Structure-based design of a novel class of autotaxin inhibitors based on endogenous allosteric modulators

Autotaxin (ATX) facilitates the hydrolysis of lysophosphatidylcholine to lysophosphatidic acid (LPA), a bioactive phospholipid, which facilitates a diverse range of cellular effects in multiple tissue types. Abnormal LPA expression can lead to the progression of diseases such as cancer and fibrosis. Previously, we identified a potent ATX steroid-derived hybrid (partially orthosteric and allosteric) inhibitor which did not form interactions with the catalytic site. Herein, we describe the design, synthesis, and biological evaluation of a focused library of novel steroid-derived analogues targeting the bimetallic catalytic site, representing an entirely unique class of ATX inhibitors of type V designation, which demonstrate significant pathway-relevant biochemical and phenotypic biological effects. The current compounds modulated LPA-mediated ATX allostery and achieved indirect blockage of LPA1 internalization, in line with the observed reduction in downstream signaling cascades and chemotaxis induction. These novel type V ATX inhibitors represent a promising tool to inactivate the ATX-LPA signaling axis

Ventriculitis and hydrocephalia secondary to meningeal cryptococcosis in a non-HIV patient: a case report in the Hospital de San Jose in 2014

La criptocococis meníngea es la infección fúngica más frecuente del sistema nervioso central; generalmente se presenta en pacientes VIH seropositivos, aunque existe una proporción considerable de paciente VIH seronegativos, siendo en estos casos su presentación más agresiva. Esta infección tiene manifestaciones neurológicas variables que son secundarias al aumento de la presión intracraneal. La ventriculitis con hidrocefalia secundaria es una de las complicaciones de mayor morbi-mortalidad especialmente en pacientes VIH seronegativos. Presentamos un reporte de caso de criptocococis meníngea género Neoformans subtipo Grubbi con ventriculitis e hidrocefalia secundaria en paciente VIH seronegativo confirmado por histopatologíaMeningeal criptocococis is the most common fungal infection of the central nervous system, occuring in HIV seropositive patients, although there is a significant proportion of HIV seronegative patients, in whom the presentation is more aggresive. This infection has variable clinical manifestations secondary to increased intracranial pressure. Ventriculitis with hydrocephalus is one of the complications with the poorest outcome and mortality especially in HIV seronegative patients. We present a case report of meningeal criptocococis Neoformans subtype Grubbi with ventriculitis and secondary hydrocephalus in HIV seronegative patient confirmed by histopatholog

Rational design of autotaxin inhibitors by structural evolution of endogenous modulators

Autotaxin produces the bioactive lipid lysophosphatidic acid (LPA), and is a drug target of considerable interest for numerous pathologies. We report the expedient, structure-guided evolution of weak physiological allosteric inhibitors (bile salts) into potent competitive Autotaxin inhibitors that do not interact with the catalytic site. Functional data confirms that our lead compound attenuates LPA mediated signalling in cells, and reduces LPA synthesis in vivo, providing a promising natural product derived scaffold for drug discovery

Structure-activity relationships of small molecule autotaxin inhibitors with a discrete binding mode

Autotaxin (ATX) is a secreted enzyme responsible for the hydrolysis of lysophosphatidylcholine (LPC) to the bioactive lysophosphatidic acid (LPA) and choline. The ATX-LPA signalling pathway is implicated in cell survival, migration, and proliferation; thus, the inhibition of ATX is a recognized therapeutic target for a number of diseases including fibrotic diseases, cancer, and inflammation, amongst others. Many of the developed synthetic inhibitors for ATX have resembled the lipid chemotype of the native ligand; however, a small number of inhibitors have been described that deviate from this common scaffold. Herein, we report the structure-activity relationships (SAR) of a previously reported small molecule ATX inhibitor. We show through enzyme kinetics studies that analogues of this chemotype are noncompetitive inhibitors, and using a crystal structure with ATX we confirm the discrete binding mode

Regulation and cell signaling of phospholipases: The light at the end of the tunnel

The focus of this thesis is on phospholipases, which specifically cleave phospholipids. These lipids are not only the basic building blocks of our cells’ membranes, but they are also potent cell signaling molecules, modulating pathways that determine a cell’s fate. In Part A of this thesis, I study the phospholipase called autotaxin (or ATX), an essential enzyme that cleaves a specific lipid type: lysophospholipids. The most physiologically relevant products of autotaxin is a series of lipids called lysophosphatidic acids (LPAs). LPAs are necessary for many life processes, such as embryonic development and blood vessel growth, but are also the drivers for many diseases, as they can trigger cancer metastasis, inflammation and fibrosis, among others. In 2011, the crystal structure of autotaxin revealed a triple interconnected binding side: the tripartite site. This site encompasses the catalytic active site, the hydrophobic pocket, and the allosteric site known as tunnel. Here, I explore the role of the tunnel to unveil a new regulatory mechanism, where the LPA product accelerates its own synthesis by binding to the tunnel. I also investigate how the tunnel acts as a lipid-binding site that facilitates receptor-specific delivery of LPA to cell-surface receptors. As different inhibitors have been designed to block autotaxin’s activity, we compare two different types in liver disease models to gain new insights into the clinical benefit of blocking the tunnel. We also analyze the binding mode of compounds used for positron emission tomography (PET), in hopes that they can be used for non-invasive detection of autotaxin. Additionally, we collaboratively discover and optimize a new hybrid series of autotaxin inhibitors binding both the tunnel and the active site. This part concludes with a discussion of the current view on the enzymatic and non-enzymatic functions of autotaxin. I also examine how clinical success of drug candidates targeting autotaxin depends on binding the tunnel, thereby inhibiting the non-enzymatic signaling functions that I uncover in my research. In Part B of this thesis, I research another family of phospholipases: the glycerophosphodiester phosphodiesterase (or GDPD) family. GDPDs recognize a complex lipid anchor (called glycosylphosphatidylinositol or GPI) that cells attach to some proteins. Cleavage of this anchor by the GDPDs will shed a specific protein off the cell surface into, for instance, the blood, where it can have a specific function, or be used as a biomarker for cancer. The emerging picture in this relatively new field is that the activity of specific GDPDs relates to a positive prognosis of breast cancer and neuroblastoma, among others. Here, I study the determinants of this behavior by removing different stretches of GDE2’s C-terminal amino acid sequence. We then analyze the impact of altered intracellular trafficking on neuroblastoma cell lines. Given the accumulated evidence of the role of GDE2 and GDE3 in malignant cancers, I also venture to identify new clinically relevant substrates they may recognize and cleave. This part concludes with a discussion of the main findings and implications of my work on the GDPD protein family, emphasizing on future research directions

Regulation and cell signaling of phospholipases: The light at the end of the tunnel

The focus of this thesis is on phospholipases, which specifically cleave phospholipids. These lipids are not only the basic building blocks of our cells’ membranes, but they are also potent cell signaling molecules, modulating pathways that determine a cell’s fate. In Part A of this thesis, I study the phospholipase called autotaxin (or ATX), an essential enzyme that cleaves a specific lipid type: lysophospholipids. The most physiologically relevant products of autotaxin is a series of lipids called lysophosphatidic acids (LPAs). LPAs are necessary for many life processes, such as embryonic development and blood vessel growth, but are also the drivers for many diseases, as they can trigger cancer metastasis, inflammation and fibrosis, among others. In 2011, the crystal structure of autotaxin revealed a triple interconnected binding side: the tripartite site. This site encompasses the catalytic active site, the hydrophobic pocket, and the allosteric site known as tunnel. Here, I explore the role of the tunnel to unveil a new regulatory mechanism, where the LPA product accelerates its own synthesis by binding to the tunnel. I also investigate how the tunnel acts as a lipid-binding site that facilitates receptor-specific delivery of LPA to cell-surface receptors. As different inhibitors have been designed to block autotaxin’s activity, we compare two different types in liver disease models to gain new insights into the clinical benefit of blocking the tunnel. We also analyze the binding mode of compounds used for positron emission tomography (PET), in hopes that they can be used for non-invasive detection of autotaxin. Additionally, we collaboratively discover and optimize a new hybrid series of autotaxin inhibitors binding both the tunnel and the active site. This part concludes with a discussion of the current view on the enzymatic and non-enzymatic functions of autotaxin. I also examine how clinical success of drug candidates targeting autotaxin depends on binding the tunnel, thereby inhibiting the non-enzymatic signaling functions that I uncover in my research. In Part B of this thesis, I research another family of phospholipases: the glycerophosphodiester phosphodiesterase (or GDPD) family. GDPDs recognize a complex lipid anchor (called glycosylphosphatidylinositol or GPI) that cells attach to some proteins. Cleavage of this anchor by the GDPDs will shed a specific protein off the cell surface into, for instance, the blood, where it can have a specific function, or be used as a biomarker for cancer. The emerging picture in this relatively new field is that the activity of specific GDPDs relates to a positive prognosis of breast cancer and neuroblastoma, among others. Here, I study the determinants of this behavior by removing different stretches of GDE2’s C-terminal amino acid sequence. We then analyze the impact of altered intracellular trafficking on neuroblastoma cell lines. Given the accumulated evidence of the role of GDE2 and GDE3 in malignant cancers, I also venture to identify new clinically relevant substrates they may recognize and cleave. This part concludes with a discussion of the main findings and implications of my work on the GDPD protein family, emphasizing on future research directions