionalization of FBC-macrocycles was also studied, and how the C-7 epoxide configuration was retentively translatedto the C-3 stereogenicity in dearomatized products under kinetic control tosecure the requisite (3S,7S,8S)-configurationsfor the bielschowskysin synthesis. Unlike previouslyspeculated, our results suggest that the most strained FBC-macrocycles bearing aE-(D7,8)-alkene moiety may stand as the truebiosynthetic precursors to bielschowskysin and several other polycyclic naturalproducts of this class.

The stunning advances in understanding the reactivity and selectivity principles of asymmetric pericyclic reactions have had a profound impact on the synthetic planning of complex natural products. Indeed, electrocyclizations, cycloadditions, and sigmatropic rearrangements enable synthetic chemists to craft highly functionalized scaffolds that would not otherwise be possible with a similar atom-, step-, and redox-economy. In this review, selected examples from the last two decades of research (2003–2020) on tandem processes combining oxa-6π electrocyclic reactions are discussed in terms of reactivity challenges, inherent reversibility, and key structural bond formation in the assembly of natural products. A particular emphasis is given to the electrocyclic ring-closures in the tandem processes featuring Knoevenagel-type condensations, Diels–Alder cycloadditions, Stille couplings, and oxidative dearomatizations. The synthetic manifolds reviewed here illustrate how oxa-6π electrocyclizations are intimately linked to the construction of complex natural product scaffolds and have inspired a number of biomimetic syntheses in the laboratory.

Nature remarkably employs posttranslational modifications of the 20 canonical alpha-amino acids to devise a far larger structural, conformational, and functional diversity found in non-proteinogenic amino acids (NPAAs), which ultimately translates into a plethora of complex biological functions. Synthetic chemists are continuously trying to reproduce and even extrapolate the repertoire of NPAA building blocks to build structural diversity into bioactive molecules and materials. The direct asymmetric functionalization of alpha-imino esters represents one of the most robust and attractive routes to NPAAs. This review summarizes the most prominent examples of bench-stable (ald)imine surrogates exploited for the synthesis of NPAAs, including our most recent results in the nucleophilic substitution of alpha-haloglycines and other alpha-haloaminals. A synopsis of kinetic studies, reaction optimizations, and enantioselective catalytic methods is also presented. 1 Introduction 2 Asymmetric Synthesis of Tertiary alpha-Substituted NPAAs 2.1 From N,O-Acetals (alpha-Hydroxy/Alkyloxy/Acetoxyglycines) 2.2 From alpha-Amido Sulfones 2.3 From alpha-Haloglycine Esters 2.4 From N,O-Bis(Boc) Hydroxyglycine 3 Asymmetric Synthesis of Acyclic Quaternary alpha,alpha-Disubstituted NPAAs 4 Concluding Remarks

The design of novel and functional biomimetic foldamers remains a major challenge in creating mimics of native protein structures. Herein, we report the stabilization of a short b -sheet by incorporating N -(hydroxy)glycine (Hyg) residues into the backbone of peptides. These short peptide−peptoid hybrids form unique parallel b -sheet structures by self-assembly. Spectroscopic and crystallographic data collected suggest that the local conformational perturbations induced by N -(hydroxy)amides are outweighed by a network of strong interstrand hydrogen-bonds.

A versatile synthetic protocol of aza-Friedel−Crafts alkylationhas been developed for the synthesis of quaternary a-aminoesters. This operationally simple alkylation proceeds under ambient conditionswith high efficiency, regioselectivity, and an exceptionally broad scope ofarene nucleophiles. A key feature of this alkylation is the role associatedwith the silver(I) salt counteranions liberated during the reaction. Takingadvantage of a phase-transfer counteranion/BrØnsted acid pair mechanism,a catalytic enantioselective version of the reaction is also reported.

A general and efficient synthesis of α-haloglycine esters from commercially available feedstock chemicals, in a single step, is reported. The reactivity of these α-haloglycine esters with various nucleophiles was studied as surrogates of α-iminoesters upon activation with hydrogen-bond donor catalysts. DFT calculations on the α-haloglycine structures (X = F, Cl, Br) accompanied by an X-ray characterization of the α-bromoglycine ester support the existence of a "generalized" anomeric effect created by hyperconjugation. This peculiar hyperconjugative effect is proposed to be responsible for the enhanced halogen nucleofugality leading to a facile halogen abstraction by hydrogen-bond donor catalysts. This reactivity was exploited with thiourea catalysts on several catalytic transformations (aza-Friedel-Crafts and Mannich reactions) for the synthesis of several types of non-proteinogenic α-amino esters.

We report a chiral-squaramide-catalyzed enantio- and diastereoselec-tive synthesis of α-allyl amino esters. The optimized protocol provides access to N-carbamoyl-protected amino esters via nucleophilic allyla-tion of readily accessible α-chloro glycinates. A variety of useful α-allyl amino esters were prepared-including crotylated products bearing vicinal stereocenters that are inaccessible through enolate alkylation-with high enantioselectivity (up to 97% ee) and diastereoselectivity (> 10:1). The reactions display first-order kinetic dependence on both the α-chloro glycinate and the nucleophile, consistent with rate-limiting C-C bond formation. Computational analysis of the uncatalyzed reaction predicts an energetically inaccessible iminium intermediate, and a lower energy concerted SN2 mechanism.

The Cover Feature shows examples of non-proteinogenic α-amino acids that can be obtained from various α-haloglycines (X = Br, Cl, F) in a practical, scalable, and enantioselective manner. Owing to a peculiar hyperconjugation effect, the innate reactivity of α-haloglycines became the key to crack open the vault, revealing new treasures of amino acids. Taking advantage of this reactivity with hydrogen-bond donor catalysts was possible due to C–X bond elongation as shown by X-ray crystal structure. More information can be found in the Full Paper by S. P. Roche and S. S. Samanta.

Rationale: While checkpoint inhibitors have revolutionized the treatment of melanoma, it is not known whether switching from one monoclonal antibody drug to another one would be justified in the case of a treatment failure. Herein, we report a case illustrating a durable response to pembrolizumab after a failure with nivolumab. Patient concerns: A 76-year-old white male noticed an enlarging papular lesion on his neck. Diagnosis: Malignant melanoma. Interventions: The patient underwent surgery in December 2013 and was found to have a B-Rapidly Accelerated Fibrosarcoma (BRAF) V600E mutated melanoma. Treatment with BRAF and MAPK/Erk kinase (MEK) inhibitors along with radiation was initiated. After 1 year, the disease progressed, and the treatment was switched to the cytotoxic T-lymphocyte antigen 4 (CTLA-4) blocking antibody, ipilimumab. As the tumor did not respond, the treatment was changed to programmed cell death receptor-1 (PD-1) blockers: nivolumab followed by pembrolizumab. Since the initial diagnosis, the tumor response was monitored by computed tomography (CT) scans. Immunohistochemistry (IHC) was also used for the assessment of programmed death ligand 1 PD-L1) expression in the neck, lung, and spleen lesions. Outcomes: The patient had an initial mixed response to nivolumab, but the disease ultimately progressed as evidenced by new metastases to the spleen, thus the treatment was switched to pembrolizumab. After 46 cycles of treatment, all sites of metastases disappeared, including a substantial shrinkage of the splenic metastasis. To gain understanding about the pharmacological differences between nivolumab and pembrolizumab, the PD-1-ligands interactions and conformational dynamics responsible for the PD-1/PD-L1 checkpoint blockade were investigated. The higher affinity of pembrolizumab might likely arise from a unique and large patch of interactions engaging the C'D loop of PD-1, thus forcing an important motion across the PD-1 immunoreceptor. Lessons: In this case report, we described the tolerance and response of a melanoma patient to a sequence of various agents, including ipilimumab, nivolumab, and pembrolizumab. To the best of our knowledge, this is the first clinical report highlighting differences between PD-1 blockers, as shown by the unexpected and durable response of the tumor to pembrolizumab, after a treatment failure with nivolumab.

4H-Pyrans (4H-Pys) and 1,4-dihydropyridines (1,4-DHPs) are important classes of heterocyclic scaffolds in medicinal chemistry. Herein, an indium(III)-catalyzed one-pot domino reaction for the synthesis of highly functionalized 4H-Pys, and a model of 1,4-DHP is reported. This alternative approach to the challenging Hantzsch 4-component reaction enables the synthesis of fused-tricyclic heterocycles, and the mechanistic studies underline the importance of an intercepted-Knoevenagel adduct to achieve higher chemoselectivity towards these types of unsymmetrical heterocycles.