Medicinal Chemistry Approaches Research Articles

Studies on diketopiperazine and dipeptide analogs as opioid receptor ligands

Using the structure of gliotoxin as a starting point, we have prepared two different chemotypes with selective affinity to the kappa opioid receptor (KOR). Using medicinal chemistry approaches and structure-activity relationship (SAR) studies, structural features required for the observed affinity were identified, and advanced molecules with favorable Multiparameter Optimization (MPO) and Ligand Lipophilicity (LLE) profiles were prepared. Using the Thermal Place Preference Test (TPPT), we have shown that compound2 blocks the antinociceptive effect of U50488, a known KOR agonist. Multiple reports suggest that modulation of KOR signaling is a promising therapeutic strategy in treating neuropathic pain (NP). As a proof-of-concept study, we tested compound 2 in a rat model of NP and recorded its ability to modulate sensory and emotional pain-related behaviors. Observed in vitro and in vivo results suggest that these ligands can be used to develop compounds with potential application as pain therapeutics.

Small-Molecule Fluorescent Ligands for the CXCR4 Chemokine Receptor.

The C-X-C chemokine receptor type 4, or CXCR4, is a chemokine receptor found to promote cancer progression and metastasis of various cancer cell types. To investigate the pharmacology of this receptor, and to further elucidate its role in cancer, novel chemical tools are a necessity. In the present study, using classic medicinal chemistry approaches, small-molecule-based fluorescent probes were designed and synthesized based on previously reported small-molecule antagonists. Here, we report the development of three distinct chemical classes of fluorescent probes that show specific binding to the CXCR4 receptor in a novel fluorescence-based NanoBRET binding assay (pKD ranging 6.6-7.1). Due to their retained affinity at CXCR4, we furthermore report their use in competition binding experiments and confocal microscopy to investigate the pharmacology and cellular distribution of this receptor.

Probing the Allosteric Modulation of P-Glycoprotein: A Medicinal Chemistry Approach Toward the Identification of Noncompetitive P-Gp Inhibitors.

A medicinal chemistry approach combining in silico and in vitro methodologies was performed aiming at identifying and characterizing putative allosteric drug-binding sites (aDBSs) at the interface of the transmembrane- and nucleotide-binding domains (TMD-NBD) of P-glycoprotein. Two aDBSs were identified, one in TMD1/NBD1 and another one in TMD2/NBD2, by means of in silico fragment-based molecular dynamics and characterized in terms of size, polarity, and lining residues. From a small library of thioxanthone and flavanone derivatives, experimentally described to bind at the TMD-NBD interfaces, several compounds were identified to be able to decrease the verapamil-stimulated ATPase activity. An IC50 of 81 ± 6.6 μM is reported for a flavanone derivative in the ATPase assays, providing evidence for an allosteric efflux modulation in P-glycoprotein. Molecular docking and molecular dynamics gave additional insights on the binding mode on how flavanone derivatives may act as allosteric inhibitors.

Therapeutic disruption of RAD52-ssDNA complexation via novel drug-like inhibitors.

RAD52 protein is a coveted target for anticancer drug discovery. Similar to poly-ADP-ribose polymerase (PARP) inhibitors, pharmacological inhibition of RAD52 is synthetically lethal with defects in genome caretakers BRCA1 and BRCA2 (∼25% of breast and ovarian cancers). Emerging structure activity relationships for RAD52 are complex, making it challenging to transform previously identified disruptors of the RAD52-ssDNA interaction into drug-like leads using traditional medicinal chemistry approaches. Using pharmacophoric informatics on the RAD52 complexation by epigallocatechin (EGC), and the Enamine in silico REAL database, we identified six distinct chemical scaffolds that occupy the same physical space on RAD52 as EGC. All six were RAD52 inhibitors (IC50 ∼23-1200 μM) with two of the compounds (Z56 and Z99) selectively killing BRCA-mutant cells and inhibiting cellular activities of RAD52 at micromolar inhibitor concentrations. While Z56 had no effect on the ssDNA-binding protein RPA and was toxic to BRCA-mutant cells only, Z99 inhibited both proteins and displayed toxicity towards BRCA-complemented cells. Optimization of the Z99 scaffold resulted in a set of more powerful and selective inhibitors (IC50 ∼1.3-8 μM), which were only toxic to BRCA-mutant cells. RAD52 complexation by Z56, Z99 and its more specific derivatives provide a roadmap for next generation of cancer therapeutics.

Novel small-molecule modulation of PIEZO1 investigated by conventional and automated patch-clamp.

PIEZO1 trimers form mechanically-activated calcium-permeable non-selective cation channels which are expressed in many types of cells. Mutations in the PIEZO1 gene are associated with generalised lymphatic dysplasia, varicose vein disease, dehydrated hereditary stomatocytosis, malarial resistance and other conditions. Pharmacological modulation of PIEZO1 has been achieved using various compounds, most notably the small-molecule agonist Yoda1 and its inhibitor Dooku1. However, improvements in the potency and pharmacokinetic properties of such compounds are required to increase the usefulness, and small-molecule inhibitors of PIEZO1 are largely lacking. We sought to identify novel small-molecule agonists and inhibitors of mouse and human PIEZO1 channels. Analogues of Yoda1 were generated by medicinal chemistry approaches and a library of 11,200 compounds was screened in a calcium assay using cells overexpressing human PIEZO1. Analogues of hits from the screen were designed and synthesised and then tested for effectiveness in cells overexpressing mouse or human PIEZO1 using an orthogonal calcium assay, conventional (manual) patch-clamp and automated patch-clamp in 384-well format (SyncroPatch 384). We identified Yoda1 analogues with better aqueous solubility and microsomal stability than Yoda1 and improved potency. We identified and validated two novel structurally distinct small-molecule inhibitors of PIEZO1. Automated patch-clamp techniques were refined for activation of PIEZO1 channels by fluid flow in 384-well format. Novel small-molecule modulators of the channels were tested in these automated assays and confirmed as PIEZO1 modulators. We gained preliminary knowledge of the structure-activity relationships and promising compounds for further development. Supported by British Heart Foundation.

Ageladine A, a Bromopyrrole Alkaloid from the Marine Sponge Agelas nakamurai

During the last three decades, secondary metabolites of marine origin have emerged as a significant source of bioactive compounds. Among the marine organisms explored, sponges offer a vast number of metabolites with unique structural diversity and a plethora of biological activities. Ageladine A, a fluorescent bromopyrrole alkaloid isolated from the marine sponge Agelas nakamurai, exhibited matrix metalloproteinase (MMP) inhibitory properties, as well as antiangiogenic activity. Due to this interesting biological profile, Ageladine A became, soon after its discovery, a target for total synthesis. In addition, a significant number of derivatives have been synthesized, and their biological activity was evaluated. The present review highlights all the successful efforts made towards the synthesis of Ageladine A. Furthermore, all the medicinal chemistry approaches to identify and assess new more potent inhibitors and to elucidate the structural features responsible for the activity are described.

Medicinal chemistry approaches to target the MNK-eIF4E axis in cancer.

Aberrant translation of proteins that promote cell proliferation is an essential factor that defines oncogenic processes and cancer. The process for ribosomal translation of proteins from mRNA requires an essential initiation step which is controlled by the protein eIF4E, which binds the RNA 5'-cap and forms the eIF4F complex that subsequently translates protein. Typically, eIF4E is activated by phosphorylation on Ser209 by MNK1 and MNK2 kinases. Substantial work has shown that eIF4E and MNK1/2 are dysregulated in many cancers and this axis has therefore become an active area of interest for developing new cancer therapeutics. This review summarizes and discusses recent work to develop small molecules that target different steps in the MNK-eIF4E axis as potential cancer therapeutics. The aim of this review is to cover the breadth of different molecular approaches being taken and the medicinal chemistry basis for their optimization and testing as new cancer therapeutics.

Antimalarials Targeting the Malaria Parasite Cation ATPase P. falciparum ATP4 (PfATP4).

Malaria, caused by parasites of the Plasmodium species and transmitted through the bites of infected female Anopheles mosquitoes, is still a fatal and dangerous disease in mainly tropical and subtropical regions. The widespread resistance of P. falciparum to antimalarial drugs forces the search for new molecules with activity against this parasite. While a large number of compounds can inhibit P. falciparum growth in vitro, unfortunately, only a limited number of targets have been identified so far. One of the most promising approaches has been the identification of effective inhibitors of P-type cation-transporter ATPase 4 (PfATP4) in P. falciparum. PfATP4 is a Na+ efflux pump that maintains a low cytosolic Na+ in the parasite. Thus, upon treatment with PfATP4 inhibitors, the parasites rapidly accumulate Na+, which triggers processes leading to parasite death. PfATP4 is present in the parasite plasma membrane but is absent in mammals; its exclusivity thus makes it a good antimalarial drug target. The current review presents PfATP4 function in the context of the pharmacological influence of its inhibitors. In addition, compounds with inhibitory activities belonging to spiroindolones, dihydroisoquinolones, aminopyrazoles, pyrazoleamides, and 4-cyano-3-methylisoquinolines, are also reviewed. Particular emphasis is placed on the results of preclinical and clinical studies in which their effectiveness was tested. PfATP4-associated antimalarials rapidly cleared parasites in mouse models and preliminary human trials. These findings highlight a fundamental biochemical mechanism sensitive to pharmacological intervention that can form a medicinal chemistry approach for antimalarial drug design to create new molecules with potent PfATP4 inhibitory activity.

Synthesis and Atimicrobial Evaluation of Deuterated Analogues of Metronidazole

The discovery of novel therapeutic molecules is always difficult, and there are a variety of methodologies that use the most diverse and innovative medicinal chemistry approaches. One such approach is the deuteration technique: Deuteration is the process of substituting deuterium for hydrogen in a molecule. When compared to the drug molecule, its deuterated analogues may retain the features of the original molecule and, in some cases, improve its pharmacological activity, with fewer side effects and lower toxicity. Metronidazole is a commonly used antibiotic to treat anaerobic bacterial infections, protozoal and microaerophilic bacterial infections. Metronidazole has a half-life of 6.5 ± 2.9 hours. A number of studies have recently been conducted on the selective substitution of hydrogen with deuterium. which increases the bond strength, increasing the biological half-life and, consequently, the drug's metabolic stability also increases. In an attempt to address metronidazole's drawbacks and clinical resistance, deuterated metronidazole was synthesised, characterised, and tested for antibacterial, antifungal, and anti-tubercular activities. The metronidazole and its deuterated compound showed equipotent antifungal activity and aerobic antibacterial activity. Also, when compared with the non-deuterated compound, deuterated metronidazole exhibited better anaerobic antibacterial and anti-tubercular activity.

Discovery and Development of a Selective Inhibitor of the ER Resident Chaperone Grp78

A recent study illustrated that a fluorescence polarization assay can be used to identify substrate-competitive Hsp70 inhibitors that can be isoform-selective. Herein, we use that assay in a moderate-throughput screen and report the discovery of a druglike amino-acid-based inhibitor with reasonable specificity for the endoplasmic reticular Hsp70, Grp78. Using traditional medicinal chemistry approaches, the potency and selectivity were further optimized through structure-activity relationship (SAR) studies in parallel assays for six of the human Hsp70 isoforms. The top compounds were all tested against a panel of cancer cell lines and disappointingly showed little effect. The top-performing compound, 8, was retested using a series of endoplasmic reticulum (ER) stress-inducing agents and found to synergize with these agents. Finally, 8 was tested in a spheroid tumor model and found to be more potent than in two-dimensional models. The optimized Grp78 inhibitors are the first reported isoform-selective small-molecule-competitive inhibitors of an Hsp70-substrate interaction.

Small-molecule metabolites in SARS-CoV-2 treatment: a comprehensive review.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has quickly spread all over the world. In this respect, traditional medicinal chemistry, repurposing, and computational approaches have been exploited to develop novel medicines for treating this condition. The effectiveness of chemicals and testing methods in the identification of new promising therapies, and the extent of preparedness for future pandemics, have been further highly advantaged by recent breakthroughs in introducing noble small compounds for clinical testing purposes. Currently, numerous studies are developing small-molecule (SM) therapeutic products forinhibiting SARS-CoV-2 infection and replication, as well as managing the disease-related outcomes. Transmembrane serine protease (TMPRSS2)-inhibiting medicinal products can thus prevent the entry of the SARS-CoV-2 into the cells, and constrain its spreading along with the morbidity and mortality due to the coronavirus disease 2019 (COVID-19), particularly when co-administered with inhibitors such as chloroquine (CQ) and dihydroorotate dehydrogenase (DHODH). The present review demonstrates that the clinical-stage therapeutic agents, targeting additional viral proteins, might improve the effectiveness of COVID-19 treatment if applied as an adjuvant therapy side-by-side with RNA-dependent RNA polymerase (RdRp) inhibitors.

Nucleobase-modified nucleosides and nucleotides: Applications in biochemistry, synthetic biology, and drug discovery.

. DNA is often referred to as the "molecule of life" since it contains the genetic blueprint for all forms of life on this planet. The core building blocks composing DNA are deoxynucleotides. While the deoxyribose sugar and phosphate group are ubiquitous, it is the composition and spatial arrangement of the four natural nucleobases, adenine (A), cytosine (C), guanine (G), and thymine (T), that provide diversity in the coding information present in DNA. The ability of DNA to function as the genetic blueprint has historically been attributed to the formation of proper hydrogen bonding interactions made between complementary nucleobases. However, recent chemical and biochemical studies using nucleobase-modified nucleotides that contain "non-hydrogen bonding" functional groups have challenged many of the dogmatic views for the necessity of hydrogen-bonding interactions for DNA stability and function. Based on years of exciting research, this area has expanded tremendously and is thus too expansive to provide a comprehensive review on the topic. As such, this review article provides an opinion highlighting how nucleobase-modified nucleotides are being applied in diverse biomedical fields, focusing on three exciting areas of research. The first section addresses how these analogs are used as mechanistic probes for DNA polymerase activity and fidelity during replication. This section outlines the synthetic logic and medicinal chemistry approaches used to replace hydrogen-bonding functional groups to examine the contributions of shape/size, nucleobase hydrophobicity, and pi-electron interactions. The second section extends these mechanistic studies to provide insight into how nucleobase-modified nucleosides are used in synthetic biology. One example is through expansion of the genetic code in which changing the composition of DNA makes it possible to site-specifically incorporate unnatural amino acids bearing unique functional groups into enzymes and receptors. The final section describes results of pre-clinical studies using nucleobase-modified nucleosides as potential therapeutic agents against diseases such as cancer.

Estrogen Receptor-α Targeting: PROTACs, SNIPERs, Peptide-PROTACs, Antibody Conjugated PROTACs and SNIPERs.

Targeting selective estrogen subtype receptors through typical medicinal chemistry approaches is based on occupancy-driven pharmacology. In occupancy-driven pharmacology, molecules are developed in order to inhibit the protein of interest (POI), and their popularity is based on their virtue of faster kinetics. However, such approaches have intrinsic flaws, such as pico-to-nanomolar range binding affinity and continuous dosage after a time interval for sustained inhibition of POI. These shortcomings were addressed by event-driven pharmacology-based approaches, which degrade the POI rather than inhibit it. One such example is PROTACs (Proteolysis targeting chimeras), which has become one of the highly successful strategies of event-driven pharmacology (pharmacology that does the degradation of POI and diminishes its functions). The selective targeting of estrogen receptor subtypes is always challenging for chemical biologists and medicinal chemists. Specifically, estrogen receptor α (ER-α) is expressed in nearly 70% of breast cancer and commonly overexpressed in ovarian, prostate, colon, and endometrial cancer. Therefore, conventional hormonal therapies are most prescribed to patients with ER + cancers. However, on prolonged use, resistance commonly developed against these therapies, which led to selective estrogen receptor degrader (SERD) becoming the first-line drug for metastatic ER + breast cancer. The SERD success shows that removing cellular ER-α is a promising approach to overcoming endocrine resistance. Depending on the mechanism of degradation of ER-α, various types of strategies of developed.

Assay Development and Small Molecule Screens for Inhibitors of an RNA Binding Protein Implicated in Hematologic Malignancies

RNA-binding proteins (RBPs) play a critical role in regulating RNA splicing, polyadenylation, and translation. RBPs typically contain discrete RNA-binding domains (RBDs) that bind to specific nucleotide motifs. Given their important role in regulating gene expression, RBPs are often mutated or aberrantly expressed in a variety of malignancies, particularly hematological cancers. As a result, RBPs appear to be excellent therapeutic targets. However, given that most RBPs lack enzymatic activity, it has been challenging to develop targeted therapies. Over the past several years we and others have used genetic and molecular biology approaches to understand the consequence of aberrant expression of the poly-C RBP, heterogeneous ribonucleoprotein K (hnRNP K), and determine the mechanisms by which it drives hematologic malignancies (Cancer Cell, 2015; NEJM, 2013, JNCI, 2020; and BioRxiv, 2021). To develop a pharmacologic strategy to target hnRNP K's RNA binding activities, we developed a robust fluorescent anisotropy (FA) assay to evaluate hnRNP K's ability to bind target transcripts that are critical for tumorigenesis; such as, BRD4, MYC, RUNX1, and 7SK. We next performed small molecule screens using ~80,000 compounds and identified 34 compounds that inhibited hnRNP K's ability to bind RNA targets. To gain a deeper understanding of whether these compounds directly interact with hnRNP K to block its RNA binding functions, we performed SYPRO-orange thermal shift assays. Here we identified compounds that bound hnRNP K directly and altered hnRNP K thermostability. To evaluate association rate, dissociation rate, and KD constants, we next performed SPR assays. These experiments identified compounds bound hnRNP K with affinities between (350nM and 20uM). We next performed cell-based studies, such as CETSA. Here we identified compounds that crossed the plasma membrane, directly engaged hnRNP K in vivo, and significantly stabilized hnRNP K. Molecular analyses revealed that cells treated with these lead compounds have reduced c-Myc expression, a direct mRNA target of hnRNP K. To develop a granular understanding of how these hnRNP K-small molecule interactions impact growth and cell survival, we performed high-throughput cell-based assays. These studies demonstrated that cells with inducible hnRNP K overexpression were sensitive to these small molecules. To begin medicinal chemistry approaches and to understand each compound's chemical requirements for binding to hnRNP K, we scanned molecules for reactive moieties. Here, we identified two compounds possessing amide groups within two carbons of either a furan or pyrrole moiety. These compounds, #20 and #23, respectively, were then chemically modified and FA assays were repeated in the presence of target mRNAs. Here, we observed that both the furan and isoxazole ring of compound #20 and pyrrole rings of compound #23 are critical for hnRNP K interactions, while the cyclopropane moiety on compound #23 is dispensable. Further analyses revealed additional structural analogs with either increased or decreased affinities and these results have been critical to our understanding of the importance of each moiety. Currently, we are using cryo-EM to evaluate positional interactions between hnRNP K (hnRNP K's RBDs) and these small molecules. These results will be used to further our medicinal chemistry approaches to generate more potent analogs. Collectively, our results demonstrate that hnRNP K functions can be directly inhibited by small molecules that block its RNA binding functions and demonstrates that the development of effective therapies for RBPs is possible. 1. hnRNP K Is a Haploinsufficient Tumor Suppressor that Regulates Proliferation and Differentiation Programs in Hematologic Malignancies. Cancer Cell, 2015. 28(4): p. 486-499. 2. Cancer Genome Atlas Research Network, Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N Engl J Med. 2013 368(22):2059-74. 3. Uncovering the Role of RNA-Binding Protein hnRNP K in B-Cell Lymphomas. JNCI: Journal of the National Cancer Institute, 2020. 4. Heterogeneous nuclear ribonucleoprotein K is overexpressed in acute myeloid leukemia and causes myeloproliferative disease in mice via altered Runx1 splicing. BioRxiv. doi: https://doi.org/10.11.02.05.429385, 2021 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

LBSAT142 Discovery And Characterization Of A Potent And Orally Bioavailable Parathyroid Hormone Receptor Type-1 (PTHR1) Antagonist For The Treatment Of Hypercalcemia

Abstract Hypercalcemia is a common disorder defined as a serum calcium concentration higher than the normal range of 8.5 - 10.5 mg/dL. The most common causes of hypercalcemia are over-secretion of parathyroid hormone (PTH) from one or more enlarged parathyroid glands, which can lead to primary hyperparathyroidism (PHPT), or over-secretion of parathyroid hormone related protein (PTHrP) from a cancerous tumor, which leads to humoral hypercalcemia of malignancy (HHM). PTH is an 84 amino acid peptide that regulates calcium and phosphate homeostasis through activation of its receptor, PTHR1. PTHrP has close homology to the N-terminal region of PTH and also activates PTHR1, inducing similar biological actions. Activation of PTHR1, a class B G-protein coupled receptor expressed in bone and kidney, leads to an increase in cAMP and PKA activation, inducing gene expression changes of important modulators of bone homeostasis. In the kidney, it increases renal phosphate excretion and calcium reabsorption. Hyperactivation of PTHR1 due to high levels of either PTH or PTHrP results in increased calcium release from the bone matrix, as well as increased calcium reabsorption in the kidney, causing hypercalcemia. Surgery is the first line therapy for PHPT, but patients that cannot or choose not to have surgery are prescribed calcimimetics and/or bisphosphonates. Calcimimetics decrease circulating calcium levels but have no effect on bone homeostasis, while bisphosphonates improve bone homeostasis but have little effect on circulating calcium levels. HHM patients are prescribed bisphosphonates or denosumab, both of which possess undesirable side effects. We hypothesize that blocking PTH/PTHrP action via a PTHR1 antagonist may provide an improved therapeutic mechanism to treat PHPT and HHM, and potentially other diseases of hypercalcemia. Using an iterative medicinal chemistry approach, Crinetics has identified several nonpeptide PTHR1 antagonists via both binding and functional in vitro assays. One of these compounds, Antagonist 1 (ANT-1), has low nanomolar binding affinity for both human and rat PTHR1 and is potent in human, monkey, rat, mouse and canine PTHR1 functional antagonist assays in vitro. ANT-1 has good oral exposure in preclinical species and desirable drug-like properties, including lack of inhibition of cytochromes P450 and the hERG ion channel, and stability in liver microsomes. In rat models of PTH- and PTHrP-induced hypercalcemia, ANT-1 dose-dependently suppresses ionized blood calcium, providing support that a nonpeptide PTH antagonist has potential use as an effective therapeutic for hypercalcemia caused by PHPT and HHM. Currently, ANT-1 and other potential candidate molecules are being evaluated in a battery of safety studies to select the optimal molecule(s) suitable for evaluation in human clinical trials. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.

Recent Drug Development and Medicinal Chemistry Approaches for the Treatment of SARS-CoV-2 Infection and COVID-19.

COVID‐19, caused by SARS‐CoV‐2 infection, continues to be a major public health crisis around the globe. Development of vaccines and the first cluster of antiviral drugs has brought promise and hope for prevention and treatment of severe coronavirus disease. However, continued development of newer, safer, and more effective antiviral drugs are critically important to combat COVID‐19 and counter the looming pathogenic variants. Studies of the coronavirus life cycle revealed several important biochemical targets for drug development. In the present review, we focus on recent drug design and medicinal chemistry efforts on small molecule drug discovery including the development of nirmatrelvir that targets viral protein synthesis and remdesivir and molnupiravir that target viral RdRp. These are recent FDA approved drugs for the treatment of COVID‐19.

Design, Synthesis, and Biological Activity of New CB2 Receptor Ligands: from Orthosteric and Allosteric Modulators to Dualsteric/Bitopic Ligands.

The design of dualsteric/bitopic agents as single chemical entities able to simultaneously interact with both the orthosteric and an allosteric binding site represents a novel approach in medicinal chemistry. Biased dualsteric/bitopic agents could enhance certain signaling pathways while diminishing the others that cause unwanted side effects. We have designed, synthesized, and functionally characterized the first CB2R heterobivalent bitopic ligands. In contrast to the parent orthosteric compound, our bitopic ligands selectively target CB2R versus CB1R and show a functional selectivity for the cAMP signaling pathway versus βarrestin2 recruitment. Moreover, the most promising bitopic ligand FD-22a displayed anti-inflammatory activity in a human microglial cell inflammatory model and antinociceptive activity in vivo in an experimental mouse model of neuropathic pain. Finally, computational studies clarified the binding mode of these compounds inside the CB2R, further confirming their bitopic nature.

Abstract 4233: Generation of a Bicycle NK-TICA™, a novel NK cell engaging molecule designed to induce targeted tumor cytotoxicity

Abstract The tumor specific activation of natural killer (NK) cells is an area of active investigation in immune oncology, but to date has relied on complex biologic modalities (e.g., antibodies, fusion proteins, or cell therapies). NK cells are highly responsive immune cells that can detect and eliminate tumor cells and bridge innate to adaptive immune responses. Bicycles® are small (ca.1.5kDa), chemically synthetic, structurally constrained peptides discovered via phage display and optimized using structure-driven design and medicinal chemistry approaches. We have applied the Bicycle platform technology to identify Bicycles® that bind specifically to the key activating receptor, NKp46. When chemically coupled to tumor antigen binding Bicycles, this results in highly potent, antigen-dependent receptor activation and NK cell activation. We term this new class of fully synthetic molecules NK-TICAs and we will describe herein their discovery and evaluation.We demonstrate potent, selective binding of our Bicycles to receptor-expressing cells and the capability of the bifunctional molecule to induce NK cell function in vitro. With Bicycle’s novel NK-TICA™ compound, we demonstrate the engagement of NK cells, the specific activation and function of NK cells, and enhanced tumor cytotoxicity in a tumor target- and dose-dependent manner.In conclusion, NK-TICAs drive NK cell-mediated tumor cell killing and cytokine production in vitro and as such have the potential to catalyze the development of durable anti-tumor immunity in tumor types not well served by current therapies. We hypothesize that utilization of Bicycle NK-TICA™ as a multifunctional immune cell engager will promote the modulation of NK cells, as well as the infiltration and anti-tumor activity of NK cells in solid tumors. The data presented here provide initial proof of concept for the application of our Bicycle technology to drive NK cell-mediated tumor immunity. Citation Format: Fay J. Dufort, Christopher J. Leitheiser, Gemma Mudd, Julia Kristensson, Alexandra Rezvaya, Katie Gaynor, Sandra Uhlenbroich, Liudvikas Urbonas, Heather Scott, Liuhong Chen, Helen Harrison, Michael Skynner, Kevin McDonnell, Philip E. Brandish, Nicholas Keen. Generation of a Bicycle NK-TICA™, a novel NK cell engaging molecule designed to induce targeted tumor cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4233.

Abstract 2138: Creating next-generation biologics using a novel chemistry platform technology

Abstract Bright Peak Therapeutics is developing a portfolio of differentiated biotherapeutics using chemistry for applications in immuno-oncology and autoimmune diseases. Our unique chemical protein synthesis and engineering platform allows us to fine-tune cytokines and other proteins to interrogate and modulate biological functions by incorporating new functional modifications. Standard recombinant bacterial or cellular expression systems used to produce proteins are largely restricted to using canonical amino acids, which limits access to diverse modifications that can bestow additional functional properties. With chemical protein synthesis technology, canonical and non-canonical modifications including conjugation handles can be easily introduced, ultimately enabling a medicinal chemistry approach for engineering cytokine structures. Enhanced cytokines with differentiated biology developed using this approach can be further elaborated by conjugating to a diverse array of molecules. We first applied our technology platform to identify BPT-143, a rationally designed enhanced IL-2 variant currently in IND-enabling studies. BPT-143 is engineered to have enhanced binding to IL2Rβ and no binding to IL2Rα for improved efficacy and safety independent of the conjugation to a half-life extending 30 kDa PEG. The chemical synthesis technology is robust, reproducible, and scalable. We are applying our platform to enhance a number of other cytokines for use in immuno-oncology and autoimmune diseases. Additionally, our synthetically engineered cytokines can be easily conjugated to monoclonal antibodies as ‘payloads’ using a distinct chemical conjugation technology. A rapid and simple chemical process allows site-selective conjugation of our engineered cytokines to existing antibodies ‘as-is’ to generate novel immunocytokines (IC). This ‘off-the-shelf’ approach is orthogonal to recombinant fusion methods to create ICs and does not require complex recombinant protein expression optimization and lengthy cell-line development. Moreover, it allows rapid screening of cytokine payloads in a structure-activity relationship (SAR) format to identify dual-targeting ICs with precisely tailored properties to generate the desired biological effect. We have prepared a number of ICs including anti-PD-1/IL-2 ICs with various drug-antibody ratio (DAR) and conjugation sites within the antibody. We will provide an overview of the platform technology and present highlights of its application for discovery and development of designer therapeutic cytokines and ICs. Citation Format: Vijaya R. Pattabiraman, Matilde Arévalo Ruiz, Régis Boehringer, Benoit Hornsperger, Roy Meoded, Robert C. Tam, Bertolt Kreft. Creating next-generation biologics using a novel chemistry platform technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2138.

Chemical approaches towards installation of rare functional groups in bacterial surface glycans.

Bacterial surface glycans perform a diverse and important set of biological roles, and have been widely used in the treatment of bacterial infectious diseases. The majority of bacterial surface glycans are decorated with diverse rare functional groups, including amido, acetamidino, carboxamido and pyruvate groups. These functional groups are thought to be important constituents for the biological activities of glycans. Chemical synthesis of glycans bearing these functional groups or their variants is essential for the investigation of structure-activity relationships by a medicinal chemistry approach. To date, a broad choice of synthetic methods is available for targeting the different rare functional groups in bacterial surface glycans. This article reviews the structures of naturally occurring rare functional groups in bacterial surface glycans, and the chemical methods used for installation of these groups.