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New antibacterial compounds are urgently needed, especially for infections caused by the top-priority Gram-negative bacteria that are increasingly difficult to treat. Lipid A is a key component of the Gram-negative outer membrane and the LpxH enzyme plays an important role in its biosynthesis, making it a promising antibacterial target. Inspired by previously reported ortho-N-methyl-sulfonamidobenzamide-based LpxH inhibitors, novel benzamide substitutions were explored in this work to assess their in vitro activity. Our findings reveal that maintaining wild-type antibacterial activity necessitates removal of the N-methyl group when shifting the ortho-N-methyl-sulfonamide to the meta-position. This discovery led to the synthesis of meta-sulfonamidobenzamide analogs with potent antibacterial activity and enzyme inhibition. Moreover, we demonstrate that modifying the benzamide scaffold can alter blocking of the cardiac voltage-gated potassium ion channel hERG. Furthermore, two LpxH-bound X-ray structures show how the enzyme-ligand interactions of the meta-sulfonamidobenzamide analogs differ from those of the previously reported ortho analogs. Overall, our study has identified meta-sulfonamidobenzamide derivatives as promising LpxH inhibitors with the potential for optimization in future antibacterial hit-to-lead programs.

In vitro screening of large compound libraries with automated high-throughput screening is expensive and time-consuming and requires dedicated infrastructures. Conversely, the selection of DNA-encoded chemical libraries (DECLs) can be rapidly performed with routine equipment available in most laboratories. In this study, we identified novel inhibitors of SARS-CoV-2 main protease (M^pro) through the affinity-based selection of the DELopen library (open access for academics), containing 4.2 billion compounds. The identified inhibitors were peptide-like compounds containing an N-terminal electrophilic group able to form a covalent bond with the nucleophilic Cys145 of M^pro, as confirmed by x-ray crystallography. This DECL selection campaign enabled the discovery of the unoptimized compound SLL11 (IC₅₀ = 30 nM), proving that the rapid exploration of large chemical spaces enabled by DECL technology allows for the direct identification of potent inhibitors avoiding several rounds of iterative medicinal chemistry. As demonstrated further by x-ray crystallography, SLL11 was found to adopt a highly unique U-shaped binding conformation, which allows the N-terminal electrophilic group to loop back to the S1′ subsite while the C-terminal amino acid sits in the S1 subsite. MP1, a close analog of SLL11, showed antiviral activity against SARS-CoV-2 in the low micromolar range when tested in Caco-2 and Calu-3 (EC₅₀ = 2.3 µM) cell lines. As peptide-like compounds can suffer from low cell permeability and metabolic stability, the cyclization of the compounds will be explored in the future to improve their antiviral activity.

Drugs targeting SARS-CoV-2 could have saved millions of lives during the COVID-19 pandemic, and it is now crucial to develop inhibitors of coronavirus replication in preparation for future outbreaks. We explored two virtual screening strategies to find inhibitors of the SARS-CoV-2 main protease in ultralarge chemical libraries. First, structure-based docking was used to screen a diverse library of 235 million virtual compounds against the active site. One hundred top-ranked compounds were tested in binding and enzymatic assays. Second, a fragment discovered by crystallographic screening was optimized guided by docking of millions of elaborated molecules and experimental testing of 93 compounds. Three inhibitors were identified in the first library screen, and five of the selected fragment elaborations showed inhibitory effects. Crystal structures of target-inhibitor complexes confirmed docking predictions and guided hit-to-lead optimization, resulting in a noncovalent main protease inhibitor with nanomolar affinity, a promising in vitro pharmacokinetic profile, and broad-spectrum antiviral effect in infected cells.

Oligopeptide boronates with a lipophilic tail are known to inhibit the type I signal peptidase in E. coli, which is a promising drug target for developing novel antibiotics. Antibacterial activity depends on these oligopeptides having a cationic modification to increase their permeation. Unfortunately, this modification is associated with cytotoxicity, motivating the need for novel approaches. The sulfonimidamide functionality has recently gained much interest in drug design and discovery, as a means of introducing chirality and an imine-handle, thus allowing for the incorporation of additional substituents. This in turn can tune the chemical and biological properties, which are here explored. We show that introducing the sulfonimidamide between the lipophilic tail and the peptide in a series of signal peptidase inhibitors resulted in antibacterial activity, while the sulfonamide isostere and previously known non-cationic analogs were inactive. Additionally, we show that replacing the sulfonamide with a sulfonimidamide resulted in decreased cytotoxicity, and similar results were seen by adding a cationic sidechain to the sulfonimidamide motif. This is the first report of incorporation of the sulfonimidamide functional group into bioactive peptides, more specifically into antibacterial oligopeptides, and evaluation of its biological effects.

Tick-borne encephalitis (TBE) is a severe neurological disorder caused by tick-borne encephalitis virus (TBEV), a member of the Flavivirus genus. Currently, two vaccines are available in Europe against TBEV. However, TBE cases have been rising in Sweden for the past twenty years, and thousands of cases are reported in Europe, emphasizing the need for antiviral treatments against this virus. The NS2B-NS3 protease is essential for flaviviral life cycle and has been studied as a target for the design of inhibitors against several well-known flaviviruses, but not TBEV. In the present study, Compound 86, a known tripeptidic inhibitor of dengue (DENV), West Nile (WNV) and Zika (ZIKV) proteases, was predicted to be active against TBEV protease using a combination of in silico techniques. Further, Compound 86 was found to inhibit recombinant TBEV protease with an IC50 = 0.92 mu M in the in vitro enzymatic assay. Additionally, two more peptidic analogues were synthetized and they displayed inhibitory activities against both TBEV and ZIKV proteases. In particular, Compound 104 inhibited ZIKV protease with an IC50 = 0.25 mu M. These compounds represent the first reported inhibitors of TBEV protease to date and provides valuable information for the further development of TBEV as well as pan-flavivirus protease inhibitors.

Of painful conditions, somatic pain of acute nociceptive origin can be effectively managed clinically, while neuropathic pain of chronic neuropathy origin is difficult to control. For molecules involved in pain sensation, substance P (SP) is algesic, exacerbating painful sensation, while its amino-terminal fragment, heptapeptide SP(1-7), confers biological activities different from its full-length parent neuropeptide precursor. We previously demonstrated SP(1-7) interaction with pain processing to alleviate chronic pain. Here we evaluated SP(1-7) and its C-terminal amidated analogue SP(1-7) amide, together with SP and opioid agonist DAMGO. We tested mouse behaviors of both acute somatic pain in tail-flick latency assay, and neuropathic pain in sciatic nerve injury model of chronic constriction injury (CCI). DAMGO produced dose-dependent analgesia for somatic pain as expected, so did both SP(1-7) and its analogue SP(1-7) amide, while SP yielded the opposite effect of algesia, in a phenomenon we termed `contrintus', meaning 'opposite from within' to denote that two peptides of the same origin (SP and its metabolic fragment SP(1-7)) produced opposite effects. In CCI model, DAMGO showed a general reduction in allodynia sensitivity for both nerve-injured and normal paws, without selective effect for neuropathic pain, consistent with clinical observation that opioids are less effective for chronic neuropathic pain. On the other hand, both SP(1-7) and SP(1-7) amide displayed dose-dependent anti-allodynia effect that is selective for neuropathic pain. These findings suggest that SP(1-7) and its analogue may be useful for developing pharmaceuticals to treat neuropathic pain.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus and infection by ZIKV Asian lineage is known to cause fetal brain anomalies and Guillain-Barrés syndrome. The WHO declared ZIKV a global public health emergency in 2016. However, currently neither vaccines nor antiviral prophylaxis/treatments are available. In this study, we report the identification of a C2-symmetric diol-based Human immunodeficiency virus type-1 (HIV) protease inhibitor active against ZIKV NS2B-NS3 protease. The compound, referred to as 9b, was identified by in silico screening of a library of 6265 protease inhibitors. Molecular dynamics (MD) simulation studies revealed that compound 9b formed a stable complex with ZIKV protease. Interaction analysis of compound 9b's binding pose from the cluster analysis of MD simulations trajectories predicted that 9b mostly interacted with ZIKV NS3. Although designed as an aspartyl protease inhibitor, compound 9b was found to inhibit ZIKV serine protease in vitro with IC₅₀ = 143.25 ± 5.45 µM, in line with the in silico results. Additionally, linear interaction energy method (LIE) was used to estimate binding affinities of compounds 9b and 86 (a known panflavivirus peptide hybrid with IC₅₀ = 1.64 ± 0.015 µM against ZIKV protease). The LIE method correctly predicted the binding affinity of compound 86 to be lower than that of 9b, proving to be superior to the molecular docking methods in scoring and ranking compounds. Since most of the reported ZIKV protease inhibitors are positively charged peptide-hybrids, with our without electrophilic warheads, compound 9b represents a less polar and more drug-like non-peptide hit compound useful for further optimization.Communicated by Ramaswamy Sarma.

Macrocyclic analogues of the linear hexapeptide, angiotensin IV (AngIV) have proved to be potent inhibitors of insulin-regulated aminopeptidase (IRAP, oxytocinase, EC 3.4.11.3). Along with higher affinity, macrocycles may also offer better metabolic stability, membrane permeability and selectivity, however predicting the outcome of particular cycle modifications is challenging. Here we describe the development of a series of macrocyclic IRAP inhibitors with either disulphide, olefin metathesis or lactam bridges and variations of ring size and other functionality. The binding mode of these compounds is proposed based on molecular dynamics analysis. Estimation of binding affinities (∆G) and relative binding free energies (∆∆G) with the linear interaction energy (LIE) method and free energy perturbation (FEP) method showed good general agreement with the observed inhibitory potency. Experimental and calculated data highlight the cumulative importance of an intact N-terminal peptide, the specific nature of the macrocycle, the phenolic oxygen and the C-terminal functionality.

Many synthetic routes have been explored to make small molecule sulfonimidamides (SIAs), however, its introduction into larger molecules such as oligopeptides has not been studied before. We herein demonstrate three alternative and complementary methods for synthesis of SIA based pseudopeptides, on solid phase, using both on and off‐resin SIA‐synthesis, via sulfonimidoyl chlorides from sulfonamides, in high conversion. Beside evaluation of various resins such as 2‐CTC, Wang, and Rink amide‐ChemMatrix, the possibilities to further N‐functionalize and cyclize the SIA functionality on solid support are shown. The diastereomers of SIA containing pseudopeptides could in most cases be separated using normal reverse phase preparative HPLC. The solid phase SIA methodology has many advantages when it comes to handling and purification as compared to in solution, and will therefore enable exploration of the SIA group as isosteric substitutions and peptidomimetic building blocks in the development of drug‐like pseudopeptides in many ways. Of particular note these approaches facilitate combinatorial library synthesis as demonstrated herein.

Herein, we report the synthesis of novel sulfonimidamides (SIAs) based on amino acid building blocks using a one-pot method from tert-butyldiphenylsilyl-protected (TBDPS) sulfonamides, as well as exploration of orthogonal deprotection strategies. Among the several protecting groups investigated, TBDPS showed higher conversion, allowed UV detection and simple diastereomeric separation; in particular in combination with amino acid tert-butyl esters. Moreover, we applied the present method to synthesize cyclic five-membered acyl sulfonimidamides in two steps. The described synthesis of SIA-based amino acid building blocks in combination with the orthogonal protection groups provide access to unnatural amino acid building blocks useful for further incorporation into larger molecules, such as peptide-based transition-state analogues and peptidomimetics. The chirality of the SIA group, as well as its additional point of diversity provided by the extra NH group, creates opportunities for the development of unique compound libraries that explore new chemical space, which is of considerable importance for the pharmaceutical and agrochemical industry.