Résumé : Les podocytes, cellules épithéliales rénales, sont nécessaires au maintien de la structure et de la fonction de filtration des glomérules rénaux. La dédifférenciation et l’apoptose des podocytes sont des évènements précoces de la néphropathie diabétique. Des études ont rapporté que l’insuline est nécessaire à la survie des podocytes puisque la délétion du récepteur à l’insuline dans les podocytes de souris entraîne une pathologie glomérulaire semblable à la néphropathie. D’autres études ont montré que la protéine tyrosine phosphatase Src homology-2 domain-containing phosphatase-1 (SHP-1) inhibe les voies de signalisation de l’insuline au niveau du foie et du muscle en déphosphorylant la sous-unité bêta du récepteur à l’insuline (IRβ) et la kinase Phosphatidylinositide 3-kinase (PI3K). Il a récemment été démontré que l’expression de SHP-1 est élevée dans les cortex rénaux de souris diabétiques. Nous avons donc émis l’hypothèse que l’expression de SHP-1 induite par l’hyperglycémie altère les actions de l’insuline dans les podocytes. Nous avons premièrement utilisé un modèle in vivo de souris diabétiques de type 1 (Ins2+/C96Y; Akita). Comparées aux souris contrôles (Ins2+/+), les souris Akita présentaient une apoptose élevée des podocytes ainsi qu’une perte des pédicelles. La phosphorylation de la protéine kinase B (Akt) et de Extracellular signal-regulated kinase 1/2 (ERK1/2), suite à une injection systémique d’insuline, était également significativement diminuée dans les cortex rénaux des souris Akita. Cette diminution correspondant à une résistance à l’insuline corrélait avec une augmentation de deux fois de l’expression de SHP-1 dans les glomérules. Nous avons ensuite utilisé une lignée immortalisée de podocytes murins en culture et avons observé que l’exposition à des concentrations élevées de glucose (HG; 25 mM) pendant 96 h, entraînait l’augmentation de l’expression de marqueurs apoptotiques et de l’activité enzymatique de caspase-3/7 en comparaison aux concentrations normales de glucose (NG; 5,6 mM). L’exposition en HG a augmenté l’expression de l’ARNm et protéique de SHP-1, en plus de réduire la signalisation de l’insuline dans les podocytes. La surexpression de la forme dominante-négative de SHP-1 dans les podocytes a permis de renverser les effets de HG et de restaurer les actions de l’insuline. Finalement, l’augmentation de l’expression de SHP-1, tant in vivo qu’in vitro, a été directement corrélée à son association avec IRβ et à la diminution de la phosphorylation de IRβ, Akt et ERK1/2 suite à une stimulation à l’insuline. En conclusion, nous avons montré que l’expression élevée de SHP-1 dans les glomérules cause une résistance à l’insuline et la mort des podocytes contribuant ainsi à la néphropathie diabétique. // Abstract : Podocytes are epithelial renal cells required to preserve glomerular structure and filtration. Their dedifferentiation and apoptosis are early events of diabetic nephropathy progression. Previous studies have shown that insulin action is critical for podocyte survival since deletion of its receptor lead to a glomerular pathology similar to nephropathy. It has also been demonstrated that Src homology-2 domain-containing phosphatase-1 (SHP-1), a protein tyrosine phosphatase, inhibits insulin signaling pathway in liver and muscle by dephosphorylating tyrosine residues on insulin receptor beta-subunit (IRβ) and the Phosphatidylinositide 3-kinase (PI3K). A recent study concluded that SHP-1 is elevated in kidney cortex of type 1 diabetic mice. We hypothesized that hyperglycemia-induced SHP-1 expression may affect insulin actions in podocytes. To confirm this hypothesis, we used type 1 diabetic Akita mice (Ins2+/C96Y). Compared to control littermate mice (Ins2+/+), Akita mice developed elevated podocyte foot process effacement and podocyte apoptosis. In contrast to control mice, insulin-stimulated protein kinase B (Akt) and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation was remarkably reduced in renal podocytes of Akita mice. This phosphorylation diminution associated to a renal insulin resistance was correlated with a two-fold increase of SHP-1 expression in the glomeruli. We then used cultured murine podocytes cell line to confirm our in vivo results. Podocytes exposed to high glucose concentration (HG; 25 mM) for 96 h exhibited high levels of apoptotic markers and caspase-3/7 enzymatic activity as compared to normal glucose concentration (NG; 5,6 mM). HG exposure raised mRNA and protein levels of SHP-1 and reduced the insulin-signaling pathway in podocytes. Overexpression of dominant-negative SHP-1 in podocytes prevented HG effects and restored insulin actions. Finally, elevated SHP-1 expression induced by high glucose levels was directly correlated to an increased association with insulin receptor-β subunit (IRβ) in vitro and in vivo. This association is therefore leading to the reduction of both IRβ phosphorylation and insulin-stimulated Akt and ERK phosphorylation. In conclusion, our results showed that high levels of SHP-1 in glomeruli cause insulin resistance and podocyte loss, thereby contributing to diabetic nephropathy.

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

SHP-2 est une tyrosine phosphatase impliquée dans la signalisation intracellulaire déclenchée par des facteurs de croissance, des cytokines pro-inflammatoires et des produits bactériens. Bien que cette phosphatase soit exprimée de manière ubiquiste et donc dans l’épithélium intestinal, son rôle dans ce tissu n'était pas connu. Afin de mieux comprendre les rôles joués par cette phosphatase dans l’intestin, nous avons généré un modèle murin de délétion conditionnelle de Shp-2 spécifiquement dans les cellules épithéliales intestinales (SHP-2[indice supérieur CEI-KO]). Nos résultats montrent que dès l'âge de 1 mois, toutes les souris expérimentales ont développé spontanément de l'inflammation au niveau du côlon. En fait, dans les cellules épithéliales intestinales, SHP-2 contrôle le niveau d’activation d’effecteurs de signalisation importants tels que les kinases ERK1/2 de même que les facteurs de transcription NFκB, STAT3 et β-caténine. En modulant ces voies de signalisation, SHP-2 contrôle des processus cellulaires primordiaux pour le maintien de l’homéostasie intestinale: la détermination des cellules à mucus et des cellules de Paneth, la composition de la flore, la perméabilité paracellulaire et la restitution épithéliale. La dérégulation de ces processus cellulaires peut expliquer l'apparition rapide d'inflammation colique chez les souris SHP-2C[indice supérieur EI-KO]. De plus, l'inflammation chronique observée chez les souris SHP-2[indice supérieur] CEI-KO entraîne avec l'âge le développement de cancer colorectal associé à la colite. Finalement, nos résultats chez l'humain montrent qu'il y a une diminution significative d'expression de SHP-2 chez les patients atteints de maladies inflammatoires intestinales comparativement aux patients témoins. Également, deux polymorphismes de PTPN11 sont retrouvés préférentiellement chez les patients atteints de colite ulcéreuse. En conclusion, nos résultats démontrent que la phosphatase SHP-2 protège l'épithélium intestinal contre l'inflammation et le cancer colorectal associé à la colite.

Shp-1 (Src homology 2 domain-containing phosphatase 1) est une tyrosine phosphatase retrouvée principalement chez les cellules hématopoïétiques, mais également chez les cellules épithéliales. Bien que Shp-1 soit reconnue comme étant un régulateur négatif de plusieurs voies de signalisation intracellulaire chez les cellules hématopoïétiques, son rôle dans les cellules épithéliales a été jusqu’ici très peu étudié. Afin de mieux comprendre son rôle dans les cellules épithéliales intestinales, nous avons généré un modèle murin de délétion conditionnelle de Shp-1 spécifiquement dans l’épithélium intestinal (Shp-1CEI-KO). De manière intéressante, dès l’âge de 6 semaines, les souris expérimentales présentent une intestinalomégalie associée à une légère augmentation de la prolifération cryptale. La taille des cellules épithéliales est également augmentée, suggérant de l’hypertrophie cellulaire chez les souris invalidées pour Shp-1. Parallèlement, la voie de signalisation PI3K/Akt/mTor est activée dans l’épithélium des souris mutantes. Nous avons également noté une production accrue de cellules caliciformes et de leurs précurseures, les cellules intermédiaires, en absence de Shp-1. Par contre, la maturation des cellules de Paneth semble grandement compromise vu la baisse importante d’expression du lysozyme et des RegIIIβ et RegIIIγ, de même que la faible densité de leurs granules de sécrétion. La comparaison du phénotype intestinal des souris Shp-1CEI-KO avec celui des souris PtenCEI-KO suggère que l’hyperactivation de la voie PI3K/Akt/mTor est responsable en partie des altérations phénotypiques observées chez la souris invalidée pour Shp-1. En conclusion, nos résultats montrent que la tyrosine phosphatase Shp-1 est un régulateur important de l’homéostasie de l’épithélium intestinal en contrôlant notamment la croissance cellulaire et la différenciation des cellules de la lignée sécrétrice.

Tuberculose (TB) é um grave problema de saúde pública, sendo a segunda maior causa de morte entre doenças infecto contagiosas. Em 2014, 9,6 milhões de casos e, aproximadamente, 1,5 milhão de mortes foram reportados. O Programa Nacional de Controle da Tuberculose preconiza para o tratamento a administração simultânea de quatro medicamentos. Contudo, casos de tratamento inadequado favorecem o surgimento de cepas multirresistentes e extensivamente resistentes aos medicamentos disponíveis. Diante disso, torna-se urgente a necessidade de investigar novos alvos moleculares e desenvolver novos fármacos que sejam úteis e eficazes para o tratamento da infecção. As proteínas tirosina fosfatases (PTPs) constituem uma grande família de enzimas responsáveis pela hidrólise do fosfato ligado aos resíduos de tirosina em proteínas. A importância destas fosfatases reside no fato de estarem envolvidas na regulação de uma série de funções celulares, tais como crescimento, interação intercelular, metabolismo, transcrição, motilidade e resposta imune. A partir da análise do genoma do Mycabacteirum tuberculosis, foram identificadas duas proteínas tirosinas fosfatases (PtpA e PtpB), responsáveis pela sua sobrevivência nos macrófagos do hospedeiro. Ambas as enzimas têm sido exploradas como alvo molecular para o desenvolvimento de novos fármacos para a tuberculose. Nessa dissertação, as sequências gênicas que codificam para as enzimas PtpA e PtpB de M. tuberculosis foram clonadas com sucesso nos vetores de expressão. A expressão solúvel das proteínas permitiu o estabelecimento de um protocolo padronizado de purificação. Ensaios de cristalização foram conduzidos e cristais de proteínas obtidos tiveram os dados cristalográficos coletados. Para a enzima PtpB foi possível determinar a estrutura cristalográfica em alta resolução em complexo com um grupo fosfato no sítio catalítico. Essa estrutura foi então utilizada na etapa posterior de descoberta de novos candidatos a inibidores. Os trabalhos computacionais conduzidos incluíram uma combinação de estratégias para a identificação de pontos de interação relevantes para o processo de reconhecimento molecular e ligação bem como para a construção de modelos farmacofóricos 3D específicos para cada enzima. Esses dados foram utilizados para a seleção de um conjunto de 8 candidatos a inibidores da PtpA e 5 candidatos a inibidores da PtpB. Portanto, estudos de biologia molecular estrutural e química medicinal foram empregados com sucesso para o estabelecimento de uma plataforma produtiva dos alvos selecionados bem como para a seleção de novos candidatos a inibidores. / Tuberculosis (TB) is a serious public health problem and the second leading cause of death among infectious diseases. In 2014, 9.6 million cases, and approximately 1.5 million deaths were reported. The National Program for Tuberculosis Control recommends the simultaneous administration of four drugs as treatment for the disease. However, inadequate treatment determines the emergence of multidrug- and extensively-resistant strains to available drugs. Therefore, new molecular targets and drugs are urgently needed for the treatment of the infection. The protein tyrosine phosphatases (PTPs) are a large family of enzymes responsible for the hydrolysis of phosphate group bound to tyrosine residues in proteins. The importance of these molecules is related to the regulation of a number of cellular functions, including growth, intercellular interaction, metabolism, transcription, motility and immune response. Based on Mycabacteirum tuberculosis genome analysis, two protein tyrosine phosphatases (PTPA and PtpB) were related to mycobacterium survival in host macrophages. Both enzymes have been explored as a molecular target for the development of new drugs for TB. In this dissertation, the gene sequences encoding the enzymes PtpA and PtpB from M. tuberculosis were successfully cloned in expression vectors. The soluble expression of the proteins allowed the establishment of a standardized purification protocol. Crystallization assays were conducted, protein crystals were obtained, and crystallographic data were collected. We determine the crystallographic structure of PtpB in complex with a phosphate group in the catalytic site at high resolution. This structure was then used in the subsequent step for the discovery of new inhibitor candidates. Computational studies included a combination of strategies for identifying interaction points relevant to the process of molecular recognition and binding as well as the construction of 3D pharmacophore models specific for each enzyme. These data were used to select a set of 8 and 5 compounds as PtpA and PtpB inhibitor candidates, respectively. Therefore, structural molecular biology and medicinal chemistry studies have been successfully conducted for the establishment of a platform aimed to the production of the selected targets as well as for the selection of new inhibitor candidates.

by Paula A. Livingston. / Thesis (M.S.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web. / Two protein tyrosine phosphatases, dual specificity phosphatase PVP and low molecular weight phosphatase WZB were purified and characterized. PVP was expressed as inclusion bodies and a suitable purification and refolding method was devised. Enzyme kinetics revealed that p-nitrophenylphosphate and (Sb(B-naphthyl phosphate were substrates with KM of 4.0mM and 8.1mM respectively. PVP showed no reactivity towards phosphoserine. Kinetic characterization of WZB showed that only pnitrophenylphosphate was a substrate with no affinity for Ç-naphthyl phosphate and phosphoserine. Optimal conditions for activity with PNPP were found at a pH of 5 with a KM of 1.1mM, kcat of 35.4s-1 and kcat/KM of 32.2s-1mM-1. Inhibition studies showed that phosphate, fluoride, and molybdate were competitive inhibitors with Ki of 3.2mM, 71.7mM, and 50.4(So(BM respectively and hydrogen peroxide abolished activity. Active site mutants of WZB Cys9Ser and Asp115Asn showed no activity.

Tuberculose (TB) é um grave problema de saúde pública, sendo a segunda maior causa de morte entre doenças infecto contagiosas. Em 2014, 9,6 milhões de casos e, aproximadamente, 1,5 milhão de mortes foram reportados. O Programa Nacional de Controle da Tuberculose preconiza para o tratamento a administração simultânea de quatro medicamentos. Contudo, casos de tratamento inadequado favorecem o surgimento de cepas multirresistentes e extensivamente resistentes aos medicamentos disponíveis. Diante disso, torna-se urgente a necessidade de investigar novos alvos moleculares e desenvolver novos fármacos que sejam úteis e eficazes para o tratamento da infecção. As proteínas tirosina fosfatases (PTPs) constituem uma grande família de enzimas responsáveis pela hidrólise do fosfato ligado aos resíduos de tirosina em proteínas. A importância destas fosfatases reside no fato de estarem envolvidas na regulação de uma série de funções celulares, tais como crescimento, interação intercelular, metabolismo, transcrição, motilidade e resposta imune. A partir da análise do genoma do Mycabacteirum tuberculosis, foram identificadas duas proteínas tirosinas fosfatases (PtpA e PtpB), responsáveis pela sua sobrevivência nos macrófagos do hospedeiro. Ambas as enzimas têm sido exploradas como alvo molecular para o desenvolvimento de novos fármacos para a tuberculose. Nessa dissertação, as sequências gênicas que codificam para as enzimas PtpA e PtpB de M. tuberculosis foram clonadas com sucesso nos vetores de expressão. A expressão solúvel das proteínas permitiu o estabelecimento de um protocolo padronizado de purificação. Ensaios de cristalização foram conduzidos e cristais de proteínas obtidos tiveram os dados cristalográficos coletados. Para a enzima PtpB foi possível determinar a estrutura cristalográfica em alta resolução em complexo com um grupo fosfato no sítio catalítico. Essa estrutura foi então utilizada na etapa posterior de descoberta de novos candidatos a inibidores. Os trabalhos computacionais conduzidos incluíram uma combinação de estratégias para a identificação de pontos de interação relevantes para o processo de reconhecimento molecular e ligação bem como para a construção de modelos farmacofóricos 3D específicos para cada enzima. Esses dados foram utilizados para a seleção de um conjunto de 8 candidatos a inibidores da PtpA e 5 candidatos a inibidores da PtpB. Portanto, estudos de biologia molecular estrutural e química medicinal foram empregados com sucesso para o estabelecimento de uma plataforma produtiva dos alvos selecionados bem como para a seleção de novos candidatos a inibidores. / Tuberculosis (TB) is a serious public health problem and the second leading cause of death among infectious diseases. In 2014, 9.6 million cases, and approximately 1.5 million deaths were reported. The National Program for Tuberculosis Control recommends the simultaneous administration of four drugs as treatment for the disease. However, inadequate treatment determines the emergence of multidrug- and extensively-resistant strains to available drugs. Therefore, new molecular targets and drugs are urgently needed for the treatment of the infection. The protein tyrosine phosphatases (PTPs) are a large family of enzymes responsible for the hydrolysis of phosphate group bound to tyrosine residues in proteins. The importance of these molecules is related to the regulation of a number of cellular functions, including growth, intercellular interaction, metabolism, transcription, motility and immune response. Based on Mycabacteirum tuberculosis genome analysis, two protein tyrosine phosphatases (PTPA and PtpB) were related to mycobacterium survival in host macrophages. Both enzymes have been explored as a molecular target for the development of new drugs for TB. In this dissertation, the gene sequences encoding the enzymes PtpA and PtpB from M. tuberculosis were successfully cloned in expression vectors. The soluble expression of the proteins allowed the establishment of a standardized purification protocol. Crystallization assays were conducted, protein crystals were obtained, and crystallographic data were collected. We determine the crystallographic structure of PtpB in complex with a phosphate group in the catalytic site at high resolution. This structure was then used in the subsequent step for the discovery of new inhibitor candidates. Computational studies included a combination of strategies for identifying interaction points relevant to the process of molecular recognition and binding as well as the construction of 3D pharmacophore models specific for each enzyme. These data were used to select a set of 8 and 5 compounds as PtpA and PtpB inhibitor candidates, respectively. Therefore, structural molecular biology and medicinal chemistry studies have been successfully conducted for the establishment of a platform aimed to the production of the selected targets as well as for the selection of new inhibitor candidates.

<p>Tyrosine phosphorylation is a crucial mechanism in cellular signaling and regulates proliferation, differentiation, migration and adhesion. The phosphorylation reaction is reversible and is governed by two families of enzymes: protein tyrosine kinases and protein tyrosine phosphatases (PTPs). This thesis investigates the role of PTPs in regulating receptor protein tyrosine kinases (RTKs), and explores a mechanism for regulation of phosphatase activity.</p><p>Most receptor tyrosine kinases are activated by ligand induced dimerization, which results in an increase in receptor phosphorylation. Preparations of ligand-stimulated dimeric PDGF β-receptors were shown to be less susceptible to dephosphorylation compared with unstimulated receptors. This revealed that reduced receptor dephosphorylation contributes to ligand-induced increase in RTK phosphorylation.</p><p>The receptor-like phosphatase DEP-1 site-selectively dephosphorylates the PDGF β-receptor. One of the most preferred sites is the PLC-γ binding phosphotyrosine pY1021, and the autoregulatory pY857 is one of the least preferred sites. By using chimeric phospho-peptides derived from these two sites as substrate for DEP-1, it was shown that a lysine residue at position +3 acts as a negative determinant for DEP-1 and that an aspartic acid residue at position –1 is a positive determinant.</p><p>The modulatory effect of TC-PTP on PDGF β-receptor signaling was explored by using mouse embryonic fibroblasts derived from TC-PTP knockout mice. PDGF β-receptors derived from knockout cells exhibited a higher level of ligand-induced phosphorylation compared to receptors from wildtype cells. The increase was unevenly distributed between different autophosphorylation sites. The PLC-γ binding site, previously implicated in chemotactic response, displayed the largest increase. Consistently, a cell migration assay revealed hyper-responsiveness to PDGF of TC-PTP knockout cells as compared to wildtype cells.</p><p>Reversible oxidation of the active site cysteine in PTPs is a mechanism, which have been postulated to regulate phosphatase specific activity. An antibody-based generic method for detection of oxidized PTPs was developed. Using this method it was revealed for the first time that UV-induced inactivation of PTPs involves oxidation of the active site cysteine.</p>

Tyrosine phosphorylation is a crucial mechanism in cellular signaling and regulates proliferation, differentiation, migration and adhesion. The phosphorylation reaction is reversible and is governed by two families of enzymes: protein tyrosine kinases and protein tyrosine phosphatases (PTPs). This thesis investigates the role of PTPs in regulating receptor protein tyrosine kinases (RTKs), and explores a mechanism for regulation of phosphatase activity. Most receptor tyrosine kinases are activated by ligand induced dimerization, which results in an increase in receptor phosphorylation. Preparations of ligand-stimulated dimeric PDGF β-receptors were shown to be less susceptible to dephosphorylation compared with unstimulated receptors. This revealed that reduced receptor dephosphorylation contributes to ligand-induced increase in RTK phosphorylation. The receptor-like phosphatase DEP-1 site-selectively dephosphorylates the PDGF β-receptor. One of the most preferred sites is the PLC-γ binding phosphotyrosine pY1021, and the autoregulatory pY857 is one of the least preferred sites. By using chimeric phospho-peptides derived from these two sites as substrate for DEP-1, it was shown that a lysine residue at position +3 acts as a negative determinant for DEP-1 and that an aspartic acid residue at position –1 is a positive determinant. The modulatory effect of TC-PTP on PDGF β-receptor signaling was explored by using mouse embryonic fibroblasts derived from TC-PTP knockout mice. PDGF β-receptors derived from knockout cells exhibited a higher level of ligand-induced phosphorylation compared to receptors from wildtype cells. The increase was unevenly distributed between different autophosphorylation sites. The PLC-γ binding site, previously implicated in chemotactic response, displayed the largest increase. Consistently, a cell migration assay revealed hyper-responsiveness to PDGF of TC-PTP knockout cells as compared to wildtype cells. Reversible oxidation of the active site cysteine in PTPs is a mechanism, which have been postulated to regulate phosphatase specific activity. An antibody-based generic method for detection of oxidized PTPs was developed. Using this method it was revealed for the first time that UV-induced inactivation of PTPs involves oxidation of the active site cysteine.

The development of reversible and irreversible inhibitors of steroid sulfatase (STS) and protein tyrosine phosphatase 1B (PTP1B) is reported herein. STS belongs to to the aryl sulfatase family of enzymes that have roles in diverse processes such as hormone regulation, cellular degradation, bone and cartilage development, intracellular communication, and signalling pathways. STS catalyzes the desulfation of sulfated steroids which are the storage forms of many steroids such as the female hormone estrone. Its crucial role in the regulation of estrogen levels has made it a therapeutic target for the treatment of estrogen-dependent cancers. Estrone sulfate derivatives bearing 2- and 4-mono- and difluoromethyl substitutions were examined as quinone methide-generating suicide inhibitors of STS with the goal of developing these small molecules as activity-based probes for proteomic profiling of sulfatases. Kinetic studies suggest that inhibition by the monofluoro derivatives is a result of a quinone methide intermediate that reacts with active-site nucleophiles. However, the main inhibition pathway of the 4-difluoromethyl derivative involved an unexpected process in which initially formed quinone methide diffuses from the active site and decomposes to an aldehyde in solution which then acts as a potent, almost irreversible STS inhibitor. This is the first example where this class of inactivator functions by in situ generation of an aldehyde. 6- and 8-mono- and difluoromethyl coumarin derivatives were also examined as quinone methide-generating suicide inhibitors of STS. The 6-monofluoromethyl derivative acted as a classic suicide inhibitor. The partition ratio of this compound was found to be very large indicating that this class of compounds is not likely suitable as an activity-based probe for proteomic profiling of sulfatases. Boronic acids derived from steroid and coumarin platforms were also examined as STS inhibitors with the goal of improving our understanding of substrate binding specificity of STS. Inhibition constants in the high nanomolar to low micromolar range were observed for the steroidal derivatives. The coumarin derivatives were poor inhibitors. These results suggest that the boronic acid moiety must be attached to a platform very closely resembling a natural substrate in order for it to impart a beneficial effect on binding affinity compared to its phenolic analog. The mode of inhibition observed was reversible and kinetic properties corresponding to the mechanism for slow-binding inhibitors were not observed. PTP1B catalyzes the dephosphorylation of phosphotyrosine residues in the insulin receptor kinase and is a key enzyme in the down regulation of insulin signaling. Inhibitors of PTP1B are considered to have potential as therapeutics for treating type II diabetes mellitus. The difluoromethylenesulfonic (DFMS) acid group, one of the best monoanionic phosphotyrosine mimics reported in the literature, was examined as a phosphotyrosine (pTyr) mimic in a non-peptidyl platform for PTP1B inhibition. The DFMS-bearing inhibitor was found to be an approximately 1000-fold poorer inhibitor than its phosphorus analogue. It was also found that the fluorines in the DFMS inhibitor contributed little to inhibitory potency. In addition, [sulfonamido(difluoromethyl)]-phenylalanine (F2Smp) was examined as a neutral pTyr mimic in commonly used hexapeptide and tripeptide platforms. F2Smp was found to be a poor pTyr mimic. These inhibition studies also revealed that the tripeptide platform is not suitable for assessing pTyr mimics for PTP1B inhibition. Taken together, the kinetic data on the inhibition of STS and PTP1B provide valuable information relevant for future design of inhibitors of these two therapeutic targets.