Synthesis and penicillin‐binding protein inhibitory assessment of dipeptidic 4‐phenyl‐β‐lactams from α‐amino acid‐derived imines

Monocyclic beta-lactams revive the research field on antibiotics, which are threatened by the emergence of resistant bacteria. A six-step synthetic route was developed, providing easy access to new 3-amino-1-carboxymethyl-4-phenyl-beta-lactams, of which the penicillin-binding protein (PBP) inhibitory potency was demonstrated biochemically

Anthranilic Acid Inhibitors of Undecaprenyl Pyrophosphate Synthase (UppS), an Essential Enzyme for Bacterial Cell Wall Biosynthesis

We report the successful implementation of virtual screening in the discovery of new inhibitors of undecaprenyl pyrophosphate synthase (UppS) from Escherichia coli. UppS is an essential enzyme in the biosynthesis of bacterial cell wall. It catalyzes the condensation of farnesyl pyrophosphate (FPP) with eight consecutive isopentenyl pyrophosphate units (IPP), in which new cis-double bonds are formed, to generate undecaprenyl pyrophosphate. The latter serves as a lipid carrier for peptidoglycan synthesis, thus representing an important target in the antibacterial drug design. A pharmacophore model was designed on a known bisphosphonate BPH-629 and used to prepare an enriched compound library that was further docked into UppS conformational ensemble generated by molecular dynamics experiment. The docking resulted in three anthranilic acid derivatives with promising inhibitory activity against UppS. Compound 2 displayed high inhibitory potency (IC50 = 25 μM) and good antibacterial activity against E. coli BW25113 ΔtolC strain (MIC = 0.5 μg/mL)

In silico identification, synthesis and biological evaluation of novel tetrazole inhibitors of MurB

In the context of antibacterial drug discovery resurgence, novel therapeutic targets and new compounds with alternative mechanisms of action are of paramount importance. We focused on UDP-N- acetylenolpyruvylglucosamine reductase (i.e. MurB), an underexploited target enzyme that is involved in early steps of bacterial peptidoglycan biosynthesis. On the basis of the recently reported crystal structure of MurB in complex with NADP+ , a pharmacopohore model was generated and used in a virtual screening campaign with combined structure-based and ligand-based approaches. In order to explore chemical space around hit compounds, further similarity search and organic synthesis was employed to obtain several compounds with micromolar IC50 values on MurB. The best inhibitors in the reported series of 5-substituted tetrazol-2-yl acetamides were compounds 13, 26 and 30 with IC50 values of 34, 28 and 25 µM, respectively. None of the reported compounds possessed in vitro antimicrobial activity against S. aureus and E. coli

Design and synthesis of amino-substituted N-arylpiperidinyl-based inhibitors of the (immuno)proteasome

The constitutive proteasome and the immunoproteasome represent validated targets for pharmacological intervention in the context of various diseases, such as cancer, inflammation, and autoimmune diseases. The development of novel chemical scaffolds of non-peptidic nature, capable of inhibiting different catalytically active subunits of both isoforms, is a viable approach against these diseases. Such compounds are also useful as leads for the development of biochemical probes that enable the studies of the roles of both isoforms in various biological contexts. Here, we present a ligand-based computational design of (immuno)proteasome inhibitors, which resulted in the amino-substituted N-arylpiperidine-based compounds that can inhibit different subunits of the (immuno)proteasome in the low micromolar range. The compounds represent a useful starting point for further structure-activity relationship studies that will, hopefully, lead to non-peptidic compounds that could be used in pharmacological and biochemical studies of both proteasomes

Ensemble Docking Coupled to Linear Interaction Energy Calculations for Identification of Coronavirus Main Protease (3CLpro) Non-Covalent Small-Molecule Inhibitors

SARS-CoV-2, or severe acute respiratory syndrome coronavirus 2, represents a new strain of Coronaviridae. In the closing 2019 to early 2020 months, the virus caused a global pandemic of COVID-19 disease. We performed a virtual screening study in order to identify potential inhibitors of the SARS-CoV-2 main viral protease (3CLpro or Mpro). For this purpose, we developed a novel approach using ensemble docking high-throughput virtual screening directly coupled with subsequent Linear Interaction Energy (LIE) calculations to maximize the conformational space sampling and to assess the binding affinity of identified inhibitors. A large database of small commercial compounds was prepared, and top-scoring hits were identified with two compounds singled out, namely 1-[(R)-2-(1,3-benzimidazol-2-yl)-1-pyrrolidinyl]-2-(4-methyl-1,4-diazepan-1-yl)-1-ethanone and [({(S)-1-[(1H-indol-2-yl)methyl]-3-pyrrolidinyl}methyl)amino](5-methyl-2H-pyrazol-3-yl)formaldehyde. Moreover, we obtained a favorable binding free energy of the identified compounds, and using contact analysis we confirmed their stable binding modes in the 3CLpro active site. These compounds will facilitate further 3CLpro inhibitor design

Ensemble Docking Coupled to Linear Interaction Energy Calculations for Identification of Coronavirus Main Protease (3CLpro) Non-Covalent Small-Molecule Inhibitors

SARS-CoV-2, or severe acute respiratory syndrome coronavirus 2, represents a new strain of Coronaviridae. In the closing 2019 to early 2020 months, the virus caused a global pandemic of COVID-19 disease. We performed a virtual screening study in order to identify potential inhibitors of the SARS-CoV-2 main viral protease (3CLpro or Mpro). For this purpose, we developed a novel approach using ensemble docking high-throughput virtual screening directly coupled with subsequent Linear Interaction Energy (LIE) calculations to maximize the conformational space sampling and to assess the binding affinity of identified inhibitors. A large database of small commercial compounds was prepared, and top-scoring hits were identified with two compounds singled out, namely 1-[(R)-2-(1,3-benzimidazol-2-yl)-1-pyrrolidinyl]-2-(4-methyl-1,4-diazepan-1-yl)-1-ethanone and [({(S)-1-[(1H-indol-2-yl)methyl]-3-pyrrolidinyl}methyl)amino](5-methyl-2H-pyrazol-3-yl)formaldehyde. Moreover, we obtained a favorable binding free energy of the identified compounds, and using contact analysis we confirmed their stable binding modes in the 3CLpro active site. These compounds will facilitate further 3CLpro inhibitor design

Naive prediction of protein backbone phi and psi dihedral angles using deep learning

Protein structure prediction represents a significant challenge in the field of bioinformatics, with the prediction of protein structures using backbone dihedral angles recently achieving significant progress due to the rise of deep neural network research. However, there is a trend in protein structure prediction research to employ increasingly complex neural networks and contributions from multiple models. This study, on the other hand, explores how a single model transparently behaves using sequence data only and what can be expected from the predicted angles. To this end, the current paper presents data acquisition, deep learning model definition, and training toward the final protein backbone angle prediction. The method applies a simple fully connected neural network (FCNN) model that takes only the primary structure of the protein with a sliding window of size 21 as input to predict protein backbone φ and ψ dihedral angles. Despite its simplicity, the model shows surprising accuracy for the φ angle prediction and somewhat lower accuracy for the ψ angle prediction. Moreover, this study demonstrates that protein secondary structure prediction is also possible with simple neural networks that take in only the protein amino-acid residue sequence, but more complex models are required for higher accuracies

Use of natural lead compounds and in silico screening of compound libraries in the design and synthesis of new antibacterials and ion channel modulators

Napetostno odvisni natrijevi kanalčki so veliki transmembranski proteinski kompleksi odgovorni za prevajanje električnih tokov v živčnih, mišičnih in endokrinih celicah in tkivih. Napake v njihovi ekspresiji in funkcijske spremembe so povezane z mnogimi patološkimi stanji. Tako se pri zdravljenju epilepsije, bolezni centralnega živčevja, bolezni srca in bolečinskih stanj uporabljajo učinkovine z modulatornim delovanjem na natrijevih kanalčkih. Le-te delujejo neselektivno na tip natrijevega kanalčka in izkazujejo širok nabor neželenih stranskih učinkov. Spojine, ki bi selektivno delovale na posameznih tipih napetostno odvisnih natrijevih kanalčkov, bi omogočile razvoj indikacijsko specifičnih učinkovin in olajšale študij vloge posamezenih tipov natrijevih kanalčkov v patofiziologiji. Ker so organizmi morskega izvora razmeroma neraziskan vir spojin vodnic, smo v okviru doktorskega študija uporabili alkaloida oroidin in klatrodin iz morskih spužev rodu Agelas kot izhodišče za načrtovanje in sintezo novih modulatorjev ionskih kanalčkov. Spojini vsebujeta terminalni 2-aminoimidazol povezan preko 3-aminopropenskega distančnika s halogeniranim ali nesubstituiranim pirolom na drugem koncu molekule. Pri načrtovanju spojin smo ohranili v oroidinu in klatrodinu prisotni aminoimidazolni strukturni element, saj predvidevamo da gre za ključni farmakoforni element. S pomočjo načrtovanja na osnovi liganda smo pripravili osredotočeno knjižnico analogov oroidina in klatrodina, kjer smo 3-aminopropenski distančnik zamenjali z delno rigidiziranim centralnim piperazinskim fragmentom. V okviru načrtovanja potencialnih modulatorjev napetostno odvisnih natrijevih kanalčkov smo preučili tudi vpliv zamenjave terminalnega pirola z različnimi aromatskimi ali heteroaromatskimi obroči na delovanje spojin. Sintetizirane spojine smo biološko ovrednotili na več tipih napetostno odvisnih natrijevih kanalčkov z metodo vpete krpice membrane. Ugotovili smo, da pripravljene spojine izkazujejo šibko jakost delovanja na tip 1.3 natrijevih kanalčkov, pri čemer je pomembna razdalja med terminalnima heteroaromatskima dušikoma. Potrdili smo domnevo, da je za delovanje ključen aminoimidazolni fragment, katerega bioizosterne zamenjave niso bile tolerirane v kontekstu delovanja na kanalčke. Opazili smo tudi ključno prisotnost karbonilne skupine ob terminalnem pirol-2-karboksilnem strukturnem fragmentu. Le-tega smo zamenjali z večjim heteroaromatskim indol-2-karbonilom in pripravili piperazinski analog klatrodina z IC50 vrednostjo 19 μM in selektivnim delovanjem na 1.3 tipu napetostno odvisnih natrijevih kanalčkov. Spojina ni izkazovala delovanja na 1.4, 1.5 ter 1.7 tipih napetostno odvisnih natrijevih kanalčkov in predstavlja dobro izhodišče za nadaljnji razvoj piperazinskih analogov klatrodina in oroidina kot selektivnih modulatorjev 1.3 tipa napetostno odvisnih natrijevih kanalčkov. Sekundarni metaboliti morskih spužev in njihovih simbiotskih organizmov nastopajo v razmnoževalnih, signalnih, regulacijskih in obrambnih vlogah ter imajo posledično polifarmakološke učinke. Raziskovana alkaloida klatrodin in oroidin iz morskih spužev Agelas sta tako poleg modulacije napetostno odvisnih natrijevih kanalčkov v literaturi opisana kot zaviralca nastanka bakterijskih biofilmov in kot protibakterijski učinkovini. Ker opažamo naraščajoči pojav bakterijske rezistence in upad učinkovitosti že uporabljanih farmakoloških pristopov, smo se v drugem delu doktorskega dela osredotočili na razvoj protibakterijskih učinkovin. Odkrili smo strukturne podobnosti med oroidinom oz. klatrodinom, njunimi piperazinskimi analogi in v literaturi prisotnimi piroloamidnimi zaviralci DNA giraze B. Bakterijska DNA giraza je esencialen encim, ki katalizira uvajanje negativnih zavojev na molekuli DNA oziroma katalizira topološko sproščanje ob podvajanju bakterijske DNA. Podenota B skrbi za hidrolizo molekule ATP in dovaja energijo za funkcionalno delovanje encima. Kjub substratu ATP, encim predstavlja ustrezno in validirano tarčo za razvoj novih protibakterijskih učinkovin. V nasprotju s ključnim aminoimidazolnim fragmentom, ki je bil potreben za modulatorno delovanje piperazinskih analogov oroidina ter klatrodina na napetostno odvisnih natrijevih kanalčkih, smo se pri načrtovanju spojin s potencialnim zaviralnim delovanjem na DNA girazi B osredotočili na piroloamidni oziroma amidni strukturni fragment, katerega smo pri načrtovanih spojinah ohranili. Po naših ugotovitvah ta predstavlja ključni strukturni element, ki omogoča zaviralcem DNA giraze B vzpostavitev vodikovih vezi z aminokislinskimi ostanki v adeninskem žepku ATP vezavnega mesta. Piroloamidna oziroma amidna struktura je pri zaviralcih DNA giraze B podobno povezana preko cikličnih, aromatskih, heteroaromatskih, bicikličnih in kondenziranih cikličnih distančnikov do drugega ključnega strukturnega elementa, ki pa poseduje polarno oziroma kislo funkcionalno skupino. Slednja omogoča drugi ključni kontakt zaviralcev DNA giraze B z bazičnimi argininskimi aminokislinskimi ostanki na vhodu v ATP vezavno mesto. Raziskovana naravna alkaloida in serijo njunih piperazinskih analogov smo uporabili pri in silico metodologiji virtualnega rešetanja in identificirali še aminopiperidinski in cikloheksanski distančnik kot primerna za pripravo knjižnice potencialnih zaviralcev DNA giraze B. Sintetizirali smo več kot 60 spojin treh strukturnih tipov in raziskovali vpliv strukture in konfiguracije uporabljenih distančnikov na zaviralno delovanje izoliranega encima DNA giraze B iz bakterijskega seva E. coli. Poleg centralnega skeleta smo poskušali preučiti tudi optimalno dolžino in velikost terminalnega strukturnega fragmenta z estrsko oziroma karboksilno funkcionalno skupino ter vpliv zamenjave ključnega piroloamidnega fragmenta z večjim indoloamidom. Identificirali in validirali smo pirolo-amino-piperidinski skelet s pripravljenimi spojinami, ki so izkazovale IC50 vrednosti na DNA girazi B v nizkem mikromolarnem območju. Spojina z alifatskim glutaratnim terminalnim fragmentom in pirolo-amino-piperidinskim skeletom pa je izkazovala zaviranje DNA giraze B z IC50 vrednostjo 480 nM. Prav tako smo identificirali primerno konfiguracijo cikloheksanskega distančnika in pripravili spojino s pirolo-1,4-trans-substituiranim cikloheksanskim skeletom in terminalnim glicinskim fragmentom ter IC50 vrednostjo 15.1 μM na DNA girazi B. Na podlagi podatkov vrednotenja encimske inhibicije smo pripravljene piperazinske, piperidinske in cikloheksanske zaviralce DNA giraze B uporabili za študij samega ATP vezavnega mesta. Z in silico metodami molekulskega sidranja in molekulske dinamike smo ocenili sposobnost vezave pripravljenih spojin v ATP vezavno mesto DNA giraze B in preučevali nastanek ključnih interakcij z Asp73 (E. coli številčenje) in bazičnimi argininskimi aminokislinski ostanki na vhodu v vezavno mesto. Preučili smo konformacijo uporabljenih distančnikov in dolžino terminalega fragmenta s karboksilno funkcionalno skupino. Prav tako smo razvili in silico pristop za identifikacijo ključnih ohranjenih molekul vode v ATP vezavnem mestu DNA giraze B ter dodatno potrdili vlogo ohranjene molekule vode pri vzpostavitvi vodikovih vezi med ligandom in adeninskim žepkom v aktivnem mestu DNA giraze B. Spojine s pirolo-amino-piperidinskim skeletom in zaviralnim delovanjem in vitro smo ovrednotili še z metodo površinske plazmonske resonance, kjer smo merili Kd vrednosti zaviralcev pri vezavi na fragment G24, ki je poznan kot najmanjši ATP-vezajoči proteinski fragment giraze B. Za spojine smo izmerili Kd vrednosti od 11 do 12 μM ter potrdili predhodne izmerjene IC50 vrednosti v nizkem mikromolarnem območju. Raziskovalno delo potrjuje obstoječe rezultate in strukturne študije potencialnih zaviralcev DNA giraze B ter omogoča trdne temelje za nadaljnji razvoj ključno potrebnih protibakterijskih učinkovin na terapevtski tarči DNA girazi B. Nadalje smo na podlagi farmakoinformatike identificirali knjižnico 1,3-tiazolidin-4-onov kot potencialnih protibakterijskih učinkovin in s pomočjo in silico podpore molekulskega modeliranja predlagali njihov mehanizem delovanja na encimski tarči undekaprenilpirofosfat sintazi. Spojine smo mikrobiološko ovrednotili ter določili minimalne zaviralne koncentracije na rast petih bakterijskih sevov in glivnega seva C. albicans. Knjižnica je izkazovala protibakterijsko delovanje pri čemer smo spojini (Z)-4-((2-(4-metil-piperidin-1-il)-4-oksotiazol-5(4H)-iliden)metil)benzonitril izmerili MIC vrednost 10 μg/mL na bakterijskem sevu P. aeruginosa.Voltage-gated sodium channels are large transmembrane protein complexes responsible for ion current transmission in nerve, muscle and endocrine cells and cell systems. Defects in their expression and function are linked to multiple pathological conditions. Treatment of these conditions such as epilepsy, central nervous system diseases, heart complications and various pain states is still challenging due to nonselective action of used drugs on multiple voltage-gated sodium channels and subsequent adverse side effects. Development of selective modulators of specific voltage-gated sodium channel isoforms would thus be beneficial and would enable profound studies on the role of individual voltage-gated sodium channel isoform in patophysiology. Due to under-explored chemical space of marine natural products we selected alkaloids oroidin and clathrodin as starting point for the discovery of novel modulators of voltage-gated sodium channels. Compounds are composed of two terminal fragments, namely 2-aminoimidazole and pyrrole-2-carboxamide, interconnected by a central 3-aminopropene linker moiety. In the scope of our research hypothesis we postulated that terminal aminoimidazole represents key pharmacophoric element, hence we primarily exchanged the central linker moiety with a sterically rigidified piperazine structure by using ligand based drug design methodology. Our next goal was a systematic study of the influence of terminal fragment exchange on potency of synthesized compounds and involvement of postulated pharmacophoric element. All compounds were biologically evaluated on multiple voltage-gated sodium channel isoforms using patch clamp technique. On some of prepared compounds we measured modest potency on voltage-gated sodium channel 1.3 isoform and determined that distance between terminal heterocyclic nitrogens is a crucial parameter. We confirmed our pharmacophoric element assumption as replacement of terminal aminoimidazole led to loss of potency. The role of heterocyclic moiety opposite of aminoimidazole was also elaborated and presence of carbony group at the heterocyclic amide identified as necessary for potency. Replacement of pyrrolo-2-carboxyl fragment with larger indolo-2-carbonyl led to piperazine clathrodin analogue with an IC50 value of 19 μM, selective on 1.3 voltage-gated sodium channel isoform. Prepared library of piperazine oroidin (clathrodin) analogues is a suitable starting point for further research on 1.3 voltage-gated sodium channel selective modulators, especially in the light of emerging new channel structural data. Secondary metabolites of sea sponges and their symbiotic organisms are involved in reproduction, signalling, regulation and defence roles in wholesome biological systems and they accordingly often display polypharmacological effects. Studied alkaloids oroidin and clathrodin from see sponges of the genus Agelas are reported in literature also as inhibitors of bacterial biofilm formation and antibacterial compounds. The latter observation is especially interesting in the light of emerging incidence of bacterial resistence and downfall of antibacterial therapy effectiveness. In the second part of research thesis we focused on antibacterial targets and identified structural similarity between oroidin lead compounds, prepared oroidin piperazine analogues and small molecule inhibitors of DNA gyrase B reported in literature. DNA Gyrase B is an essential bacterial enzyme responsible for the introduction of negativive supercoils or topological relaxation of DNA molecule during replication. It is composed of two subunits where subunit A performs operations on DNA molecule and subunit B hydrolises ATP supplying the required free energy. In spite of ATP as the substrate of DNA gyrase B, this enzyme subunit is a validated target for discovery of antibacterial compounds. In contrast with key aminoimidazole fragment, that was essential for potency of piperazine oroidin (clathrodin) analogues on voltage-gated sodium channels, we focused on second pyrrole-2-carboxamide terminal fragment in the design of potential inhibitors of DNA gyrase B. The latter fragment was reported as a structure element that enables crucial interactions with amino acid residues in adenine pocket of ATP binding site on DNA gyrase B. Pyrrole-2-carboxamide or amide moiety is similarly connected to the second pharmacophoric element via a cyclic, aromatic, heteroaromatic, bicyclic, condensed bicyclic or other similar linker in small molecule DNA gyrase B inhibitors reported in literature. Aforementioned second pharmacophoric element is where our initially observed similarities end. Here a structural fragment incorporating a polar or preferably acidic group is imperative as it enables key interactions towards arginine residues at the ATP binding site entrance of DNA gyrase B. We employed our leads and their piperazine analogues in the in silico virtual screening campaign only to identify additional aminopiperidine and cyclohexane central linkers for the development of focused library of potential DNA gyrase B inhibitors. We synthesized more than 60 compounds of three structural types in order to evaluate the influence of central linker and suitability of various simple aliphatic and aromatic terminal fragments incorporating a carboxyl or ester group on the inhibitory potency of compounds on isolated DNA gyrase B from E. coli. We also investigated the effects of pyrrole-2-carboxamide / indole-2-carboxamide pharmacophore exchange. We identified and validated pyrrolo-aminopiperidine scaffold in combination with prepared compounds that displayed IC50 values in low micromolar range. Compound with pyrrolo-aminopiperidine scaffold and glutaryl terminal acidic fragment possessed an IC50 value of 480 nM. Secondly, we recognised a suitable 1,4-substituted cyclohexane central linker. Representative pyrrolo-1,4-trans-cyclohexane scaffold compound incorporating a terminal glycine fragment displayed an IC50 value of 15.1 μM on DNA gyrase B. With the biological evaluation data in hand, we used prepared compounds to study the possible binding modes. With in silico methodology of molecular docking and molecular dynamics the possible binding into ATP active site was confirmed. Furthermore, previously published binding mode of small molecule DNA gyrase B inhibitors was corroborated and our compounds were found capable of interacting with Asp73 (E. coli numbering) at the adenine binding pocket and arginine residues near the ATP binding site entrance. We also developed an in silico approach towards identification of key conserved water molecules in ATP binding site and substantiated previous reports on water-mediated hydrogen bonds between ligand and amino acid residues in adenine pocket of DNA gyrase B ATP binding site. Compounds of the pyrrolo-aminopiperidine structural class were also evaluated using surface plasmon resonance where binding to the G24 protein known as the smallest ATP binding fragment of DNA gyrase B. Most potent compounds displayed affinity to the G24 protein with the Kd values of 11–12 μM, a result in agreement with our IC50 experiments. Our research confirms preceding results and strucutral studies on DNA gyrase B inhibitors and enables a solid foundation for futere design and development of DNA gyrase B as potential antibacterial compounds. In the additional investigation, we selected a library of 1,3-tiazolidin-4-one compounds and with the help of pharmacoinformatics and in silico molecluar modelling proposed their mechanism of action on the bacterial enzyme undecaprenylpyrophosphate synthase. Compounds were biologically evaluated and minimal inhibitory concentrations were measured on 5 bacterial strains and C albicans yeast. The library displayed modest antibacterial activity where (Z)-4-((2-(4-methyl-piperidin-1-yl)-4-oxothiazol-5(4H)-ylidene)methyl)benzonitrile was the most potent with a MIC value of 10 μg/mL on P. aeruginosa bacterial strain

Design of Tetra-Peptide Ligands of Antibody Fc Regions Using In Silico Combinatorial Library Screening

Abstract Peptides, or short chains of amino-acid residues, are becoming increasingly important as active ingredients of drugs and as crucial probes and/or tools in medical, biotechnological, and pharmaceutical research. Situated at the interface between small molecules and larger macromolecular systems, they pose a difficult challenge for computational methods. We report an in silico peptide library generation and prioritization workflow using CmDock for identifying tetrapeptide ligands that bind to Fc regions of antibodies that is analogous to known in vitro recombinant peptide libraries’ display and expression systems. The results of our in silico study are in accordance with existing scientific literature on in vitro peptides that bind to antibody Fc regions. In addition, we postulate an evolving in silico library design workflow that will help circumvent the combinatorial problem of in vitro comprehensive peptide libraries by focusing on peptide subunits that exhibit favorable interaction profiles in initial in silico peptide generation and testing

Neuropilin (NRPs) related pathological conditions and their modulators

Neuropilin 1 (NRP1) represents one of the two homologous neuropilins (NRP, splice variants of neuropilin 2 are the other) found in all vertebrates. It forms a transmembrane glycoprotein distributed in many human body tissues as a (co)receptor for a variety of different ligands. In addition to its physiological role, it is also associated with various pathological conditions. Recently, NRP1 has been discovered as a coreceptor for the SARS-CoV-2 viral entry, along with ACE2, and has thus become one of the COVID-19 research foci. However, in addition to COVID-19, the current review also summarises its other pathological roles and its involvement in clinical diseases like cancer and neuropathic pain. We also discuss the diversity of native NRP ligands and perform a joint analysis. Last but not least, we review the therapeutic roles of NRP1 and introduce a series of NRP1 modulators, which are typical peptidomimetics or other small molecule antagonists, to provide the medicinal chemistry community with a state-of-the-art overview of neuropilin modulator design and NRP1 druggability assessment