Maurizio Brunori

It was a Lucky Strike to be working with Eraldo Antonini on hemoglobin and myoglobin when Jeffries Wyman arrived in Rome in 1961. I found myself connected with a number of creative scientists when the concept of allosteric control was conceived and gifted to the life science community. In retrospect, this was a demonstration of the skill and imagination of a few intelligent scientists that I happened to be close to. Those talents demonstrated the power of creativity as pictured by the motto “Mens agitat molem”; a celebration of humanism and intellect that paved the way to novel discoveries in the field of structure function relationships in proteins. I have presented hereby some of the events and the people as emerged from my memory over three decades of exciting scientific life.

Allostery arises when a ligand-induced change in shape of a binding site of a protein is coupled to a tertiary/quaternary conformational change with a consequent modulation of functional properties. The two-state allosteric model of Monod, Wyman and Changeux [J. Mol. Biol. 1965; 12, 88–118] is an elegant and effective theory to account for protein regulation and control. Tetrameric hemoglobin (Hb), the oxygen transporter of all vertebrates, has been for decades the ideal system to test for the validity of the MWC theory. The small ligands affecting Hb’s behavior (organic phosphates, protons, bicarbonate) are produced by the red blood cell during metabolism. By binding to specific sites, these messengers make Hb sensing the environment and reacting consequently. HbI and HbIV from trout and human HbA are classical cooperative models, being similar yet different. They share many fundamental features, starting with the globin fold and the quaternary assembly, and reversible cooperative O₂ binding. Nevertheless, they differ in ligand affinity, binding of allosteric effectors, and stability of the quaternary assembly. Here, we recollect essential functional properties and correlate them to the tertiary and quaternary structures available in the protein databank to infer on the molecular basis of the evolution of oxygen transporters.

“Can order spring from Chaos?” is the title of an extensive Report on Italian science published by NATURE on 12 May 1983 and written by Robert Walgate, the Chief European Correspondent. It is a twenty pages complete paper touching all aspects of the struggle of Italian scientists to work in the “curious amalgam of ingenuity and muddle, a reflection of the political system”. (Nature, 1983; 303: 109–128). To read it after four decades is interesting but somewhat depressing since the main problems unfolded in the paper have not been solved, starting with the largely insufficient support of fundamental curiosity driven research. At page 114 you could find a item called: ITALY’s TOP SCIENTISTS: Four in the top one thousand. The Author refers to the data reported by the ISI (Institute of Scientific Information) that took two years to scan 3,000 major journals over the period 1965–78 and covered 5 millions articles and 67 millions references. The four top Italian scientists working in Italy were: Eraldo Antonini (3127 citations), Enrico Clementi (4001), Silvio Garattini (2833), and Giorgio Giacomelli (2483); 3 out of four were 52 years old, and one 55. Antonini did not see the Report since he passed away on March 18, 1983. However the information leaked before the publication of Nature because I remember the Messaggero of Rome reporting a whole page with the ranking of the four Italians, and even a picture of Eraldo. The students of the first year Medical course, his Class, welcomed the Professor with a standing ovation. After a short time the Board of the SIB (Società Italiana di Biochimica) casted a unanimous vote in favour of the motion of President Noris Siliprandi to begin the annual Congress with an Antonini Lecture, forever. As reported below, the tradition began immediately at the Congress in Saint-Vicent, Italy, and is continuing. In this paper I report an account of the Eraldo Antonini Lectures that I attended over the years and until September 2019, a few months before the pandemics lock down.

The study of the mechanisms whereby proteins achieve their native functionally competent conformation has been a key issue in molecular biosciences over the last 6 decades. Nevertheless, there are several debated issues and open problems concerning some aspects of this fundamental problem. By considering the emerging complexity of the so-called “native state,” we attempt hereby to propose a personal account on some of the key topics in the field, ranging from the relationships between misfolding and diseases to the significance of protein disorder. Finally, we briefly describe the recent and exciting advances in predicting protein structures from their amino acid sequence.

Seventy years ago, we learned from Chris Anfinsen that the stereochemical code necessary to fold a protein is embedded into its amino acid sequence. In water, protein morphogenesis is a spontaneous reversible process leading from an ensemble of disordered structures to the ordered functionally competent protein; conforming to Aristotle's definition of substance, the synolon of matter and form. The overall process of folding is generally consistent with a two state transition between the native and the denatured protein: not only the denatured state is an ensemble of several structures, but also the native protein populates distinct functionally relevant conformational (sub)states. This two-state view should be revised, given that any globular protein can populate a peculiar third state called amyloid, characterized by an overall architecture that at variance with the native state, is by-and-large independent of the primary structure. In a nut shell, we should accept that beside the folded and unfolded states, any protein can populate a third state called amyloid which gained center stage being the hallmark of incurable neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases as well as others. These fatal diseases are characterized by clear-cut clinical differences, yet display some commonalities such as the presence in the brain of amyloid deposits constituted by one misfolded protein specific for each disease. Some aspects of this complex problem are summarized here as an excursus from the prion's fibrils observed in the brain of aborigines who died of Kuru to the amyloid detectable in the cortex of Alzheimer's patients.

Background: Behçet’s syndrome is a chronic, multi-system inflammatory disorder characterized by painful, recurrent oral ulcers (OU) and genital ulcers (GU).¹ The GU associated with Behçet’s syndrome can contribute to difficulties with sexual activity, walking, and sitting²; may cause scarring¹; and may impair quality of life.^1,2 Apremilast (APR), an oral phosphodiesterase 4 inhibitor, has demonstrated efficacy in the treatment of the OU associated with Behçet’s syndrome in the phase III, randomized RELIEF study (BCT-002).³ Objectives: To describe the efficacy of APR for the treatment of GU associated with active Behçet’s syndrome in the RELIEF study and in a pooled data analysis of RELIEF and the phase II study. Methods: Adult patients (≥18 years of age) with active Behçet’s syndrome and ≥3 OU at randomization or ≥2 OU at screening and randomization, without active major organ involvement, were randomized (1:1) to APR 30 mg twice daily or placebo (PBO). In RELIEF, clinical improvement in GU was assessed by evaluating the time to the first GU recurrence after loss of complete response, the mean number of GU in patients without GU at baseline, and the proportion of patients who were GU-free (complete response) at Week 12 (regardless of baseline GU status). A pooled analysis of patients in RELIEF and a randomized, phase II study⁴ were conducted to assess achievement of GU complete response in patients with GU at baseline. In patients with GU complete response before Week 12, the median time to the first GU recurrence after loss of complete response was based on Kaplan-Meier estimates. The mean number of GU was summarized descriptively using data as observed. Between-group differences in the proportion of patients who were GU-free at Week 12 were analyzed by Cochran-Mantel-Haenszel test using non-responder imputation to handle missing data. Statistical tests were 2 sided (α=0.05). Results: A total of 207 patients were randomized and received ≥1 dose of study medication (APR: n=104; PBO: n=103). In all, 17 patients in the APR group and 17 in the PBO group had GU at baseline, with mean GU counts of 2.9 (APR) and 2.6 (PBO). Among patients with GU at baseline in RELIEF, 12/17 (70.6% [APR]) and 7/17 (41.2% [PBO]) achieved GU complete response at Week 12 (P=0.110). The median time to first GU recurrence in these patients occurred earlier with PBO (6.1 weeks) vs. APR (not calculable). In the pooled analysis of RELIEF and the phase II study, a significantly greater proportion of patients with GU at baseline achieved GU complete response at Week 12 with APR vs. PBO (21/27 [77.8%] vs. 9/23 [39.1%]; P=0.011) (Figure 1). The proportion of patients who were GU-free was significantly greater with APR (92/104 [88.5%]) vs. PBO (72/101 [71.3%]), regardless of baseline number of GU (P=0.002) (Figure 2). Conclusion: The number of patients with GU was low, but the totality of the data shows a favorable trend in the treatment effect of APR on GU. Greater proportions of APR-treated patients were GU-free at Week 12 vs. patients receiving PBO, and the time to the first GU recurrence occurred earlier with PBO vs. APR. References: [1]Kokturk A. Patholog Res Int. 2012;2012:690390. 2. Senusi A, et al. Orphanet J Rare Dis. 2015;10:117. 3. Hatemi G, et al. N Engl J Med. 2019;381:1918-1928. 4. Hatemi G, et al. N Engl J Med. 2015;372:1510-1518. Disclosure of Interests: Gulen Hatemi Grant/research support from: BMS, Celgene Corporation, Silk Road Therapeutics – grant/research support, Consultant of: Bayer, Eli Lilly – consultant, Speakers bureau: AbbVie, Mustafa Nevzat, Novartis, UCB – speaker, Alfred Mahr Consultant of: Celgene, Speakers bureau: Roche, Chugai, Mitsuhiro Takeno Speakers bureau: Esai, Tanabe-Mitsubishi – speaker; Celgene Corporation – advisory board, Doyoung Kim: None declared, Melike Melikoglu: None declared, Sue Cheng Employee of: Amgen Inc. – employment; Celgene Corporation – employment at the time of study conduct, Shannon McCue Employee of: Amgen Inc. – employment; Celgene Corporation – employment at the time of study conduct, Sven Richter Employee of: Amgen Inc. – employment; Celgene Corporation – employment at the time of study conduct, Michele Brunori Employee of: Amgen Inc. – employment; Celgene Corporation – employment at the time of study conduct, Maria Paris Employee of: Amgen Inc. – employment; Celgene Corporation – employment at the time of study conduct, Mindy Chen Employee of: Amgen Inc. – employment; Celgene Corporation – employment at the time of the conduct, Yusuf Yazici Consultant of: BMS, Celgene Corporation, Genentech, Sanofi – consultant, Consultant of: BMS, Celgene Corporation, Genentech, Sanofi – consultant

Background: Oral ulcers (OU) associated with Behçet’s syndrome are often painful, may interfere with the ability to eat and can negatively affect quality of life.^1,2 Apremilast (APR), an oral phosphodiesterase 4 inhibitor, demonstrated efficacy in the treatment of OU associated with Behçet’s syndrome in a phase III, multicenter, randomized, double-blind, placebo (PBO)-controlled study (RELIEF; BCT-002).³ Objectives: To describe the efficacy of APR treatment in improving OU pain associated with Behçet’s syndrome in RELIEF. Methods: Patients were randomized (1:1) to APR 30 mg twice daily (APR 30 BID) or PBO twice daily for a 12-week PBO-controlled phase, followed by a 52-week active treatment extension. Eligible patients were ≥18 years of age and had active Behçet’s syndrome with ≥3 OU at randomization or ≥2 OU at screening and randomization and without active major organ involvement. Clinical improvement in OU was evaluated by the area under the curve for the number of OU through Week 12 (AUC_Wk0-12; primary efficacy endpoint) and by assessments of OU number. Patient-reported OU pain was evaluated by the 100-mm visual analogue scale (VAS). The statistical tests were 2-sided (α=0.05). The proportions of patients achieving the minimal clinically important difference (MCID) and higher rates of improvement, defined as ≥10-mm,⁴ ≥30-mm (3-fold MCID), ≥50-mm (5-fold MCID) improvements in OU pain VAS scores, respectively, were analyzed through Week 12. An ANCOVA model was used to analyze the primary endpoint and assessments of OU number and OU pain (VAS). The proportion of patients achieving improvement in OU pain VAS scores at Week 12 were summarized descriptively. Results: A total of 207 patients were randomized and received ≥1 dose of study medication (APR: n=104; PBO: n=103). At baseline, the mean (SD) number of OU was 4.2 (3.7) in the APR 30 BID group and 3.9 (2.7) in the PBO group, and the mean (SD) OU pain VAS scores were 61.2 (27.6) and 60.8 (26.9), respectively. At Week 12, significantly greater improvements were observed with APR 30 BID vs. PBO in AUC_Wk0-12 (least-squares [LS] mean [SE]: 129.5 [15.9] vs. 222.1 [15.9]; PP=0.0003) and OU pain VAS scores (LS mean [SE] change from baseline: −40.7 [3.3] vs. −15.9 [3.3]; PConclusion: For patients with active Behçet’s syndrome, APR 30 BID provided significantly greater improvements vs. PBO in OU number and OU pain at Week 12, including the greater proportion of patients achieving MCID and 3- and 5-fold MCID of OU pain in the APR 30 BID group vs. the PBO group. These results indicate a clinically meaningful treatment effect of APR 30 BID on the OU associated with Behçet’s syndrome. References: [1]Kokturk A. Patholog Res Int. 2012;2012:690390. [2]Hatemi G, et al. Ann Rheum Dis. 2008;67:1656-1662. [3]Hatemi G, et al. N Engl J Med. 2019;381:1918-1928. 4. Dworkin RH, et al. J Pain. 2008;9:105-121. Disclosure of Interests: Gulen Hatemi Grant/research support from: BMS, Celgene Corporation, Silk Road Therapeutics – grant/research support, Consultant of: Bayer, Eli Lilly – consultant, Speakers bureau: AbbVie, Mustafa Nevzat, Novartis, UCB – speaker, Alfred Mahr Consultant of: Celgene, Speakers bureau: Roche, Chugai, Mitsuhiro Takeno Speakers bureau: Esai, Tanabe-Mitsubishi – speaker; Celgene Corporation – advisory board, Doyoung Kim: None declared, Melike Melikoglu: None declared, Sue Cheng Employee of: Amgen Inc. – employment; Celgene Corporation – employment at the time of study conduct, Shannon McCue Employee of: Amgen Inc. – employment; Celgene Corporation – employment at the time of study conduct, Sven Richter Employee of: Amgen Inc. – employment; Celgene Corporation – employment at the time of study conduct, Michele Brunori Employee of: Amgen Inc. – employment; Celgene Corporation – employment at the time of study conduct, Maria Paris Employee of: Amgen Inc. – employment; Celgene Corporation – employment at the time of study conduct, Mindy Chen Employee of: Amgen Inc. – employment; Celgene Corporation – employment at the time of the conduct, Yusuf Yazici Consultant of: BMS, Celgene Corporation, Genentech, Sanofi – consultant, Consultant of: BMS, Celgene Corporation, Genentech, Sanofi – consultant