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α-Crystallin chaperone mimetic drugs inhibit lens γ-crystallin aggregation: Potential role for cataract prevention

2022; Elsevier BV; Volume: 298; Issue: 10 Linguagem: Inglês

10.1016/j.jbc.2022.102417

1083-351X

Sidra Islam, T. Michael, Brett S. Frank, Grant L. Hom, Samuel Wheeler, Hisashi Fujioka, Benlian Wang, Geeta Minocha, David R. Sell, Xingjun Fan, Kirsten J. Lampi, Vincent M. Monnier,

Advanced Glycation End Products research

Γ-Crystallins play a major role in age-related lens transparency. Their destabilization by mutations and physical chemical insults are associated with cataract formation. Therefore, drugs that increase their stability should have anticataract properties. To this end, we screened 2560 Federal Drug Agency–approved drugs and natural compounds for their ability to suppress or worsen H2O2 and/or heat-mediated aggregation of bovine γ-crystallins. The top two drugs, closantel (C), an antihelminthic drug, and gambogic acid (G), a xanthonoid, attenuated thermal-induced protein unfolding and aggregation as shown by turbidimetry fluorescence spectroscopy dynamic light scattering and electron microscopy of human or mouse recombinant crystallins. Furthermore, binding studies using fluorescence inhibition and hydrophobic pocket–binding molecule bis-8-anilino-1-naphthalene sulfonic acid revealed static binding of C and G to hydrophobic sites with medium-to-low affinity. Molecular docking to HγD and other γ-crystallins revealed two binding sites, one in the "NC pocket" (residues 50–150) of HγD and one spanning the "NC tail" (residues 56–61 to 168–174 in the C-terminal domain). Multiple binding sites overlap with those of the protective mini αA-crystallin chaperone MAC peptide. Mechanistic studies using bis-8-anilino-1-naphthalene sulfonic acid as a proxy drug showed that it bound to MAC sites, improved Tm of both H2O2 oxidized and native human gamma D, and suppressed turbidity of oxidized HγD, most likely by trapping exposed hydrophobic sites. The extent to which these drugs act as α-crystallin mimetics and reduce cataract progression remains to be demonstrated. This study provides initial insights into binding properties of C and G to γ-crystallins. Γ-Crystallins play a major role in age-related lens transparency. Their destabilization by mutations and physical chemical insults are associated with cataract formation. Therefore, drugs that increase their stability should have anticataract properties. To this end, we screened 2560 Federal Drug Agency–approved drugs and natural compounds for their ability to suppress or worsen H2O2 and/or heat-mediated aggregation of bovine γ-crystallins. The top two drugs, closantel (C), an antihelminthic drug, and gambogic acid (G), a xanthonoid, attenuated thermal-induced protein unfolding and aggregation as shown by turbidimetry fluorescence spectroscopy dynamic light scattering and electron microscopy of human or mouse recombinant crystallins. Furthermore, binding studies using fluorescence inhibition and hydrophobic pocket–binding molecule bis-8-anilino-1-naphthalene sulfonic acid revealed static binding of C and G to hydrophobic sites with medium-to-low affinity. Molecular docking to HγD and other γ-crystallins revealed two binding sites, one in the "NC pocket" (residues 50–150) of HγD and one spanning the "NC tail" (residues 56–61 to 168–174 in the C-terminal domain). Multiple binding sites overlap with those of the protective mini αA-crystallin chaperone MAC peptide. Mechanistic studies using bis-8-anilino-1-naphthalene sulfonic acid as a proxy drug showed that it bound to MAC sites, improved Tm of both H2O2 oxidized and native human gamma D, and suppressed turbidity of oxidized HγD, most likely by trapping exposed hydrophobic sites. The extent to which these drugs act as α-crystallin mimetics and reduce cataract progression remains to be demonstrated. This study provides initial insights into binding properties of C and G to γ-crystallins. Cataract prevalence increases with age and affects more than 60% of US citizens over the age of 79. Globally, it is estimated that more than 150 million eyes have visions less than 6/60 because of cataract (1Foster A. Vision 2020: the cataract challenge.Community Eye Health. 2000; 13: 17-19PubMed Google Scholar). Its incidence results in more than 18 million blinded individuals worldwide (2Weikel K.A. Garber C. Baburins A. Taylor A. Nutritional modulation of cataract.Nutr. Rev. 2014; 72: 30-47Crossref PubMed Scopus (78) Google Scholar). While good vision is generally obtained with intraocular lens implant, up to 10% of individuals develop complications such as posterior subcapsular opacification (3Konopinska J. Mlynarczyk M. Dmuchowska D.A. Obuchowska I. Posterior capsule opacification: a review of experimental studies.J. Clin. Med. 2021; 10: 2847Crossref PubMed Scopus (22) Google Scholar). However, cataract is insidious because blurred vision constitutes a long prodromal period prior to cataract surgery that is associated with increased car accidents, depression, risk of falls, and mortality (4Buckley J.G. Heasley K. Scally A. Elliott D.B. The effects of blurring vision on medio-lateral balance during stepping up or down to a new level in the elderly.Gait Posture. 2005; 22: 146-153Crossref PubMed Scopus (53) Google Scholar, 5Heasley K. Buckley J.G. Scally A. Twigg P. Elliott D.B. Falls in older people: Effects of age and blurring vision on the dynamics of stepping.Invest. Ophthalmol. Vis. Sci. 2005; 46: 3584-3588Crossref PubMed Scopus (38) Google Scholar, 6McGwin Jr., G. Owsley C. Gauthreaux S. The association between cataract and mortality among older adults.Ophthalmic Epidemiol. 2003; 10: 107-119Crossref PubMed Scopus (28) Google Scholar). Thus, there is an unmet pharmacological need as to the development of anticataract agents, whereby it has been estimated that delaying the onset of cataract by 10 years would halve the need for cataract surgery (7Brian G. Taylor H. Cataract blindness--challenges for the 21st century.Bull World Health Organ. 2001; 79: 249-256PubMed Google Scholar). A number of mechanisms contribute to the extraordinary longevity and transparency of the human lens, which include a quasi-anoxic milieu and reducing environment linked to high glutathione levels and antioxidant defenses (8Fan X. Monnier V.M. Whitson J. Lens glutathione homeostasis: discrepancies and gaps in knowledge standing in the way of novel therapeutic approaches.Exp. Eye Res. 2017; 156: 103-111Crossref PubMed Scopus (49) Google Scholar), the presence of UV filters (9Wood A.M. Truscott R.J. UV filters in human lenses: tryptophan catabolism.Exp. Eye Res. 1993; 56: 317-325Crossref PubMed Scopus (128) Google Scholar), and above all, the unique composition and supramolecular organization of its structural proteins, the α-, β-, and γ-crystallins. The concentration of the latter reaches 400 mg/ml in the nuclear part of the lens (10Vendra V.P. Khan I. Chandani S. Muniyandi A. Balasubramanian D. Gamma crystallins of the human eye lens.Biochim. Biophys. Acta. 2016; 1860: 333-343Crossref PubMed Scopus (67) Google Scholar), putting it at risk of spontaneous crystallization. This is however prevented by α-crystallins that are members of the small heat shock family of proteins, which act as molecular chaperones onto the βγ-crystallin family, thus shielding them for physical–chemical insults such as photo-oxidation and thermal stress (11Horwitz J. Alpha-crystallin can function as a molecular chaperone.Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10449-10453Crossref PubMed Scopus (1763) Google Scholar, 12Wang K. Spector A. Alpha-crystallin can act as a chaperone under conditions of oxidative stress.Invest. Ophthalmol. Vis. Sci. 1995; 36: 311-321PubMed Google Scholar). With advancing age, however, there is a progressive breakdown of all defenses, and crystallins are increasingly deamidated (13Vetter C.J. Thorn D.C. Wheeler S.G. Mundorff C. Halverson K. Wales T.E. et al.Cumulative deamidations of the major lens protein gammaS-crystallin increase its aggregation during unfolding and oxidation.Protein Sci. 2020; 29: 1945-1963Crossref PubMed Scopus (20) Google Scholar), oxidized (14Skouri-Panet F. Bonnete F. Prat K. Bateman O.A. Lubsen N.H. Tardieu A. Lens crystallins and oxidation: the special case of gammaS.Biophys. Chem. 2001; 89: 65-76Crossref PubMed Scopus (26) Google Scholar), glycated (15Fan X. Sell D.R. Hao C. Liu S. Wang B. 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Morimoto Y. et al.Age-related changes of alpha-crystallin aggregate in human lens.Amino Acids. 2007; 32: 87-94Crossref PubMed Scopus (41) Google Scholar), fragmentation, and generation of peptide fragments that enhance formation of light scattering aggregates (19Srivastava O.P. Srivastava K. Chaves J.M. Gill A.K. Post-translationally modified human lens crystallin fragments show aggregation in vitro.Biochem. Biophys. Rep. 2017; 10: 94-131PubMed Google Scholar). In this work, we have focused on finding small molecules that might protect the gamma crystallins from destabilization and aggregation. These proteins are thought to play a particular role in the transparency of lens nucleus. They are small monomeric densely packed proteins with an N- and C-terminal domain each consisting of two Greek keys making them quite resistant to thermal and other stresses (20Serebryany E. King J.A. The betagamma-crystallins: native state stability and pathways to aggregation.Prog. Biophys. Mol. Biol. 2014; 115: 32-41Crossref PubMed Scopus (81) Google Scholar). When such stress is applied, the N-terminal domain tends to unfold first (21Mills I.A. Flaugh S.L. Kosinski-Collins M.S. King J.A. Folding and stability of the isolated Greek key domains of the long-lived human lens proteins gammaD-crystallin and gammaS-crystallin.Protein Sci. 2007; 16: 2427-2444Crossref PubMed Scopus (88) Google Scholar), a process that is enhanced with certain cataract-prone mutations, such as the W43R mutation (22Serebryany E. King J.A. Wild-type human gammaD-crystallin promotes aggregation of its oxidation-mimicking, misfolding-prone W42Q mutant.J. Biol. Chem. 2015; 290: 11491-11503Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). By now, more than 31 and 15 cataract-associated SNPs and mutations have been described in mouse and human γ-crystallin, respectively (23Graw J. Mouse models of cataract.J. Genet. 2009; 88: 469-486Crossref PubMed Scopus (73) Google Scholar), and the list keeps growing (24Fu C. Xu J. Jia Z. Yao K. Chen X. Cataract-causing mutations L45P and Y46D promote gammaC-crystallin aggregation by disturbing hydrogen bonds network in the second Greek key motif.Int. J. Biol. Macromol. 2021; 167: 470-478Crossref PubMed Scopus (13) Google Scholar). In the end, the pharmacological challenge is to find ways to prevent the formation of the light scattering aggregates. However, the types and mechanisms of aggregation are heterogenous and fall into at least three categories (25Rocha M.A. Sprague-Piercy M.A. Kwok A.O. Roskamp K.W. Martin R.W. Chemical properties determine solubility and stability in betagamma-crystallins of the eye lens.Chembiochem. 2021; 22: 1329-1346Crossref PubMed Scopus (18) Google Scholar). The first consists of amyloid fibril formation that can be induced, for example, by exposing γD crystallins to low pH or photo-oxidative stress (26Wu J.W. Chen M.E. Wen W.S. Chen W.A. Li C.T. Chang C.K. et al.Comparative analysis of human gammaD-crystallin aggregation under physiological and low pH conditions.PLoS One. 2014; 9e112309Crossref Scopus (30) Google Scholar, 27Papanikolopoulou K. Mills-Henry I. Thol S.L. Wang Y. Gross A.A. Kirschner D.A. et al.Formation of amyloid fibrils in vitro by human gammaD-crystallin and its isolated domains.Mol. Vis. 2008; 14: 81-89PubMed Google Scholar). However, there is currently only limited evidence in support of amyloid fibril presence by EM in the aging human lens and cataract (M.J. Costello, personal communication, 2022) (28Alperstein A.M. Ostrander J.S. Zhang T.O. Zanni M.T. Amyloid found in human cataracts with two-dimensional infrared spectroscopy.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 6602-6607Crossref PubMed Scopus (51) Google Scholar). In contrast, amorphous aggregates are probably dominant in both age-related cataract (29Sharma K.K. Santhoshkumar P. Lens aging: Effects of crystallins.Biochim. Biophys. Acta. 2009; 1790: 1095-1108Crossref PubMed Scopus (255) Google Scholar)as well as heat and UV-induced aggregation models (30Roskamp K.W. Montelongo D.M. Anorma C.D. Bandak D.N. Chua J.A. Malecha K.T. et al.Multiple aggregation pathways in human gammaS-crystallin and its aggregation-prone G18V variant.Invest. Ophthalmol. Vis. Sci. 2017; 58: 2397-2405Crossref PubMed Scopus (16) Google Scholar). However, these amorphous aggregates may have multiple mechanisms of formation such as metal bridging (31Dominguez-Calva J.A. Perez-Vazquez M.L. Serebryany E. King J.A. Quintanar L. Mercury-induced aggregation of human lens gamma-crystallins reveals a potential role in cataract disease.J. Biol. Inorg. Chem. 2018; 23: 1105-1118Crossref PubMed Scopus (23) Google Scholar, 32Quintanar L. Dominguez-Calva J.A. Serebryany E. Rivillas-Acevedo L. Haase-Pettingell C. Amero C. et al.Copper and zinc ions specifically promote nonamyloid aggregation of the highly stable human gamma-D crystallin.ACS Chem. Biol. 2016; 11: 263-272Crossref PubMed Scopus (45) Google Scholar), domain swapping (33Das P. King J.A. Zhou R. Aggregation of gamma-crystallins associated with human cataracts via domain swapping at the C-terminal beta-strands.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 10514-10519Crossref PubMed Scopus (109) Google Scholar), and disulfide exchange (34Serebryany E. Yu S. Trauger S.A. Budnik B. Shakhnovich E.I. Dynamic disulfide exchange in a crystallin protein in the human eye lens promotes cataract-associated aggregation.J. Biol. Chem. 2018; 293: 17997-18009Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). In addition, native protein aggregates may also occur in which certain mutant proteins fall out of solution and precipitate or even crystallize out without major structural changes, as reported for the R58H and R36S mutants of human gamma D (HγD) (35Pande A. Pande J. Asherie N. Lomakin A. Ogun O. King J. et al.Crystal cataracts: human genetic cataract caused by protein crystallization.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6116-6120Crossref PubMed Scopus (218) Google Scholar). Based on the aforementioned understanding of the field, we have chosen to screen for compounds that can prevent a generic mixture of bovine gamma crystallins from forming amorphous aggregates upon exposure to thermal stress, with or without prior exposure to oxidant stress, followed by testing of the most active compounds against various recombinant native and mutant γ-crystallins. For the initial screen, a microtiter plate assay was developed in which bovine crystallins (2 mg/ml of 50 mM K3PO4 buffer, pH 7.2 [buffer A]) were exposed to H2O2 at varying concentrations (1–50 mM) for 12 to 36 h, upon which turbidity developed (at 50 mM H2O2 concentration) in both β- and γ-crystallins (Fig. 1, A and B). About 2560 compounds from the Microsource Discovery Library were initially tested at 500 μM in 5% dimethyl sulfoxide (DMSO) in buffer A for their ability to prevent H2O2 and heat mediated aggregation of purified bovine gamma crystallins (2 mg/ml) over 12 to 48 h. This was determined by turbidimetry at 600 nm (Fig. 1C) and confirmed using visual assessment of solution transparency over a fine text as well as the presence of cloudy aggregates visible by dark field microscopy (Fig. 1D). The drug:protein stoichiometric ratio was about 5:1. The wavelength of 600 nm was chosen to minimize interference by colored compounds, many of which absorb below 500 nm (Fig. 1E). A fitness plot was generated that resulted in a narrower choice of 135 potential anticataract drugs and 241 compounds with marked destabilizing and therefore potential cataractogenic effect (Fig. 1C). A table identifying all compounds with antiaggregation (n = 135) and proaggregation (n = 241) activity from Figure 1C is included (Table S10). Candidate compounds were rescreened using thermal denaturation upon heating at 72 °C for 20 min according to Chen et al. (36Chen Y. Zhao H. Schuck P. Wistow G. Solution properties of gamma-crystallins: compact structure and low frictional ratio are conserved properties of diverse gamma-crystallins.Protein Sci. 2014; 23: 76-87Crossref PubMed Scopus (23) Google Scholar) in order to select for compounds with chaperone rather than antioxidant activity since a large number of the latter already exist in the cataract field (37Abdelkader H. Alany R.G. Pierscionek B. Age-related cataract and drug therapy: opportunities and challenges for topical antioxidant delivery to the lens.J. Pharm. Pharmacol. 2015; 67: 537-550Crossref PubMed Scopus (54) Google Scholar). A subscreen involving 80 compounds (Fig. 1E) tested at 150 μM concentration resulted in nine compounds with thermal denaturation inhibitory activity. These include hematoporphyrin (H), tetrasodium sulfate (T), avocadene (V), chaulmoogric acid (M), hexachlorophene (X), bixin (B), closantel (C), gambogic acid (G), and dihydrogambogic acid (D). For refinement of the aforementioned screen, these compounds were tested at concentrations varying from 50 to 500 μM and 1.5 mg/ml concentration of recombinant HγD, human gamma S (HγS), its deamidation mutants γS_N14, γS_N76, γS_N143 and its triple mutant γSTM (Fig. 1F). In addition, other drugs and compounds that tested positive in the initial screen were also included, such as escin (E), hematein (I), citric acid (CA), docusate sodium (DS), and sennoside A (S) (Fig. S1B). Table 1 provides structural and other information about the most active candidate drugs, and Table 2 provides a summary of most active candidate drugs against thermal aggregation of γ-crystallins and their deamidation mutants. In these experiments, pool 1 (purified high–molecular weight [MW] bovine α-crystallins) was used as a positive control and found to potently inhibit both pool 4 protein (bovine γ-crystalline-rich fraction) and HγD aggregation, as similarly reported by multiple authors. For a review, see the study by Roskamp et al. (38Roskamp K.W. Paulson C.N. Brubaker W.D. Martin R.W. Function and aggregation in structural eye lens crystallins.Acc. Chem. Res. 2020; 53: 863-874Crossref PubMed Scopus (26) Google Scholar). Using the data generated in Figures 1F and S1B, mean inhibitory activity was calculated for all drugs across all γ-crystallins tested in order to determine the top six most active aggregation inhibitors. Based on 69, 64, 56, 54, 49, and 42% inhibition of γ-crystallin aggregation against thermal denaturation by drugs C, G, X, D, B, and M respectively, ranking was assigned in the order C > G > X > D > B > M with C being strongest and M weakest inhibitors of thermal aggregation (Fig. 1G and Table S1). Thus, drug C and drug G emerged as the strongest antidenaturation/aggregation and potential anticataract drugs. For the mechanistic studies below these two compounds together with either drug M, chosen mostly as negative control, or the entire set of six compounds were used for comparison purpose, whereby the words "drug" or "compound" are used interchangeably in the studies later.Table 1Summary of tested drugs displaying antiaggregation activity toward bovine γ-crystallin-rich proteins exposed to oxidant stress for 24 h and/or thermal stress at 72 °C for 20 minCompound nameMW (g)UsagePropertiesAntiaggregation activityProvenience, appearanceBixin (drug B)394.5Food colorAntineoplasticMedium heat shock inhibitorOrange colored solidClosantel (drug C)Two enantiomers C(R) and C(S)663.1Clinical drugAntihelminthicMedium strong dual inhibitorSynthetic, clear solid, colorlessChaulmoogric acid (drug M)280.4Experimental drugAntibacterial, antileproticDual heat shock inhibitor + antioxidantNatural product, colorless, oilyGambogic acid (drug G)628.7Experimental drugAnti-inflammatory, anticancer, multiple actionsStrong heat shock inhibitorNatural product, yellow solidDihydrogambogic (drug D)Acid (reduced G)630.8Experimental drugUndetermined activityStrong heat shock inhibitorModified natural product, solidHexachlorophene (drug X)406.9Clinical drugTopical antimicrobialMedium heat shock inhibitorSynthetic, solid, colorless Open table in a new tab Table 2Summary of the most active candidate drugs with antiaggregation activity against various γ-crystallins exposed to heating at 72 °C for 20 minCrystallin typeDrug codes in order of efficacyStoichiometric drug:protein ratio% SuppressionBovine γ-crystallinsaDenotes results from separate experiments.1.CGMDVX1.7:160/48/40/40/36/302.GCXBVD1.7:175/73/55/54/53/44HγDaDenotes results from separate experiments.1.GDCBXM1.7:169/66/65/63/61/382.DGCMBX1.7:178/75/73/73/60/47HγSCXDBGT1.7:171/68/61/60/46/40HγS_N14DCDGXBM1.7:177/76/73/60/51/48HγS_N76DCXDBMG1.7:170/60/59/57/55/55HγS_N143DCXGTDM1.7:152/50/48/42/30/25HγS_N14/76/143DGCXBMD1.7:189/76/71/46/46/32The proteins were tested at 75 μM concentration (1.5 mg/ml of 50 mM K+PO43−, pH 7.2) incubated for 20 min at 72 °C with 125 μM or less inhibitor concentration. This corresponds to 1.7:1 stoichiometric drug:protein ratio. For drug codes, refer to Table 1.a Denotes results from separate experiments. Open table in a new tab The proteins were tested at 75 μM concentration (1.5 mg/ml of 50 mM K+PO43−, pH 7.2) incubated for 20 min at 72 °C with 125 μM or less inhibitor concentration. This corresponds to 1.7:1 stoichiometric drug:protein ratio. For drug codes, refer to Table 1. His-tag free recombinant HγD crystallin produced in Escherichia coli using pET3d or pET17b expression vector was used to investigate the effects of compounds C and G against thermal-induced unfolding and aggregate formation. The antiaggregation potency of drug C and G was reconfirmed by dynamic light scattering (DLS) (Fig. 2A). Drug C and G showed excellent inhibition against aggregate formation as revealed by radius of aggregates of samples with and without drugs. There is a decrease in the size of aggregates from as high as 7261 nm without drugs to 30.5 nm with drug C and 84.9 nm with drug G. Drug M has moderate activity with mean aggregate size of 569 nm radius (Table S2). It should be noted that in comparison to the radius of unheated HγD crystallin (2.3 nm, 99%), the radius size was slightly increased with drug C (30.5) and G (84.9), suggesting that these drugs tend to prevent formation of larger size aggregates better than small ones. A DLS temperature scan from 30 to 90 °C confirmed the results with a hint that equimolar amounts of drug C and G combination are equally effective as either of them alone (Fig. 2B). Furthermore, we investigated whether these drugs can prevent thermal unfolding of HγD (Fig. 2C). The fluorescence ratio at 360 nm over 330 nm was used to determine the Tm. It is the temperature at which 50% of the protein unfolds. The Tm of HγD was found to be elevated by 4 °C, that is, from 81 °C for the control to 85 °C for both C- and G-treated protein (Fig. 2D). This suggests that these drugs are tentatively stabilizing the tertiary structure of protein and resisting thermal unfolding to a significant extent. Drug M showed no resistance to unfolding of HγD. The impact of drugs C and G on the morphology of the aggregates at 72 °C was assessed by transmission electron microscopy. Amorphous aggregates, similar to those previously reported in various mutant proteins (39Boatz J.C. Whitley M.J. Li M. Gronenborn A.M. van der Wel P.C.A. Cataract-associated P23T gammaD-crystallin retains a native-like fold in amorphous-looking aggregates formed at physiological pH.Nat. Commun. 2017; 815137Crossref Scopus (57) Google Scholar), were detected to various extents in all images. No amyloid fibers were found at 100 nm resolution (images available upon request). Using dark field microscopy, granular precipitates observed at 10 min (Fig. 2E) formed thick fibers at 30 min in the absence of drugs (see later, images available upon request). Bis-8-anilino-1-naphthalene sulfonic acid (bis-ANS)–binding studies showed that compounds C and G were most efficacious in competing with bis-ANS fluorescence, an indicator of hydrophobic surfaces in HγD (40Banerjee P.R. Puttamadappa S.S. Pande A. Shekhtman A. Pande J. Increased hydrophobicity and decreased backbone flexibility explain the lower solubility of a cataract-linked mutant of gammaD-crystallin.J. Mol. Biol. 2011; 412: 647-659Crossref PubMed Scopus (19) Google Scholar) (Fig. 2F). Table S3 provides details about percent inhibition in binding of bis-ANS by our selected drugs. The order in which these compounds suppress bis-ANS fluorescence matches the order of their antiaggregation activity. The ability of the drugs to stabilize mutants of HγD was then studied for W43R, R14C, and R58H (Fig. 2,G–I). These mutants were generated in our laboratory by site-directed mutagenesis using the primers listed in Table S4. After confirmation of the mutations by Sanger sequencing, the proteins were expressed, purified, and identified using SDS-PAGE (Fig. S2A) and Western blot (Fig. S2B), respectively. W43R is a very unstable mutant of HγD that is associated with autosomal dominant congenital cataract (41Ji F. Jung J. Koharudin L.M. Gronenborn A.M. The human W42R gammaD-crystallin mutant structure provides a link between congenital and age-related cataracts.J. Biol. Chem. 2013; 288: 99-109Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Among all four tryptophan residues, the one at position 43 is the most critical for its stability (22Serebryany E. King J.A. Wild-type human gammaD-crystallin promotes aggregation of its oxidation-mimicking, misfolding-prone W42Q mutant.J. Biol. Chem. 2015; 290: 11491-11503Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Drug G (400 and 500 μM), though not drug C, protected the mutant against aggregation when kept at 42 °C for 200 min. Drug C at all concentrations worsened the aggregation of this mutant (−34%, Fig. 2J), whereas drug M showed no effect at any of the concentrations (Fig. 2G). R14C is a well-studied mutant that is associated with juvenile onset hereditary cataract (42Pande A. Gillot D. Pande J. The cataract-associated R14C mutant of human gamma D-crystallin shows a variety of intermolecular disulfide cross-links: a Raman spectroscopic study.Biochemistry. 2009; 48: 4937-4945Crossref PubMed Scopus (28) Google Scholar). Because of the presence of an additional cysteine residue at position 14, it forms intermolecular disulfide crosslinked aggregates at physiological temperature and pH. Hence, to keep this protein stable, we added 5 mM DTT to the buffer. End-point turbidity assay (72 °C, 20 min) shows 68% prevention of thermal aggregation by drug C and 55% by drug G (Fig. 2,I and J). It should be noted that without DTT, the mutant was unstable even in the presence of drugs ruling out the possibility of using them to prevent the formation of disulfide linkages (data not shown). Drugs C and G were also found to protect R58H-HγD mutant (Fig. 2, H and J), which is associated with aceuliform cataract (43Basak A. Bateman O. Slingsby C. Pande A. Asherie N. Ogun O. et al.High-resolution X-ray crystal structures of human gammaD crystallin (1.25 A) and the R58H mutant (1.15 A) associated with aculeiform cataract.J. Mol. Biol. 2003; 328: 1137-1147Crossref PubMed Scopus (181) Google Scholar), against thermal aggregation (72 °C, 20 min) by 55 and 83%, respectively (Fig. 2J). The percent aggregation inhibition of the mutants of HγD by 500 μM of the drugs at the end point of turbidity assay is shown in Figure 2J. Upon heating HγD crystallin at 72 °C for 20 min, little change was observed in the far- and near-UV CD spectra suggesting that aggregation at that temperature is mostly unrelated to changes in secondary and tertiary structure (Fig. S2, C and D). In preparation for possible in vivo testing, the drugs were tested against native mouse γ-crystallins. Total gamma crystallins (2.5 mg/ml) were incubated with H2O2 for 18 h at 37 °C with increasing concentrations of drugs X, C, and G, or a combination thereof. Oxidative stress–mediated aggregation at 600 nm was suppressed by drug C but poorly by drug G (Fig. S4A, left panel). Drug C in combination with G or X was most effective, but these effects were observed only above 100 μM (Fig. S4A, right panel). The baseline increase at 50 μM could reflect a destabilizing effect of the drugs because of a pro-oxidative mechanism followed by stabilizing chaperone-like effect. Systematic comparison of recombinant WT mouse S (MγS) and its cataract-prone OPJ/F9S mutant (OPJ-MγS) showed that drugs C and G were potent aggregation suppressors at or above 50 μM concentration (Fig. S4, B–G). End-point heat-mediated turbidity assay (72 °C, 20 min) shows that drugs C, G, and D have antiaggregation activity toward both WT- and OPJ-MγS (Fig. S4B, left and right panels; Table S5). There is 52.6% more aggregation of OPJ-MγS in comparison to WT-MγS under same conditions of thermal stress. Similarly, DLS showed strong suppression of radius increase by C and G for both WT-MγS and OPJ-MγS (Fig. S4C,