Defining the core structure of the alpha-lactalbumin molten globule state.

Molten globules are partially folded states of proteins which are generally believed to mimic structures formed during the folding process. In order to determine the minimal requirements for the formation of a molten globule state, we have prepared a set of peptide models of the molten globule state of human alpha-lactalbumin (alphaLA). A peptide consisting of residues 1-38 crosslinked, via the native 28-111 disulfide bond, to a peptide corresponding to residues 95-120 forms a partially folded state at pH 2.8 which has all of the characteristics of the molten globule state of alphaLA as judged by near and far UV CD, fluorescence, ANS binding and urea denaturation experiments. The structure of the peptide construct is the same at pH 7.0. Deletion of residues 95-100 from the construct has little effect. Thus, less than half the sequence is required to form a molten globule. Further truncation corresponding to the selective deletion of the A (residues 1-19) or D (residues 101-110) helices or the C-terminal 310 helix (residues 112-120) leads to a significant loss of structure. The loss of structure which results from the deletion of any of these three regions is much greater than that which would be expected based upon the non-cooperative loss of local helical structure. Deletion of residues corresponding to the region of the D helix or C-terminal 310 helix region results in a peptide construct which is largely unfolded and contains no more helical structure than is expected from the sum of the helicity of the two reduced peptides. These experiments have defined the minimum core structure of the alphaLA molten globule state.

An exceptionally stable helix from the ribosomal protein L9: implications for protein folding and stability.

The ribosomal protein L9 has an unusual structure comprising two compact globular domains connected by a 34 residue alpha-helix. The middle 17 residues of the helix are exposed to solvent while the first seven pack against and form part of the N-terminal domain, and the last ten form part of the C-terminal domain. Here we report results which show that a peptide corresponding to the central helix of L9 is monomeric in aqueous solution and >85% helical at 1 degrees C and 68(+/-7)% helical at 25 degrees C. This is considerably more helical than any other protein fragment studied to date. Another peptide corresponding to the middle 17 residues of the helix is monomeric and is 41(+/-4)% helical at 1 degrees C. Because the central helix has high intrinsic stability the globular N and C-terminal domains will likely be stabilized by their interactions with the helix. Therefore, the stability of the two terminal domains should not be completely independent because both domains gain stability from a shared structural element, the central helix. Also, the ability of the central helix to form native-like structure in isolation highlights a potential role for the helix in the early stages of the folding process.

Cooperative folding of a protein mini domain: the peripheral subunit-binding domain of the pyruvate dehydrogenase multienzyme complex.

The peripheral subunit-binding domain from the dihydrolipoamide acetyltransferase (E2) component of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus is stably folded, despite its short sequence of only 43 amino acid residues. A 41 residue peptide derived from this domain, psbd41, undergoes a cooperative thermal unfolding transition with a tm of 54 degrees C. This three-helix protein is monomeric as judged by ultracentrifugation and concentration-dependent CD measurements. Peptides corresponding to the individual helices are largely unstructured both alone and in combination, indicating that the unusual stability of this protein does not arise solely from unusually stable alpha-helices. Chemical denaturation by guanidine hydrochloride is also cooperative with a delta GH2O of 3.1 kcal mol-1 at pH 8.0 and 25 degrees C. The chemical denaturation is broad with an m-value of 760 cal mol-1 M-1. psbd41 contains a buried aspartate residue at position 34 that may provide stability and specificity to the fold. A mutant peptide, psbd41Asn was synthesized in which the buried aspartate residue was mutated to asparagine. This peptide still folds cooperatively and it is monomeric, but is much less thermostable than the wild-type with a tm of only 31 degrees C. Chemical denaturations at 4 degrees C give an m-value of 740 cal mol-1 M-1, similar to the wild-type, but the stability delta GH2O is only 1.4 kcal mol-1. Both the wild-type and the mutant unfold at extremes of pH, but at 4 degrees C psbd41Asn is folded over a narrower pH range than the wild-type. Although the mutant unfolds cooperatively by thermal and by chemical denaturation, its NMR spectrum is significantly broader than that of the wild-type and it binds ANS. These results show that Asp34 is vital for the stability and specificity of this structure, the second smallest natural sequence known to fold in the absence of disulfide bonds or metal or ligand-binding sites.

A randomized, double-blind, placebo-controlled, sixteen-week study of subcutaneous golimumab in patients with active nonradiographic axial spondyloarthritis.

OBJECTIVE: Axial spondyloarthritis (SpA) is a chronic inflammatory disease characterized by back pain and stiffness. The objective of this study was to determine whether golimumab is superior to placebo in patients with nonradiographic axial SpA. METHODS: This phase III, double-blind, randomized, placebo-controlled trial was performed to evaluate subcutaneous golimumab (50 mg) versus placebo in patients ages ≥18 years to ≤45 years who had active nonradiographic axial SpA according to the Assessment of SpondyloArthritis international Society (ASAS) criteria for ≤5 years since diagnosis, high disease activity, and an inadequate response to or intolerance of nonsteroidal antiinflammatory drugs. Patients were randomized 1:1 to receive golimumab or placebo subcutaneously every 4 weeks. The primary end point was 20% improvement according to the ASAS criteria (ASAS20) at week 16. Key secondary end points were an ASAS40 response, ASAS partial remission, 50% improvement in the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), and change in the Spondyloarthritis Research Consortium of Canada (SPARCC) magnetic resonance imaging (MRI) index for sacroiliac (SI) joint inflammation (SPARCC score). RESULTS: Of the 198 patients randomized, 197 were treated (97 received golimumab, and 100 received placebo). The mean age of the patients was 31 years, and 57.1% were male. At baseline, the mean ± SD BASDAI was 6.5 ± 1.5, the mean ± SD ASDAS was 3.5 ± 0.9, and the mean ± SD SPARCC score was 11.3 ± 14.0. The primary end point, an ASAS20 response, was achieved by significantly more patients in the golimumab group compared with the placebo group (71.1% versus 40.0%; P < 0.0001). An ASAS40 response was also achieved by significantly more patients in the golimumab group compared with the placebo group (56.7% versus 23.0%; P < 0.0001). The incidence of adverse events did not differ meaningfully between groups. CONCLUSION: Patients with active nonradiographic axial SpA treated with golimumab had significantly greater improvement in symptoms compared with patients treated with placebo. Golimumab was well tolerated and had a favorable risk/benefit profile.

Structural characterization of the tumor suppressor p16, an ankyrin-like repeat protein.

The p16 protein has been identified as a tumor suppressor that functions by inhibiting the cyclin-dependent kinases CDK4 and CDK6. Deletions or point mutations in the p16 gene have been found in a number cancers, emphasizing its importance in regulating cell cycle progression. Inhibition by p16 occurs through protein-protein interactions with its targets. This is not surprising, since p16 is thought to contain ankyrin-like repeats, motifs implicated in protein-protein interactions. Our goal was to identify structural characteristics of p16 not only as an important step towards understanding CDK4 inhibition but also to explore the role of ankyrin repeats in the p16 structure, as no detailed structure of any protein containing these motifs has been reported. We have expressed, refolded, and purified p16 from E. coli and have shown it to be functionally active by specific binding to CDK4. Analytical ultracentrifugation has shown that p16 weakly self-associates to form dimers with a Kd = 270 microM. The CD spectrum indicates that the protein is composed of 33% alpha-helix, 22% beta-sheet, 19% beta-turn, and 27% other (which includes aromatic and random coil contributions). Further CD experiments suggest that p16 exhibits low structural stability with a delta G of -2.3 kcal/mol. This weak stability is a consequence of a highly dynamic structure as measured by ANS-binding, NMR hydrogen-deuterium exchange, and fluorescence. It is possible that a well-defined tertiary structure is imparted upon the binding of p16 to CDK4.

Mixed connective tissue disease in children and adolescents.

Since the recognition of mixed connective tissue disease in 1972, a small number of pediatric and adolescent patients with this disorder have been described in detail. Four additional patients, two male and two female, are reported in this study. Age range at onset of symptoms was 11 to 18 years. All had arthritis or arthralgia, cervical lymphadenopathy, and hypergammaglobulinemia. Skin rashes and restrictive pulmonary disease were present in three; Raynaud phenomenon, skeletal myopathy, elevation of serum IgE, and neurologic abnormalities were present in two of the four patients. One patient had cardiomyopathy which was progressive over three years. Renal function was normal in all four patients. Most manifestations of the disease in these patients appeared to respond to variable doses of corticosteroids. A review of 234 reported patients with mixed connective tissue disease, including 50 pediatric patients, suggests a higher prevalence of renal and cardiac disease in affected children and adolescents. A multicenter cooperative study of patients with mixed connective tissue disease is strongly recommended to determine the natural history and the effects of therapeutic interventions in this disease.

Further assessment of the clinically effective dose range of etoricoxib: a randomized, double-blinded, placebo-controlled trial in rheumatoid arthritis.

OBJECTIVE: To further assess the clinically active dose range of etoricoxib, a COX-2 selective inhibitor, in rheumatoid arthritis (RA). METHODS: RA patients were randomized to etoricoxib 10, 30, 60, or 90 mg or placebo in a double-blind, 12-week study. DMARDs (methotrexate, biologics) or low-dose corticosteroids were allowed in stable doses. The primary endpoint was the proportion of patients completing the study and achieving an American College of Rheumatology 20% (ACR20) response. Secondary endpoints included individual components of the ACR index and Patient Global Assessment of Pain. Safety was assessed by physical exam and adverse experiences (AEs) occurrences. RESULTS: Etoricoxib 90 mg was the only dose to reach a statistically significant difference from placebo (p < 0.001) on the primary endpoint; etoricoxib 60 mg approached significance (p = 0.057). Significant pain improvement vs. placebo was observed with etoricoxib 90 mg (p < 0.001), 60 mg (p = 0.018), and 30 mg (p = 0.017). Despite the use of background biologics and corticosteroids, a dose response was still apparent. A higher proportion of etoricoxib 60 and 90 mg patients had renovascular AEs (i.e., edema and hypertension) compared with placebo, although discontinuations for renovascular AEs were rare. Etoricoxib 90 mg had a higher incidence of serious AEs (n = 5; 1 was considered drug-related) versus placebo (n = 0). LIMITATIONS: The present study was not powered to detect differences in cardiovascular or gastrointestinal safety by dose. Additionally, further research is needed to clarify the role of doses less than the etoricoxib 90 mg dose for pain management in RA patients. CONCLUSION: Etoricoxib 90 mg demonstrated statistically superior efficacy (ACR20) compared with placebo and numerical superiority over the other doses of etoricoxib studied. Etoricoxib 30 and 60 mg demonstrated significant pain improvement versus placebo, suggesting utility for some patients.

A randomized placebo-controlled trial comparing the efficacy of etoricoxib 30 mg and ibuprofen 2400 mg for the treatment of patients with osteoarthritis.

OBJECTIVE: We compared the efficacy of etoricoxib 30 mg to placebo and ibuprofen 2400 mg for the treatment of osteoarthritis (OA) of the hip and knee. DESIGN: In this 12-week, randomized, double-blind, placebo- and active-comparator-controlled trial, 548 patients (median age 63 years) with OA of the hip or knee were randomized to receive placebo, etoricoxib 30 mg q.d., or ibuprofen 800 mg t.i.d. Demonstration of etoricoxib's efficacy vs placebo and comparison of its efficacy to ibuprofen were assessed using three co-primary endpoints: Western Ontario and McMaster's University Osteoarthritis Index (WOMAC) Pain Subscale (WOMAC-PS); WOMAC Physical Function Subscale (WOMAC-PFS); and Patient Global Assessment of Disease Status (PGADS). Each primary endpoint utilizes a 0-100 mm visual analog scale. To demonstrate comparable efficacy of etoricoxib vs ibuprofen, the 95% confidence intervals (CIs) for the difference in the least squares (LS) mean change over 12 weeks for all three co-primary endpoints had to fall within +/-10 mm. Safety and tolerability data were collected throughout the study. RESULTS: Mean baseline values for the three co-primary endpoints ranged from 62.52 to 70.14 mm. Both etoricoxib and ibuprofen demonstrated superior (P< or =0.002) efficacy for all primary endpoints. The LS mean (mm) changes (95% CI) over 12 weeks for etoricoxib and ibuprofen, respectively, compared to placebo were given as follows: WOMAC-PS: -11.66 (-16.31, -7.01) and -7.62 (-12.30, -2.94); WOMAC-PFS: -10.15 (-14.74, -5.57) and -7.23 (-11.85, -2.61); PGADS: -11.65 (-16.81, -6.50) and -8.11 (-13.30, -2.92). The efficacy of etoricoxib 30 mg was comparable to ibuprofen 2400 mg. All treatments were similarly well tolerated. CONCLUSION: Treatment with etoricoxib 30 mg q.d. provides superior efficacy vs placebo and comparable clinical efficacy vs ibuprofen 2400 mg (800 mg t.i.d.) for the treatment of OA of the hip and knee.

A mutational study of the peptide-binding domain of Hsc70 guided by secondary structure prediction.

The abundant, cytoplasmic molecular chaperones of eukaryotic cells, of which mammalian Hsc70 is a member, have central roles in protein folding pathways in cells. Although substantial information is now available on substrate interactions and ATPase activity, neither the crystal structure of the intact Hsc70 molecule nor its isolated peptide-binding domain is known. Recently, the crystal structure of the isolated peptide-binding domain of an evolutionary relative of mammalian Hsc70, the DnaK protein of Escherichia coli, was solved. We have generated several rat Hsc70 mutants using site-directed and cassette mutagenesis guided by secondary structure predictions to test the hypothesis that the peptide-binding domains of mammalian Hsc70 and DnaK have similar molecular structures. Biochemical properties along with the ATPase and peptide binding activities of the resulting recombinant proteins were determined. Biochemical analyses included one- and two-dimensional gel electrophoresis, electrospray mass spectrometry, and N-terminal amino acid sequencing. The results of our study suggest that the DnaK molecular structure is a useful working model for the mammalian Hsc70 peptide-binding domain. Evidence is provided that (i) small additions to the N terminus of Hsc70 alter the function of the peptide-binding domain, (ii) alterations in the C-terminal tetrapeptide EEVD result in dramatic increases in basal ATPase activity, (iii) polyalanine substitution of a helical connector segment compensates for changes at the C terminus to restore near-normal function, (iv) specific side chain interactions involving this connector segment are not required for peptide-stimulated ATPase activity, and (v) disruption of the cap homologue region inhibits peptide binding by Hsc70.

Calcium binding peptides from alpha-lactalbumin: implications for protein folding and stability.

The calcium binding protein alpha-lactalbumin folds via a molten globule intermediate. Calcium does not bind strongly to the unfolded protein or the molten globule, but does bind to the transition state between the molten globule and the native protein. Of interest are the structures formed in the transition state that promote calcium binding. To study the importance of local secondary structure on calcium binding, we have synthesized two peptides corresponding to the calcium binding site that include the flanking C-helix and 3(10)-helix. The first peptide, elbow-A, consists of residues 72-100 from bovine alpha-lactalbumin, but with Cys 73, Cys 77, and Cys 91 replaced by alanines. In the second peptide, denoted elbow, the cysteines at position 73 and 91 are included and the nativelike disulfide bond is formed. Both peptides are monomeric and unstructured in aqueous solution and bind calcium weakly with apparent K(d)'s on the order of 10(-2) M. In 50% trifluoroethanol (v/v), the peptides are 45% helical as judged by CD. NMR studies performed on elbow and elbow-A in TFE indicate that the helical structure is confined to the C-helix. In this solvent system elbow binds calcium one-to-one with a K(d) of 50 microM. Removing the disulfide bond reduces, but does not eliminate calcium binding (K(d) = 170 microM in 50% TFE). These results suggest that formation of the C-helix promotes calcium binding and may be a key determinant of calcium binding in the transition state.

Prevalence of back pain and joint problems in a manufacturing company.

To estimate prevalence of back pain and joint problems in employees of a chemical manufacturing company, a questionnaire was administered during medical surveillance examinations between 1987 and 1989. Among 5903 employees completing the questionnaire 35.4% reported back or joint pain during the past year. Back pain lasting 30 days or more occurred in 5.3% of employees, while joint pain and/or swelling occurred in 19.3% of employees. A physician visit was involved for 10.5% and 11.1% of employees reporting back pain and joint problems respectively. A trend of increasing prevalence with increasing age was significant (P less than .001) for all musculoskeletal outcomes. Unadjusted prevalence of back pain and joint problems was significantly higher among men and among whites. After adjusting for age, race, and occupation using logistic regression, the difference in prevalence for the two sexes was diminished or reversed. Similarly, differences in race were diminished when other variables were controlled. Differences in prevalence by occupation were attenuated after adjustment for age, gender, and race. Back pain tended to be reported more frequently for managers, back pain and joint problems for technicians, and back pain requiring physician visit for craftsmen. Self-reported back pain and joint problems during the previous year vary more by age and occupation and less by gender and race in this employed population.

An ex vivo angiogenesis assay utilizing commercial porcine carotid artery: modification of the rat aortic ring assay.

The study of angiogenesis as a therapeutic target requires a reliable, physiologically relevant, and technically straightforward assay. An ex vivo assay bridges the gap between cell-based assays, which may not realistically represent the complex process of vessel sprouting, and in vivo assays, which are time consuming and expensive. Porcine carotid arteries provide an ideal tissue source for angiogenesis inhibitor screens due to their availability, physiological relevance and large size. 1.5 mm2 fragments of porcine carotid arteries were incubated in 48-well culture plates and sandwiched between two 100 microliters layers of Matrigel. Sprouting was observed from the explants and quantitated, using a digital imaging system, after two weeks of incubation. Histological analysis using Factor VIII-related antigen (von Willebrand Factor) as an endothelial cell-specific marker identified these sprouts, which were consistent with endothelial cell morphology, supporting the system as a model of angiogenesis. Accordingly, the angiogenesis inhibitors suramin, 2-methoxyestradiol, and the matrix metalloprotease inhibitor Batimastat were shown to completely inhibit sprouting at 50, 0.5, and 5.0 micrograms/ml, respectively and to have ED50 values of 23, 0.15, and 0.14 microgram/ml. This assay shows good reproducibility and eliminates animal to animal variation. The system should prove adaptable to other forms of angiogenic stimulation, ultimately making a variety of assays for angiogenesis available to laboratories of limited resources.

Biochemical characterization of WrbA, founding member of a new family of multimeric flavodoxin-like proteins.

The protein WrbA had been identified as an Escherichia coli stationary-phase protein that copurified and coimmunoprecipitated with the tryptophan repressor. Sequences homologous to WrbA have been reported in several species of yeast and plants. We previously showed that this new family of proteins displays low but structurally significant sequence similarity with flavodoxins and that its members are predicted to share the alpha/beta core of the flavodoxin fold but with a short conserved insertion unique to the new family, which could account for reports that some family members may be dimeric in solution. The general sequence similarity to flavodoxins suggests that the members of the new family might bind FMN, but their wide evolutionary distribution indicates that, unlike the flavodoxins, these proteins may be ubiquitous. In this paper, we report the purification and biochemical characterization of WrbA, demonstrating that the protein binds FMN specifically and is a multimer in solution. The FMN binding constant is weaker than for many flavodoxins, being approximately 2 microM at 25 degreesC in 0. 1 mM sodium phosphate, pH 7.2. The protein participates in a dimer-tetramer equilibrium over a wide range of solution conditions, with a midpoint at approximately 1.4 microM. One FMN binds per monomer and has no apparent effect on the multimerization equilibrium. WrbA has no effect on the affinity or mode of DNA binding by the tryptophan repressor; thus, its physiological role remains unclear. Although many proteins with flavodoxin-like domains are known to be multimers, WrbA is apparently the first characterized case in which multimerization is associated directly with the flavodoxin-like domain itself.

Thermodynamic analysis of a designed three-stranded coiled coil.

The study and successful design of coiled-coil protein structural motifs have provided much insight into the rules governing protein folding and stability. In this work we use a thermodynamic approach to quantitate the rules that govern the specific oligomerization of coiled coils. We have designed a highly stable trimeric coiled coil by placing valine residues at each a position and leucine residues at each d position of the heptad repeating unit. The peptide forms a very stable trimer as determined by sedimentation equilibrium, and the concentration dependence of its circular dichroism spectrum follows a cooperative monomer/dimer/trimer equilibrium with the dimer state as a highly unstable intermediate. Its guanidinium chloride denaturation curve was collected at several peptide concentrations, and analysis of the data confirms the cooperativity of the trimerization process and provides a free energy of stabilization of - 18.4 kcal mol-1 for the trimer. The heat capacity, delta Cp, was measured by global analysis of thermal unfolding data collected at a number of guanidinium chloride concentrations. Guanidinium chloride induces cold denaturation in the thermal unfolding curves, providing a reasonably well-determined value for delta Cp of 750 cal deg-1 mol-1. This translates to a delta Cp of 8.6 cal deg-1 mol-1 per residue and corresponds well to that expected of a coiled coil with a well-defined tertiary structure.

Structure and stability of the N-terminal domain of the ribosomal protein L9: evidence for rapid two-state folding.

The N-terminal domain, residues 1-56, of the ribosomal protein L9 has been chemically synthesized. The isolated domain is monomeric as judged by analytical ultracentrifugation and concentration-dependent CD. Complete 1H chemical shift assignments were obtained using standard methods. 2D-NMR experiments show that the isolated domain adopts the same structure as seen in the full-length protein. It consists of a three-stranded antiparallel beta-sheet sandwiched between two helixes. Thermal and urea unfolding transitions are cooperative, and the unfolding curves generated from different experimental techniques, 1D-NMR, far-UV CD, near-UV CD, and fluorescence, are superimposable. These results suggest that the protein folds by a two-state mechanism. The thermal midpoint of folding is 77 +/- 2 degrees C at pD 8.0, and the domain has a delta G degree folding = 2.8 +/- 0.8 kcal/mol at 40 degrees C, pH 7.0. Near the thermal midpoint of the unfolding transition, the 1D-NMR peaks are significantly broadened, indicating that folding is occurring on the intermediate exchange time scale. The rate of folding was determined by fitting the NMR spectra to a two-state chemical exchange model. Similar folding rates were measured for Phe 5, located in the first beta-strand, and for Tyr 25, located in the short helix between strands two and three. The domain folds extremely rapidly with a folding rate constant of 2000 s-1 near the midpoint of the equilibrium thermal unfolding transition.

Comparison of bax, waf1, and IMP dehydrogenase regulation in response to wild-type p53 expression under normal growth conditions.

Recently, we demonstrated that downregulation of inosine-5'-monophosphate dehydrogenase (IMPD; IMP:NAD oxidoreductase, EC 1.2.1.14), the rate-limiting enzyme for guanine nucleotide biosynthesis, is required for p53-dependent growth suppression. These studies were performed with cell lines derived from immortal, nontumorigenic fibroblasts that express wild-type p53 conditionally by virtue of a metal-responsive promoter. Here, the p53-dependent properties of the original "p53-inducible" fibroblasts are presented in detail and compared to related properties of epithelial cells that also express wild-type p53 conditionally, but by virtue of a temperature-responsive promoter. Both types of p53-inducible cells were designed to approximate normal physiologic relationships between the host cell and the regulated p53 protein. Together, they were used to investigate expression relationships between IMPD and other p53-responsive genes proposed as mediators of p53-dependent growth suppression. In both types of cells, IMPD activity, protein, and mRNA were consistently coordinately reduced in response to p53 expression. In contrast, mRNAs for waf1, bax, and mdm2 showed disparate patterns of expression, being induced in one conditional cell type, but not the other. This distinction in regulation pattern suggests that under normal growth conditions, unlike IMPD downregulation, bax and waf1 induction is not a rate-determining event for p53-dependent growth suppression.

Mimicry of erythropoietin by a nonpeptide molecule.

Erythropoietin (EPO) controls the proliferation and differentiation of erythroid progenitor cells into red blood cells. EPO induces these effects by dimerization of the EPO receptors (EPOR) present on these cells. To discover nonpeptide molecules capable of mimicking the effects of EPO, we identified a small molecule capable of binding to one chain of EPOR and used it to synthesize molecules capable of inducing dimerization of the EPOR. We first identified compound 1 (N-3-[2-(4-biphenyl)-6-chloro-5-methyl]indolyl-acetyl-L-lysine methyl ester) by screening the in-house chemical collection for inhibitors of EPO binding to human EPOR and then prepared compound 5, which contains eight copies of compound 1 held together by a central core. Although both compounds inhibited EPO binding of EPOR, only compound 5 induced dimerization of soluble EPOR. Binding of EPO to its receptor in cells results in activation of many intracellular signaling molecules, including transcription factors like signal transducer and activator of transcription (STAT) proteins, leading to growth and differentiation of these cells. Consistent with its ability to induce dimerization of EPOR in solution, compound 5 exhibited much of the same biological activities as EPO, such as (i) the activation of a STAT-dependent luciferase reporter gene in BAF3 cells expressing human EPOR, (ii) supporting the proliferation of several tumor cell lines expressing the human or mouse EPOR, and (iii) the in vitro differentiation of human progenitor cells into colonies of erythrocytic lineage. These data demonstrate that a nonpeptide molecule is capable of inducing EPOR dimerization and mimicking the biological activities of EPO.