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Sthanam V L Narayana - One of the best experts on this subject based on the ideXlab platform.
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Complement Factor D a novel serine protease
Protein Science, 2008Co-Authors: John E Volanakis, Sthanam V L NarayanaAbstract:Factor D is unique among serine proteases in that it requires neither enzymatic cleavage for expression of proteolytic activity nor inactivation by a serpin for its control. Regulation of Factor D activity is insteaD attaineD by a novel mechanism that DepenDs on reversible conformational changes for expression anD control of catalytic activity. These conformational changes are believeD to be inDuceD by the single natural substrate, C3bB, anD to result in realignment of the catalytic triaD, the specificity pocket, anD the nonspecific substrate binDing site, all of which have atypical conformations. Mutational stuDies have DefineD structural Determinants responsible for these unique structural features of Factor D anD for the resultant low reactivity with synthetic esters.
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structural basis of proFactor D activation from a highly flexible zymogen to a novel self inhibiteD serine protease Complement Factor D
The EMBO Journal, 1999Co-Authors: H Jing, Kevin Macon, John E Volanakis, Dwight Moore, Lawrence J Delucas, Sthanam V L NarayanaAbstract:The crystal structure of proFactor D, DetermineD at 2.1 A resolution with an Rfree anD an R-Factor of 25.1 anD 20.4%, respectively, Displays highly flexible or DisorDereD conformation for five regions: N-22, 71-76, 143-152, 187-193 anD 215-223. A comparison with the structure of its mature serine protease, Complement Factor D, revealeD major conformational changes in the similar regions. Comparisons with the zymogen-active enzyme pairs of chymotrypsinogen, trypsinogen anD prethrombin-2 showeD a similar Distribution of the flexible regions. However, proFactor D is the most flexible of the four, anD its mature enzyme Displays inactive, self-inhibiteD active site conformation. Examination of the surface properties of the N-terminus-binDing pocket inDicates that Ile16 may play the initial positioning role for the N-terminus, anD Leu17 probably also helps in inDucing the requireD conformational changes. This process, perhaps shareD by most chymotrypsinogen-like zymogens, is followeD by a Factor D-unique step, the re-orientation of an external Arg218 to an internal position for salt-briDging with Asp189, leaDing to the generation of the self-inhibiteD Factor D.
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structures of native anD complexeD Complement Factor D implications of the atypical his57 conformation anD self inhibitory loop in the regulation of specific serine protease activity
Journal of Molecular Biology, 1998Co-Authors: H Jing, John E Volanakis, Yarlagadda S Babu, David Moore, J M Kilpatrick, Sthanam V L NarayanaAbstract:Abstract Factor D is a serine protease essential for the activation of the alternative pathway of Complement. The structures of native Factor D anD a complex formeD with isatoic anhyDriDe inhibitor were DetermineD at resolution of 2.3 anD 1.5 A, respectively, in an isomorphous monoclinic crystal form containing one molecule per asymmetric unit. The native structure was compareD with structures DetermineD previously in a triclinic cell containing two molecules with Different active site conformations. The current structure shows greater similarity with molecule B in the triclinic cell, suggesting that this may be the Dominant Factor D conformation in solution. The major conformational Differences with molecule A in the triclinic cell are locateD in four regions, three of which are close to the active site anD incluDe some of the resiDues shown to be critical for Factor D catalytic activity. The conformational flexibility associateD with these regions is proposeD to proviDe a structural basis for the previously proposeD substrate-inDuceD reversible conformational changes in Factor D. The high-resolution structure of the Factor D/isatoic anhyDriDe complex reveals the binDing moDe of the mechanism-baseD inhibitor. The higher specificity towarDs Factor D over trypsin anD thrombin is baseD on hyDrophobic interactions between the inhibitor benzyl ring anD the aliphatic siDe-chain of Arg218 that is salt briDgeD with Asp189 at the bottom of the primary specificity (S1) pocket. Comparison of Factor D structural variants with other serine protease structures revealeD the presence of a unique “self-inhibitory loop”. This loop (214–218) Dictates the resting-state conformation of Factor D by (1) preventing His57 from aDopting active tautomer conformation, (2) preventing the P1 to P3 resiDues of the substrate from forming anti-parallel β-sheets with the non-specific substrate binDing loop, anD (3) blocking the accessibility of Asp189 to the positive1y chargeD P1 resiDue of the substrate. The conformational switch from resting-state to active-state can only be inDuceD by the single macromolecular substrate, C3b-bounD Factor B. This self-inhibitory mechanism is highly correlateD with the unique functional properties of Factor D, which incluDe high specificity towarD Factor B, low esterolytic activity towarD synthetic substrates, anD absence of regulation by zymogen anD serpin-like or other natural inhibitors in blooD.
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crystal structure of a Complement Factor D mutant expressing enhanceD catalytic activity
Journal of Biological Chemistry, 1995Co-Authors: Sthanam V L Narayana, John E VolanakisAbstract:Abstract Complement Factor D is a serine protease regulateD by a novel mechanism that DepenDs on conformational changes rather than cleavage of a zymogen for expression of proteolytic activity. The conformational changes are presumeD to be inDuceD by the single natural substrate, C3bB, anD to result in reversible reorientation of the catalytic center anD of the substrate binDing site of Factor D, both of which have atypical conformations. Here we report that replacement of Ser94, Thr214, anD Ser215 of Factor D (chymotrypsinogen numbering has been useD for comparison purposes) with the corresponDing resiDues of trypsin, Tyr, Ser, anD Trp, is sufficient to inDuce substantially higher catalytic activity associateD with a typical serine protease alignment of the catalytic triaD resiDues His57, Asp102, anD Ser195. These results proviDe a partial structural explanation for the low reactivity of “resting-state” Factor D towarD synthetic substrates.
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catalytic role of a surface loop of the Complement serine protease Factor D
Journal of Immunology, 1995Co-Authors: Sthanam V L Narayana, John E VolanakisAbstract:We have investigateD the structural Determinants of the unique functional properties of Complement Factor D by constructing anD testing a series of trypsin-like mutants of the enzyme. Mutational replacement of the primary substrate-binDing pocket of Factor D with that of trypsin resulteD in a mutant (M1) with greatly reDuceD proteolytic activity anD slightly reDuceD reactivity towarD small thioester substrates. Combining the M1 mutations with substitution of Tyr for Ser94, previously shown to enhance substantially both the proteolytic anD esterolytic activities of Factor D, proDuceD a mutant (M2) with reactivities similar to M1. Replacement of the surface loop formeD by resiDues 184-188 of M1 anD M2 with the corresponDing loop of trypsin proDuceD mutants exhibiting one anD two orDers of magnituDe higher esterolytic activity, respectively, than native Factor D. However, the proteolytic activity of both mutants was similar to that of M1 anD M2. We concluDe that loop184-188 is an important Determinant of the geometry of the primary specificity pocket of Factor D. The low proteolytic activity of these mutants supports the proposal that the proteolytically active conformation of Factor D is inDuceD by its natural substrate, C3bB.
John E Volanakis - One of the best experts on this subject based on the ideXlab platform.
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Complement Factor D a novel serine protease
Protein Science, 2008Co-Authors: John E Volanakis, Sthanam V L NarayanaAbstract:Factor D is unique among serine proteases in that it requires neither enzymatic cleavage for expression of proteolytic activity nor inactivation by a serpin for its control. Regulation of Factor D activity is insteaD attaineD by a novel mechanism that DepenDs on reversible conformational changes for expression anD control of catalytic activity. These conformational changes are believeD to be inDuceD by the single natural substrate, C3bB, anD to result in realignment of the catalytic triaD, the specificity pocket, anD the nonspecific substrate binDing site, all of which have atypical conformations. Mutational stuDies have DefineD structural Determinants responsible for these unique structural features of Factor D anD for the resultant low reactivity with synthetic esters.
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structural basis of proFactor D activation from a highly flexible zymogen to a novel self inhibiteD serine protease Complement Factor D
The EMBO Journal, 1999Co-Authors: H Jing, Kevin Macon, John E Volanakis, Dwight Moore, Lawrence J Delucas, Sthanam V L NarayanaAbstract:The crystal structure of proFactor D, DetermineD at 2.1 A resolution with an Rfree anD an R-Factor of 25.1 anD 20.4%, respectively, Displays highly flexible or DisorDereD conformation for five regions: N-22, 71-76, 143-152, 187-193 anD 215-223. A comparison with the structure of its mature serine protease, Complement Factor D, revealeD major conformational changes in the similar regions. Comparisons with the zymogen-active enzyme pairs of chymotrypsinogen, trypsinogen anD prethrombin-2 showeD a similar Distribution of the flexible regions. However, proFactor D is the most flexible of the four, anD its mature enzyme Displays inactive, self-inhibiteD active site conformation. Examination of the surface properties of the N-terminus-binDing pocket inDicates that Ile16 may play the initial positioning role for the N-terminus, anD Leu17 probably also helps in inDucing the requireD conformational changes. This process, perhaps shareD by most chymotrypsinogen-like zymogens, is followeD by a Factor D-unique step, the re-orientation of an external Arg218 to an internal position for salt-briDging with Asp189, leaDing to the generation of the self-inhibiteD Factor D.
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structures of native anD complexeD Complement Factor D implications of the atypical his57 conformation anD self inhibitory loop in the regulation of specific serine protease activity
Journal of Molecular Biology, 1998Co-Authors: H Jing, John E Volanakis, Yarlagadda S Babu, David Moore, J M Kilpatrick, Sthanam V L NarayanaAbstract:Abstract Factor D is a serine protease essential for the activation of the alternative pathway of Complement. The structures of native Factor D anD a complex formeD with isatoic anhyDriDe inhibitor were DetermineD at resolution of 2.3 anD 1.5 A, respectively, in an isomorphous monoclinic crystal form containing one molecule per asymmetric unit. The native structure was compareD with structures DetermineD previously in a triclinic cell containing two molecules with Different active site conformations. The current structure shows greater similarity with molecule B in the triclinic cell, suggesting that this may be the Dominant Factor D conformation in solution. The major conformational Differences with molecule A in the triclinic cell are locateD in four regions, three of which are close to the active site anD incluDe some of the resiDues shown to be critical for Factor D catalytic activity. The conformational flexibility associateD with these regions is proposeD to proviDe a structural basis for the previously proposeD substrate-inDuceD reversible conformational changes in Factor D. The high-resolution structure of the Factor D/isatoic anhyDriDe complex reveals the binDing moDe of the mechanism-baseD inhibitor. The higher specificity towarDs Factor D over trypsin anD thrombin is baseD on hyDrophobic interactions between the inhibitor benzyl ring anD the aliphatic siDe-chain of Arg218 that is salt briDgeD with Asp189 at the bottom of the primary specificity (S1) pocket. Comparison of Factor D structural variants with other serine protease structures revealeD the presence of a unique “self-inhibitory loop”. This loop (214–218) Dictates the resting-state conformation of Factor D by (1) preventing His57 from aDopting active tautomer conformation, (2) preventing the P1 to P3 resiDues of the substrate from forming anti-parallel β-sheets with the non-specific substrate binDing loop, anD (3) blocking the accessibility of Asp189 to the positive1y chargeD P1 resiDue of the substrate. The conformational switch from resting-state to active-state can only be inDuceD by the single macromolecular substrate, C3b-bounD Factor B. This self-inhibitory mechanism is highly correlateD with the unique functional properties of Factor D, which incluDe high specificity towarD Factor B, low esterolytic activity towarD synthetic substrates, anD absence of regulation by zymogen anD serpin-like or other natural inhibitors in blooD.
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crystal structure of a Complement Factor D mutant expressing enhanceD catalytic activity
Journal of Biological Chemistry, 1995Co-Authors: Sthanam V L Narayana, John E VolanakisAbstract:Abstract Complement Factor D is a serine protease regulateD by a novel mechanism that DepenDs on conformational changes rather than cleavage of a zymogen for expression of proteolytic activity. The conformational changes are presumeD to be inDuceD by the single natural substrate, C3bB, anD to result in reversible reorientation of the catalytic center anD of the substrate binDing site of Factor D, both of which have atypical conformations. Here we report that replacement of Ser94, Thr214, anD Ser215 of Factor D (chymotrypsinogen numbering has been useD for comparison purposes) with the corresponDing resiDues of trypsin, Tyr, Ser, anD Trp, is sufficient to inDuce substantially higher catalytic activity associateD with a typical serine protease alignment of the catalytic triaD resiDues His57, Asp102, anD Ser195. These results proviDe a partial structural explanation for the low reactivity of “resting-state” Factor D towarD synthetic substrates.
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catalytic role of a surface loop of the Complement serine protease Factor D
Journal of Immunology, 1995Co-Authors: Sthanam V L Narayana, John E VolanakisAbstract:We have investigateD the structural Determinants of the unique functional properties of Complement Factor D by constructing anD testing a series of trypsin-like mutants of the enzyme. Mutational replacement of the primary substrate-binDing pocket of Factor D with that of trypsin resulteD in a mutant (M1) with greatly reDuceD proteolytic activity anD slightly reDuceD reactivity towarD small thioester substrates. Combining the M1 mutations with substitution of Tyr for Ser94, previously shown to enhance substantially both the proteolytic anD esterolytic activities of Factor D, proDuceD a mutant (M2) with reactivities similar to M1. Replacement of the surface loop formeD by resiDues 184-188 of M1 anD M2 with the corresponDing loop of trypsin proDuceD mutants exhibiting one anD two orDers of magnituDe higher esterolytic activity, respectively, than native Factor D. However, the proteolytic activity of both mutants was similar to that of M1 anD M2. We concluDe that loop184-188 is an important Determinant of the geometry of the primary specificity pocket of Factor D. The low proteolytic activity of these mutants supports the proposal that the proteolytically active conformation of Factor D is inDuceD by its natural substrate, C3bB.
Gary S Gilkeson - One of the best experts on this subject based on the ideXlab platform.
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Effects of Complement Factor D Deficiency on the renal Disease of MRL/lpr mice
Kidney International, 2020Co-Authors: Margaret K Elliott, Tambi Jarmi, Phillip Ruiz, Yuanyuan Xu, V. Michael Holers, Gary S GilkesonAbstract:Effects of Complement Factor D Deficiency on the renal Disease of MRL/ lpr mice. BackgrounD The alternative Complement pathway (AP) is activateD in inDiviDuals with lupus nephritis anD in murine moDels of systemic lupus erythematosus, incluDing MRL/ lpr mice. A previous stuDy from our laboratory evaluateD the Development of renal Disease in MRL/ lpr mice genetically Deficient in Factor B (Bf−/−), a protein necessary for AP activation. MRL/ lpr Bf−/− mice DevelopeD less renal Disease anD haD improveD survival; however, these mice were also a Different major histocompatibility complex (MHC) haplotype (H-2 b ) than their wilD-type littermates (H-2 k ) Due to the gene for Bf being locateD in the MHC gene complex. We unDertook the current stuDy to Determine if the DecreaseD renal Disease in MRL/ lpr Bf−/− mice was Due to the lack of AP activation or the H-2 b haplotype by stuDying the effects of Factor D (Df) Deficiency, a critical protein for AP activation, on Disease Development in MRL/ lpr mice. MethoDs Df-Deficient mice were backcrosseD with MRL/ lpr mice for four to nine generations. MRL/ lpr H-2 k Df−/−, Df+/-, anD Df+/+ littermates were evaluateD for Disease Development. Lack of AP activation in MRL/ lpr Df−/− mice was DetermineD by the zymosan assay. Serum creatinine levels were measureD using a creatinine kit. Proteinuria anD autoantiboDy levels were DetermineD by enzyme-linkeD immunosorbent assay (ELISA). Sections from one kiDney were staineD with fluorescein isothiocyanate (FITC) α-murine C3 or α-murine IgG to Detect C3 anD IgG Deposition. The remaining kiDney was cut in half with one half fixeD, sectioneD, anD staineD with hematoxylin anD eosin anD perioDic aciD-Schiff (PAS) to evaluate pathology anD another half fixeD in glutaralDehyDe anD examineD via electron microscopy. Results MRL/ lpr Df−/− mice haD similar glomerular IgG Deposition, proteinuria anD autoantiboDy levels, as Df+/+ anD Df+/- littermates. However, glomerular C3 Deposition, serum creatinine levels, anD pathologic renal Disease were significantly reDuceD in Df−/− mice. Despite the lack of renal Disease in Df−/− mice, life span was not impacteD by Factor D Deficiency. Conclusion The absence of Df anD AP activation is protective against the Development of proliferative renal Disease in MRL/ lpr mice suggesting the similar effect of Bf Deficiency in MRL/ lpr mice was also Due to the lack of AP activation.
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eliminating Complement Factor D reDuces photoreceptor susceptibility to light inDuceD Damage
Investigative Ophthalmology & Visual Science, 2007Co-Authors: Ba Rbel Rohrer, Kannan Kunchithapautham, Gary S GilkesonAbstract:PURPOSE. Genetic risk Factors such as variations in Complement Factors H (CFH) anD B (CFB) have been implicateD in the etiology of age-relateD macular Degeneration. It has been hypothesizeD that inaDequate control of Complement-Driven inflammation may be a major Factor in Disease pathogenesis. The authors testeD the involvement of the Complement system in an experimental moDel for oxiDative stress-meDiateD photoreceptor Degeneration, the light-Damage mouse moDel. METHODS. Changes in gene expression were assesseD in BALB/c retinas in response to constant-light (CL) exposure using microarrays anD real-time PCR. Susceptibility to CL exposure was testeD in CFD -/- mice on a BALB/c backgrounD. Eyes were analyzeD using electrophysiologic anD histologic techniques. RESULTS. Genes encoDing for proteins involveD in Complement activation were significantly upregulateD after CL. The altereD gene profiles were similar to proteins accumulateD in Drusen anD to genes iDentifieD in the retina anD RPE/choroiD of patients with age-relateD macular Degeneration. Cyclic-light reareD CFD -/- anD CFD +/+ mice haD inDistinguishable roD function anD number; however, after CL challenge, CFD -/- photoreceptors were significantly protecteD. CONCLUSIONS. These results suggest that roD Degeneration in the CL-DamageD retina involves the activity of the alternative Complement pathway anD that eliminating the alternative pathway is neuroprotective. Thus, the light Damage albino mouse moDel may be a gooD moDel to stuDy Complement-meDiateD photoreceptor Degeneration.
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effects of Complement Factor D Deficiency on the renal Disease of mrl lpr mice
Kidney International, 2004Co-Authors: Margaret K Elliott, Tambi Jarmi, Phillip Ruiz, Yuanyuan Xu, Michael V Holers, Gary S GilkesonAbstract:Effects of Complement Factor D Deficiency on the renal Disease of MRL/ lpr mice. BackgrounD The alternative Complement pathway (AP) is activateD in inDiviDuals with lupus nephritis anD in murine moDels of systemic lupus erythematosus, incluDing MRL/ lpr mice. A previous stuDy from our laboratory evaluateD the Development of renal Disease in MRL/ lpr mice genetically Deficient in Factor B (Bf−/−), a protein necessary for AP activation. MRL/ lpr Bf−/− mice DevelopeD less renal Disease anD haD improveD survival; however, these mice were also a Different major histocompatibility complex (MHC) haplotype (H-2 b ) than their wilD-type littermates (H-2 k ) Due to the gene for Bf being locateD in the MHC gene complex. We unDertook the current stuDy to Determine if the DecreaseD renal Disease in MRL/ lpr Bf−/− mice was Due to the lack of AP activation or the H-2 b haplotype by stuDying the effects of Factor D (Df) Deficiency, a critical protein for AP activation, on Disease Development in MRL/ lpr mice. MethoDs Df-Deficient mice were backcrosseD with MRL/ lpr mice for four to nine generations. MRL/ lpr H-2 k Df−/−, Df+/-, anD Df+/+ littermates were evaluateD for Disease Development. Lack of AP activation in MRL/ lpr Df−/− mice was DetermineD by the zymosan assay. Serum creatinine levels were measureD using a creatinine kit. Proteinuria anD autoantiboDy levels were DetermineD by enzyme-linkeD immunosorbent assay (ELISA). Sections from one kiDney were staineD with fluorescein isothiocyanate (FITC) α-murine C3 or α-murine IgG to Detect C3 anD IgG Deposition. The remaining kiDney was cut in half with one half fixeD, sectioneD, anD staineD with hematoxylin anD eosin anD perioDic aciD-Schiff (PAS) to evaluate pathology anD another half fixeD in glutaralDehyDe anD examineD via electron microscopy. Results MRL/ lpr Df−/− mice haD similar glomerular IgG Deposition, proteinuria anD autoantiboDy levels, as Df+/+ anD Df+/- littermates. However, glomerular C3 Deposition, serum creatinine levels, anD pathologic renal Disease were significantly reDuceD in Df−/− mice. Despite the lack of renal Disease in Df−/− mice, life span was not impacteD by Factor D Deficiency. Conclusion The absence of Df anD AP activation is protective against the Development of proliferative renal Disease in MRL/ lpr mice suggesting the similar effect of Bf Deficiency in MRL/ lpr mice was also Due to the lack of AP activation.
Xiangzhu Wang - One of the best experts on this subject based on the ideXlab platform.
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novel small molecule inhibitors targeting Complement Factor D for therapy of paroxysmal nocturnal hemoglobinuria
Blood, 2014Co-Authors: Paul B Morgan, Steven D. Podos, Jane A. Thanassi, Avinash Phadke, Venkat Gadhachanda, Godwin Pais, Akihiro Hashimoto, Qiuping Wang, Dawei Chen, Xiangzhu WangAbstract:INTRODUCTION: The Complement system is a pivotal player in multiple hematological conDitions that incluDe paroxysmal nocturnal hemoglobinuria (PNH). The current stanDarD of care for PNH is intravenous infusion of eculizumab, a humanizeD monoclonal antiboDy that targets the terminal Complement protein C5 anD thereby efficiently impairs intravascular hemolysis. However, a significant fraction of PNH patients in clinical practice responD incompletely to eculizumab Due to extravascular hemolysis by immune cells as eculizumab Does not prevent erythrocyte opsonization. ADDitionally, a non-responsive sub-population has been iDentifieD with a rare genetic polymorphism in C5 that renDers the variant incapable of binDing eculizumab. Therefore, an unmet meDical neeD remains for regimens that improve efficacy anD that can be aDministereD orally. To achieve this goal, we initiateD a Discovery program for small molecule inhibitors of Complement Factor D, a serine protease that is the rate limiting enzyme of the alternative Complement pathway. We have obtaineD high resolution (1.5 A) crystal structure of inhibitor-bounD Factor D. Through structure-guiDeD optimization, we have DiscovereD novel inhibitors of Factor D that possess high potency anD specificity as well as pharmacokinetic properties suitable for oral aDministration. Herein, we present a biochemical characterization of leaD compounDs anD stuDies of their activities in various Complement-meDiateD processes. METHODS: The binDing affinity to Factor D was DetermineD by surface plasmon resonance (Biacore) analysis. The inhibitory effect on Factor D protease was evaluateD biochemically using both a nonspecific synthetic substrate anD a natural substrate consisting of C3b anD the Complement Factor B. The alternative pathway (AP) hemolytic assay was conDucteD with unsensitizeD rabbit erythrocytes, 8% human or non-human primate serum, anD 10 mM MgEGTA. The classical pathway (CP) hemolytic assay was conDucteD with antiboDy-sensitizeD sheep erythrocytes anD 0.5% human serum. To assess inhibition of CP activation through the AP-DepenDent amplification loop, inhibition of terminal Complement complex (TCC) formation by 50% human serum was quantitateD following activation by the CP-specific activator HAIGG. Inhibition of Complement opsonization anD lysis of PNH-like erythrocytes exposeD to aciDifieD serum (Ham test) was assesseD ex vivo. Finally, selecteD leaD compounDs were aDministereD to non-human primates to evaluate the efficacy of Factor D inhibitors for blockaDe of alternative pathway-meDiateD events. RESULTS: LeaD compounDs obtaineD through optimization bounD to Factor D with KD values below 1 nM. Tight binDing to Factor D of the compounDs resulteD in potent inhibition of Factor D proteolytic activity assesseD using a synthetic nonspecific substrate as well as a natural substrate. When evaluateD in an AP hemolytic assay with human serum, the most potent compounDs inhibiteD cell lysis with EC50 values below 10 nM. Similar inhibitory activity was observeD when compounDs were assesseD in AP hemolytic assays with non-human primate serum, but not with roDent serum. The compounDs showeD no activity in a CP hemolytic assay using highly DiluteD human serum anD antiboDy-coateD erythrocytes, inDicating specificity for Factor D. When evaluateD in the presence of 50% human serum anD with TCC formation as an enDpoint, however, the compounDs blockeD approximately 75% of TCC formation upon CP activation, a consequence of blockage of the AP-DepenDent amplification loop. Furthermore, the compounDs preventeD both opsonisation anD lysis of PNH-like cells in the Ham test. Finally, when selecteD compounDs were aDministereD to non-human primates, potent inhibition of TCC formation following ex vivo AP activation was observeD with EC50 values below 50 nM. CONCLUSIONS: Highly potent anD specific Factor D inhibitors that block both the terminal pathway anD opsonization were DiscovereD using structure-guiDeD Design. These orally Deliverable inhibitors holD promise as a novel therapeutic approach for treatment of Complement-meDiateD hematological conDitions incluDing PNH. Disclosures Morgan: SweDish Orphan Biovitrum: Consultancy, Research FunDing; Glaxo Smith Kline: Consultancy; Achillion Pharmaceuticals: Consultancy, Research FunDing. Thanassi: Achillion Pharmaceuticals: Employment. PoDos: Achillion Pharmaceuticals : Employment. PhaDke: Achillion Pharmaceuticals: Employment. GaDhachanDa: Achillion Pharmaceuticals: Employment. Pais: Achillion Pharmaceuticals: Employment. Hashimoto: Achillion Pharmaceuticals: Employment. Wang: Achillion Pharmaceuticals: Employment. Chen: Achillion Pharmaceuticals: Employment. Wang: Achillion Pharmaceuticals: Employment. Agarwal: Achillion Pharmaceuticals: Employment. DeshpanDe: Achillion Pharmaceuticals: Employment. Huang: Achillion Pharmaceuticals: Employment. Wiles: Achillion Pharmaceuticals: Employment. Huang: Achillion Pharmaceuticals: Employment.
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preclinical evaluation of orally bioavailable small molecule inhibitors of Complement Factor D as a potential treatment for paroxysmal nocturnal hemoglobinuria
Blood, 2014Co-Authors: Jason Allan Wiles, Steven D. Podos, Jane A. Thanassi, Avinash Phadke, Venkat Gadhachanda, Godwin Pais, Akihiro Hashimoto, Qiuping Wang, Dawei Chen, Xiangzhu WangAbstract:BACKGROUND: Complement Factor D, a serine protease, plays an essential role in the activation of the alternative Complement pathway anD proviDes important amplification of the classical anD lectin Complement pathways. Cleavage of Factor B by Factor D generates C3 convertase that leaDs to opsonization of targeteD surfaces with Complement activation fragments anD to the formation of the terminal Complement complex (TCC); both events leaD to cell lysis. Complement Dysregulation unDerlies multiple hematological DisorDers incluDing paroxysmal nocturnal hemoglobinuria (PNH), which is characterizeD by Complement-meDiateD lysis of clonal populations of erythrocytes that lack glycophosphatiDylinositol-anchoreD Complement regulators. The current treatment for PNH is intravenous infusion of the anti-C5 monoclonal antiboDy eculizumab. Although eculizumab lessens intravascular hemolysis, it Does not prevent opsonization of erythrocytes anD subsequent extravascular hemolysis by immune cells. In contrast, Factor D inhibitors are expecteD to inhibit both terminal Complement pathway activation as well as opsonization anD shoulD, therefore, be well-positioneD to potentially serve this unmet meDical neeD. Herein, we present the preclinical evaluation of our small-molecule inhibitors of Factor D incluDing potency, off-target activities, metabolism, anD pharmacokinetic properties. METHODS & RESULTS: Initial inhibitors were DiscovereD through laboratory anD virtual screening efforts. A high-resolution (1.5 A) X-ray structure of an early proprietary inhibitor co-crystallizeD with Factor D aiDeD our optimization campaign that culminateD in compounDs with IC50 values below 100 nM in biochemical protease assays using natural anD non-specific substrates. The potent inhibitory effect of the compounDs on Factor D protease activity translateD to inhibition of cell lysis in an alternative pathway hemolytic assay with EC50 values as low as single-Digit nM. These small molecules are highly selective for Factor D anD DisplayeD no significant inhibitory effect on a panel of human serine proteases. In accorDance with the high selectivity, the inhibitors showeD minimal cellular toxicity, no effect on multiple human receptor-liganD interactions, anD no inhibition of hERG potassium channel current. Finally, oral anD intravenous aDministration of selecteD leaD compounDs to preclinical animal species showeD pharmacokinetic properties that suggest their suitability for oral Dosing in humans. CONCLUSIONS: We have DiscovereD highly active small-molecule inhibitors of Factor D that Demonstrate oral bioavailability anD low off-target activities. These key attributes, anD the potential to inhibit both intravascular anD extravascular hemolysis through inhibition of both terminal Complement pathway anD opsonization, position these inhibitors as promising Development canDiDates for the oral treatment of PNH. Disclosures Wiles:Achillion Pharmaceuticals: Employment. PoDos:Achillion Pharmaceuticals : Employment. Thanassi:Achillion Pharmaceuticals: Employment. PhaDke:Achillion Pharmaceuticals: Employment. GaDhachanDa:Achillion Pharmaceuticals: Employment. Pais:Achillion Pharmaceuticals: Employment. Hashimoto:Achillion Pharmaceuticals: Employment. Wang:Achillion Pharmaceuticals: Employment. Chen:Achillion Pharmaceuticals: Employment. Wang:Achillion Pharmaceuticals: Employment. Agarwal:Achillion Pharmaceuticals: Employment. Rivera:Achillion Pharmaceuticals: Employment. Elliot:Achillion Pharmaceuticals: Employment. Marlor:Achillion Pharmaceuticals: Employment. Zhang:Achillion Pharmaceuticals: Employment. DeshpanDe:Achillion Pharmaceuticals: Employment. Huang:Achillion Pharmaceuticals: Employment. Huang:Achillion Pharmaceuticals: Employment.
H Jing - One of the best experts on this subject based on the ideXlab platform.
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structural basis of proFactor D activation from a highly flexible zymogen to a novel self inhibiteD serine protease Complement Factor D
The EMBO Journal, 1999Co-Authors: H Jing, Kevin Macon, John E Volanakis, Dwight Moore, Lawrence J Delucas, Sthanam V L NarayanaAbstract:The crystal structure of proFactor D, DetermineD at 2.1 A resolution with an Rfree anD an R-Factor of 25.1 anD 20.4%, respectively, Displays highly flexible or DisorDereD conformation for five regions: N-22, 71-76, 143-152, 187-193 anD 215-223. A comparison with the structure of its mature serine protease, Complement Factor D, revealeD major conformational changes in the similar regions. Comparisons with the zymogen-active enzyme pairs of chymotrypsinogen, trypsinogen anD prethrombin-2 showeD a similar Distribution of the flexible regions. However, proFactor D is the most flexible of the four, anD its mature enzyme Displays inactive, self-inhibiteD active site conformation. Examination of the surface properties of the N-terminus-binDing pocket inDicates that Ile16 may play the initial positioning role for the N-terminus, anD Leu17 probably also helps in inDucing the requireD conformational changes. This process, perhaps shareD by most chymotrypsinogen-like zymogens, is followeD by a Factor D-unique step, the re-orientation of an external Arg218 to an internal position for salt-briDging with Asp189, leaDing to the generation of the self-inhibiteD Factor D.
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structures of native anD complexeD Complement Factor D implications of the atypical his57 conformation anD self inhibitory loop in the regulation of specific serine protease activity
Journal of Molecular Biology, 1998Co-Authors: H Jing, John E Volanakis, Yarlagadda S Babu, David Moore, J M Kilpatrick, Sthanam V L NarayanaAbstract:Abstract Factor D is a serine protease essential for the activation of the alternative pathway of Complement. The structures of native Factor D anD a complex formeD with isatoic anhyDriDe inhibitor were DetermineD at resolution of 2.3 anD 1.5 A, respectively, in an isomorphous monoclinic crystal form containing one molecule per asymmetric unit. The native structure was compareD with structures DetermineD previously in a triclinic cell containing two molecules with Different active site conformations. The current structure shows greater similarity with molecule B in the triclinic cell, suggesting that this may be the Dominant Factor D conformation in solution. The major conformational Differences with molecule A in the triclinic cell are locateD in four regions, three of which are close to the active site anD incluDe some of the resiDues shown to be critical for Factor D catalytic activity. The conformational flexibility associateD with these regions is proposeD to proviDe a structural basis for the previously proposeD substrate-inDuceD reversible conformational changes in Factor D. The high-resolution structure of the Factor D/isatoic anhyDriDe complex reveals the binDing moDe of the mechanism-baseD inhibitor. The higher specificity towarDs Factor D over trypsin anD thrombin is baseD on hyDrophobic interactions between the inhibitor benzyl ring anD the aliphatic siDe-chain of Arg218 that is salt briDgeD with Asp189 at the bottom of the primary specificity (S1) pocket. Comparison of Factor D structural variants with other serine protease structures revealeD the presence of a unique “self-inhibitory loop”. This loop (214–218) Dictates the resting-state conformation of Factor D by (1) preventing His57 from aDopting active tautomer conformation, (2) preventing the P1 to P3 resiDues of the substrate from forming anti-parallel β-sheets with the non-specific substrate binDing loop, anD (3) blocking the accessibility of Asp189 to the positive1y chargeD P1 resiDue of the substrate. The conformational switch from resting-state to active-state can only be inDuceD by the single macromolecular substrate, C3b-bounD Factor B. This self-inhibitory mechanism is highly correlateD with the unique functional properties of Factor D, which incluDe high specificity towarD Factor B, low esterolytic activity towarD synthetic substrates, anD absence of regulation by zymogen anD serpin-like or other natural inhibitors in blooD.