RESUMEN
Antimicrobial resistance (AMR) poses a serious threat to our society from both the medical and economic point of view, while the antibiotic discovery pipeline has been dwindling over the last decades. Targeting non-essential bacterial pathways, such as those leading to antibiotic persistence, a bacterial bet-hedging strategy, will lead to new molecular entities displaying low selective pressure, thereby reducing the insurgence of AMR. Here, we describe a way to target (p)ppGpp (guanosine tetra- or penta-phosphate) signaling, a non-essential pathway involved in the formation of persisters, with a structure-based approach. A superfamily of enzymes called RSH (RelA/SpoT Homolog) regulates the intracellular levels of this alarmone. We virtually screened several fragment libraries against the (p)ppGpp synthetase domain of our RSH chosen model RelSeq, selected three main chemotypes, and measured their interaction with RelSeq by thermal shift assay and STD-NMR. Most of the tested fragments are selective for the synthetase domain, allowing us to select the aminobenzoic acid scaffold as a hit for lead development.
Asunto(s)
Antibacterianos , Guanosina Pentafosfato , Antibacterianos/farmacología , Bacterias/metabolismo , Guanosina Pentafosfato/metabolismoRESUMEN
Here, two conformationally constrained sialyl analogues were synthesized and characterized in their interaction with the inhibitory Siglec, human CD22 (h-CD22). An orthogonal approach, including biophysical assays (SPR and fluorescence), ligand-based NMR techniques, and molecular modelling, was employed to disentangle the interaction mechanisms at a molecular level. The results showed that the Sialyl-TnThr antigen analogue represents a promising scaffold for the design of novel h-CD22 inhibitors. Our findings also suggest that the introduction of a biphenyl moiety at position 9 of the sialic acid hampers canonical accommodation of the ligand in the protein binding pocket, even though the affinity with respect to the natural ligand is increased. Our results address the search for novel modifications of the Neu5Ac-α(2-6)-Gal epitope, outline new insights for the design and synthesis of high-affinity h-CD22 ligands, and offer novel prospects for therapeutic intervention to prevent autoimmune diseases and B-cell malignancies.
Asunto(s)
Linfocitos B , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico , Humanos , Ligandos , Ácido N-Acetilneuramínico , Unión Proteica , Lectina 2 Similar a Ig de Unión al Ácido Siálico/metabolismoRESUMEN
Hsp90 (i.e., Heat shock protein 90) is a well-established therapeutic target for several diseases, ranging from misfolding-related disfunctions to cancer. In this framework, we have developed in recent years a family of benzofuran compounds that act as Hsp90 allosteric modulators. Such molecules can interfere with the stability of some relevant Hsp90 client oncoproteins, showing a low µM cytotoxic activity in vitro in cancer cell lines. Here we identify the target profile of these chemical probes by means of chemical proteomics, which established MDH2 (mitochondrial malate dehydrogenase) as an additional relevant cellular target that might help elucidate the molecular mechanism of their citotoxicity. Western blotting, DARTS (i.e., Drug Affinity Responsive Target Stability) and enzymatic assays data confirmed a dose-dependent interaction of MDH2 with several members of the benzofuran Hsp90 modulators family and a computational model allowed to interpret the observed interactions.
Asunto(s)
Antineoplásicos/farmacología , Benzofuranos/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Malato Deshidrogenasa/antagonistas & inhibidores , Regulación Alostérica/efectos de los fármacos , Antineoplásicos/química , Benzofuranos/química , Relación Dosis-Respuesta a Droga , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Malato Deshidrogenasa/metabolismo , Modelos Moleculares , Estructura Molecular , Relación Estructura-ActividadRESUMEN
The efficient spread of SARS-CoV-2 resulted in a unique pandemic in modern history. Despite early identification of ACE2 as the receptor for viral spike protein, much remains to be understood about the molecular events behind viral dissemination. We evaluated the contribution of C-type lectin receptors (CLRS) of antigen-presenting cells, widely present in respiratory mucosa and lung tissue. DC-SIGN, L-SIGN, Langerin and MGL bind to diverse glycans of the spike using multiple interaction areas. Using pseudovirus and cells derived from monocytes or T-lymphocytes, we demonstrate that while virus capture by the CLRs examined does not allow direct cell infection, DC/L-SIGN, among these receptors, promote virus transfer to permissive ACE2+ Vero E6 cells. A glycomimetic compound designed against DC-SIGN, enable inhibition of this process. These data have been then confirmed using authentic SARS-CoV-2 virus and human respiratory cell lines. Thus, we described a mechanism potentiating viral spreading of infection.
Asunto(s)
COVID-19/transmisión , Lectinas Tipo C/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Animales , Antígenos CD/metabolismo , COVID-19/prevención & control , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Chlorocebus aethiops , Humanos , Células Jurkat , Pulmón/metabolismo , Lectinas de Unión a Manosa/metabolismo , Manósidos/farmacología , Unión Proteica/efectos de los fármacos , Receptores de Superficie Celular/metabolismo , Mucosa Respiratoria/metabolismo , Células VeroRESUMEN
Bacteria have developed different bet hedging strategies to survive hostile environments and stressful conditions with persistency being maybe the most elegant yet still poorly understood one. Persisters' temporary tolerance to antibiotic treatment hints at their role not only in chronic and recurrent infections but also in the insurgence of resistant strains. Therefore, hampering persisters formation might represent an innovative strategy in the quest for new effective antimicrobial compounds. Among the molecular mechanisms postulated for the persister phenotypic switch, we decided to focus our attention on the stringent response and, in particular, on the upstream triggering step that is the accumulation of guanosine tetra- and pentaphosphate, collectivity called (p)ppGpp. Intracellular levels of (p)ppGpp are regulated by a superfamily of enzymes called RSH (RelA/SpoT homologue) that are able to promote its synthesis via pyrophosphate transfer from an ATP molecule to the 3' position of either GDP or GTP. These enzymes are classified based on the structural domain(s) present (only synthetase, only hydrolase, or both). Here we present our work on Rel Seq (from S. equisimilis), still the only bifunctional Rel protein for which a GDP-bound "synthetase-ON" structure is available. Analysis of the synthetase site, occupied only by GDP, revealed a partially active state, where the supposed ATP binding region is not conformationally apt to accommodate it. In order to achieve a protein model that gets closer to a fully active state, we generated a chimera structure of Rel Seq by homology modeling, starting from the crystal structure of the catalytically competent state of RelP, a smaller, single-domain, Rel protein from S. aureus. Molecular dynamics simulations allowed verifying the stability of the generated chimera model. Virtual screening and ligand design studies are underway.
RESUMEN
Siglecs (sialic acid binding immunoglobulin (Ig)-like lectins) constitute a group of 15 human and 9 murine cell-surface transmembrane receptors belonging to the I-type lectin family, mostly expressed on innate immune cells and characterized by broadly similar structural features. Here, the prominent inhibitory CD22 (Siglec-2), well known in maintaining tolerance and preventing autoimmune responses on B cells, is studied in its human and murine forms in complex with sialoglycans. In detail, the role of the N-glycolyl neuraminic acid (Neu5Gc) moiety in the interaction with both orthologues was explored. The analysis of the binding mode was carried out by the combination of NMR spectroscopy, computational approaches, and CORCEMA-ST calculations. Our findings provide a first model of Neu5Gc recognition by h-CD22 and show a comparable molecular recognition profile by h- and m-CD22. These data open the way to innovative diagnostic and/or therapeutic methodologies to be used in the modulation of the immune responses.
Asunto(s)
Betacoronavirus , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/epidemiología , Pandemias , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/epidemiología , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/uso terapéutico , Alanina/análogos & derivados , Alanina/uso terapéutico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Betacoronavirus/efectos de los fármacos , Betacoronavirus/genética , Investigación Biomédica/estadística & datos numéricos , COVID-19 , Química/educación , Ensayos Clínicos como Asunto , Infecciones por Coronavirus/diagnóstico , Humanos , Italia/epidemiología , Laboratorios/estadística & datos numéricos , Filogenia , Neumonía Viral/diagnóstico , SARS-CoV-2 , Tratamiento Farmacológico de COVID-19RESUMEN
Chemical modification of pseudo-dimannoside ligands guided by fragment-based design allowed for the exploitation of an ammonium-binding region in the vicinity of the mannose-binding site of DC-SIGN, leading to the synthesis of a glycomimetic antagonist (compound 16) of unprecedented affinity and selectivity against the related lectin langerin. Here, the computational design of pseudo-dimannoside derivatives as DC-SIGN ligands, their synthesis, their evaluation as DC-SIGN selective antagonists, the biophysical characterization of the DC-SIGN/16 complex, and the structural basis for the ligand activity are presented. On the way to the characterization of this ligand, an unusual bridging interaction within the crystals shed light on the plasticity and potential secondary binding sites within the DC-SIGN carbohydrate recognition domain.
RESUMEN
Guanosine penta- or tetraphosphate (pppGpp or ppGpp, respectively) is a nucleotide signalling molecule with a marked effect on bacterial physiology during stress. Its accumulation slows down cell metabolism and replication, supposedly leading to the formation of the antibiotic-tolerant persister phenotype. A specifically tailored fluorescent chemosensor, PyDPA, allows the detection of (p)ppGpp in solution with high selectivity, relative to that of other nucleotides. Herein, an optimised synthetic approach is presented that improves the overall yield from 9 to 67 % over 7â steps. The simplicity and robustness of this approach will allow groups investigating the many facets of (p)ppGpp easy access to this probe.
Asunto(s)
Complejos de Coordinación/síntesis química , Colorantes Fluorescentes/síntesis química , Guanosina Pentafosfato/análisis , Guanosina Tetrafosfato/análisis , Pirenos/síntesis química , Fluorescencia , Guanosina Pentafosfato/química , Guanosina Tetrafosfato/química , Zinc/químicaRESUMEN
Molecular chaperones are pivotal in folding and degradation of the cellular proteome but their impact on the conformational dynamics of near-native membrane proteins with disease relevance remains unknown. Here we report the effect of chaperone activity on the functional conformation of the temperature-sensitive mutant cystic fibrosis channel (∆F508-CFTR) at the plasma membrane and after reconstitution into phospholipid bilayer. Thermally induced unfolding at 37 °C and concomitant functional inactivation of ∆F508-CFTR are partially suppressed by constitutive activity of Hsc70 and Hsp90 chaperone/co-chaperone at the plasma membrane and post-endoplasmic reticulum compartments in vivo, and at single-molecule level in vitro, indicated by kinetic and thermodynamic remodeling of the mutant gating energetics toward its wild-type counterpart. Thus, molecular chaperones can contribute to functional maintenance of ∆F508-CFTR by reshaping the conformational energetics of its final fold, a mechanism with implication in the regulation of metastable ABC transporters and other plasma membrane proteins activity in health and diseases.The F508 deletion (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) is the most common CF causing mutation. Here the authors show that cytosolic chaperones shift the F508del channel conformation to the native fold by kinetic and thermodynamic remodelling of the gating energetics towards that of wild-type CTFR.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/metabolismo , Proteínas del Choque Térmico HSC70/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Fibrosis Quística/genética , Proteínas del Choque Térmico HSC70/genética , Proteínas HSP90 de Choque Térmico/genética , Humanos , Chaperonas Moleculares/genética , Mutación , Pliegue de Proteína , TemperaturaRESUMEN
The small RNA ReaL of the opportunistic pathogen Pseudomonas aeruginosa has been characterized. Our results indicate that ReaL contributes to P. aeruginosa virulence. In the Galleria mellonella infection model, reaL gene deletion resulted in decreased virulence, while ReaL overexpression resulted in a hyper-virulent phenotype. We also demonstrate that ReaL is embedded in the P. aeruginosa quorum sensing (QS) with the role of linking las to pqs systems. We show that ReaL is negatively regulated by the las regulator LasR and impacts positively the synthesis of the pqs quinolone signal PQS by a positive post-transcriptional effect on the pqsC gene. Perturbations of ReaL levels affect pyocyanin synthesis, biofilm formation and swarming motility, processes that are known to be influenced by PQS synthesis. In addition to being regulated by LasR, ReaL is also responding to infection relevant cues that P. aeruginosa can experience in mammalian hosts such as temperature and oxygen availability. Furthermore, ReaL shows a growth phase-dependent pattern of expression, being up-regulated in stationary phase, due to the activity of the alternative σ factor RpoS. Together, these regulations of ReaL expression are expected to contribute to the fine co-modulation of PQS synthesis and, ultimately, virulence.
Asunto(s)
Regulación Bacteriana de la Expresión Génica/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Percepción de Quorum/genética , ARN Nuclear Pequeño/genética , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Eliminación de Gen , Mariposas Nocturnas/microbiología , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/patología , Piocianina/metabolismo , Quinolonas/metabolismo , Factor sigma/metabolismo , Transducción de Señal/genética , Transactivadores/genética , VirulenciaRESUMEN
The synthesis and conformational analysis of pseudo-thio-1,2-dimannoside are described. This molecule mimics mannobioside (Manα(1,2)Man) and is an analog of pseudo-1,2-dimannoside, with expected increased stability to enzymatic hydrolysis. A short and efficient synthesis was developed based on an epoxide ring-opening reaction by a mannosyl thiolate, generated in situ from the corresponding thioacetate. NMR-NOESY studies supported by MM3∗ calculations showed that the pseudo-thio-1,2-dimannoside shares the conformational behavior of the pseudo-1,2-dimannoside and is a structural mimic of the natural disaccharide. Its affinity for DC-SIGN was measured by SPR and found to be comparable to the corresponding O-linked analog, offering good opportunities for further developments.
Asunto(s)
Moléculas de Adhesión Celular/antagonistas & inhibidores , Lectinas Tipo C/antagonistas & inhibidores , Manósidos/química , Manósidos/farmacología , Receptores de Superficie Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/metabolismo , Diseño de Fármacos , Humanos , Lectinas Tipo C/metabolismo , Modelos Moleculares , Receptores de Superficie Celular/metabolismo , Resonancia por Plasmón de Superficie , Tioglicósidos/química , Tioglicósidos/farmacologíaRESUMEN
Allosteric compounds that stimulate Hsp90 adenosine triphosphatase (ATPase) activity were rationally designed, showing anticancer potencies in the low micromolar to nanomolar range. In parallel, the mode of action of these compounds was clarified and a quantitative model that links the dynamic ligand-protein cross-talk to observed cellular and in vitro activities was developed. The results support the potential of using dynamics-based approaches to develop original mechanism-based cancer therapeutics.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Antineoplásicos/farmacología , Proteínas HSP90 de Choque Térmico/metabolismo , Adenosina Trifosfatasas/química , Regulación Alostérica , Antineoplásicos/química , Diseño de Fármacos , Proteínas HSP90 de Choque Térmico/química , Ligandos , Unión ProteicaRESUMEN
The initial steps of viral infections are mediated by interactions between viral proteins and cellular receptors. Blocking the latter with high-affinity ligands may inhibit infection. DC-SIGN, a C-type lectin receptor expressed by immature dendritic cells and macrophages, mediates human immunodeficiency virus (HIV) infection by recognizing mannose clusters on the HIV-1 gp120 envelope glycoprotein. Mannosylated glycodendrimers act as HIV entry inhibitors thanks to their ability to block this receptor. Previously, an amphoteric, but prevailingly cationic polyamidoamine named AGMA1 proved effective as infection inhibitor for several heparan sulfate proteoglycan-dependent viruses, such as human papilloma virus HPV-16 and herpes simplex virus HSV-2. An amphoteric, but prevailingly anionic PAA named ISA23 proved inactive. It was speculated that the substitution of mannosylated units for a limited percentage of AGMA1 repeating units, while imparting anti-HIV activity, would preserve the fundamentals of its HPV-16 and HSV-2 infection inhibitory activity. In this work, four biocompatible linear PAAs carrying different amounts of mannosyl-triazolyl pendants, Man-ISA7, Man-ISA14, Man-AGMA6.5 and Man-AGMA14.5, were prepared by reaction of 2-(azidoethyl)-α-D-mannopyranoside and differently propargyl-substituted AGMA1 and ISA23. All mannosylated PAAs inhibited HIV infection. Both Man-AGMA6.5 and Man-AGMA14.5 maintained the HPV-16 and HSV-2 activity of the parent polymer, proving broad-spectrum, dual action mode virus infection inhibitors.
Asunto(s)
Antivirales/farmacología , Materiales Biocompatibles/farmacología , Manosa/farmacología , Poliaminas/farmacología , Enfermedades de Transmisión Sexual/tratamiento farmacológico , Antivirales/síntesis química , Antivirales/química , Antivirales/uso terapéutico , Materiales Biocompatibles/síntesis química , Materiales Biocompatibles/química , Materiales Biocompatibles/uso terapéutico , Bioensayo , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Células HeLa , Herpesvirus Humano 2/efectos de los fármacos , Papillomavirus Humano 16/efectos de los fármacos , Humanos , Manosa/síntesis química , Manosa/química , Manosa/uso terapéutico , Peso Molecular , Poliaminas/síntesis química , Poliaminas/química , Poliaminas/uso terapéutico , Enfermedades de Transmisión Sexual/virologíaRESUMEN
Controlling biochemical pathways through chemically designed modulators may provide novel opportunities to develop therapeutic drugs and chemical tools. The underlying challenge is to design new molecular entities able to act as allosteric chemical switches that selectively turn on/off functions by modulating the conformational dynamics of their target protein. We examine the origins of the stimulation of ATPase and closure kinetics in the molecular chaperone Hsp90 by allosteric modulators through atomistic molecular dynamics (MD) simulations and analysis of protein-ligand interactions. In particular, we focus on the cross-talk between allosteric ligands and protein conformations and its effect on the dynamic properties of the chaperone's active state. We examine the impact of different allosteric modulators on the stability, structural and internal dynamics properties of Hsp90 closed state. A critical aspect of this study is the development of a quantitative model that correlates Hsp90 activation to the presence of a certain compound, making use of information on the dynamic adaptation of protein conformations to the presence of the ligand, which allows to capture conformational states relevant in the activation process. We discuss the implications of considering the conformational dialogue between allosteric ligands and protein conformations for the design of new functional modulators.
Asunto(s)
Proteínas HSP90 de Choque Térmico/química , Proteínas de Saccharomyces cerevisiae/química , Adenosina Trifosfatasas/metabolismo , Regulación Alostérica , Proteínas HSP90 de Choque Térmico/metabolismo , Cinética , Ligandos , Modelos Químicos , Modelos Moleculares , Chaperonas Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Microbial adhesion is an essential step in infection and is mediated primarily by protein-carbohydrate interactions. Antagonists of such interactions have become a promising target for anti-adhesive therapy in several infective diseases. Monovalent protein-sugar interactions are often weak, and most successful anti-adhesive materials consist of multivalent glycoconjugates. Although often very effective in hampering microbial adhesion, natural epitopes often show limited resistance to enzymatic degradation. The use of carbohydrate mimics (glycomimetics) as a replacement for natural sugars potentially allows higher metabolic stability and also higher selectivity towards the desired protein target. In this review we describe the state of the art in the design and synthesis of glycoconjugates and glycomimetics employed for the construction of anti-adhesive biomaterials.
Asunto(s)
Antibacterianos/administración & dosificación , Adhesión Bacteriana/efectos de los fármacos , Adhesión Bacteriana/fisiología , Materiales Biomiméticos/administración & dosificación , Glicoconjugados/administración & dosificación , Materiales Biomiméticos/química , Relación Dosis-Respuesta a Droga , Glicoconjugados/químicaRESUMEN
Antagonists of mannose binding lectin (MBL) have shown a protective role against brain reperfusion damage after acute ischemic stroke. Here we describe the design and streamlined synthesis of glycomimetic MBL antagonists based on a new tetravalent dendron scaffold. The dendron was developed by optimisation of a known polyester structure previously demonstrated to be very efficient for ligand presentation to MBL. Replacement of a labile succinyl ester bond with a more robust amide functionality, use of a longer and more hydrophilic linker, fast modular synthesis and orthogonal functionalisation at the focal point are the main features of the new scaffold. The glycoconjugate constructs become stable to silica gel chromatography and to water solutions at physiological pH, while preserving water solubility and activity in an SPR assay against the murine MBL-C isoform. Higher-order constructs were easily assembled, as demonstrated by the synthesis of a 16-valent dendrimer, which leads to two orders of magnitude increase in activity over the tetravalent version against MBL-C.
Asunto(s)
Isquemia Encefálica/fisiopatología , Dendrímeros/química , Glicoconjugados/química , Lectina de Unión a Manosa/deficiencia , Accidente Cerebrovascular/patología , Animales , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Glicoconjugados/metabolismo , Ligandos , Lectina de Unión a Manosa/fisiología , RatonesRESUMEN
Hsp90 is a molecular chaperone of pivotal importance for multiple cell pathways. ATP-regulated internal dynamics are critical for its function and current pharmacological approaches block the chaperone with ATP-competitive inhibitors. Herein, a general approach to perturb Hsp90 through design of new allosteric ligands aimed at modulating its functional dynamics is proposed. Based on the characterization of a first set of 2-phenylbenzofurans showing stimulatory effects on Hsp90 ATPase and conformational dynamics, new ligands were developed that activate Hsp90 by targeting an allosteric site, located 65â Å from the active site. Specifically, analysis of protein responses to first-generation activators was exploited to guide the design of novel derivatives with improved ability to stimulate ATP hydrolysis. The molecules' effects on Hsp90 enzymatic, conformational, co-chaperone and client-binding properties were characterized through biochemical, biophysical and cellular approaches. These designed probes act as allosteric activators of the chaperone and affect the viability of cancer cell lines for which proper functioning of Hsp90 is necessary.
Asunto(s)
Adenosina Trifosfatasas/química , Adenosina Trifosfato/química , Benzofuranos/química , Chaperoninas/química , Proteínas HSP90 de Choque Térmico/química , Adenosina Trifosfatasas/metabolismo , Sitio Alostérico , Fenómenos Bioquímicos , Línea Celular Tumoral , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Hidrólisis , Ligandos , Unión Proteica , Conformación ProteicaRESUMEN
DC-SIGN is a dendritic cell-specific C-type lectin receptor that recognizes highly glycosylated ligands expressed on the surface of various pathogens. This receptor plays an important role in the early stages of many viral infections, including HIV, which makes it an interesting therapeutic target. Glycomimetic compounds are good drug candidates for DC-SIGN inhibition due to their high solubility, resistance to glycosidases, and nontoxicity. We studied the structural properties of the interaction of the tetrameric DC-SIGN extracellular domain (ECD), with two glycomimetic antagonists, a pseudomannobioside (1) and a linear pseudomannotrioside (2). Though the inhibitory potency of 2, as measured by SPR competition experiments, was 1 order of magnitude higher than that of 1, crystal structures of the complexes within the DC-SIGN carbohydrate recognition domain showed the same binding mode for both compounds. Moreover, when conjugated to multivalent scaffolds, the inhibitory potencies of these compounds became uniform. Combining isothermal titration microcalorimetry, analytical ultracentrifugation, and dynamic light scattering techniques to study DC-SIGN ECD interaction with these glycomimetics revealed that 2 is able, without any multivalent presentation, to cluster DC-SIGN tetramers leading to an artificially overestimated inhibitory potency. The use of multivalent scaffolds presenting 1 or 2 in HIV trans-infection inhibition assay confirms the loss of potency of 2 upon conjugation and the equal efficacy of chemically simpler compound 1. This study documents a unique case where, among two active compounds chemically derived, the compound with the lower apparent activity is the optimal lead for further drug development.