Drug Discovery & Vaccine Development

Drug discovery & vaccine development

Martino Bolognesi Dept. of BioSciences University of Milano, Italy [email protected] http://users.unimi.it/biolstru/Home.html

1 Macromolecular crystallography relies on the earlier discovery that inorganic crystals could diffract X‐rays, a new phenomenon, not observed before 1912 in nature ...

Max von Laue 1914 Physics Nobel Laureate

... and on the discovery –dating back to the 19th century ‐ that proteins could assemble into crystals, a phenomenon occasionally occurring in nature, but rarely observed.

2 3 However, it was only in the 1920’s that the true proteic content of these crystals was proven.

The Nobel Prize in Chemistry 1946 was divided, one half awarded to James Sumner "for his discovery that enzymes can be crystallized", the other half jointly to John Howard Northrop and Wendell Meredith Stanley "for their preparation of enzymes and virus proteins in a pure form". 4 5

It was shortly after (1930‐40’s) that a pioneering effort in the UK highlighted the possibility of applying crystallography to complex molecules such as insulin and hemoglobin

M.F. Perutz J.D. Bernal

D. Crowfoot Hodgkin 7 Nobel Laureates linked to X‐ray protein structural biology • Perutz & Kendrew, 1962 for Hb and Mb • Hodgkin, 1964 for VitB12 ... • Deisenhofer, Michel & Huber, 1988 for photosynthetic reaction center

• Walker, 1997 for F0F1 ATP synthase • Mac Kinnon, 2003 for K+ channels • Ramakrishnan, Steitz, Yonat, 2009 for Ribosome • Kobilka & Lefkowictz, 2012 for GPCR • ( ...Lipscomb (1976), Klug (1982), Hauptam & Karle (1985), Kornberg (2006) ...)

8 2009 Chemistry Nobel Laureates

V. Ramakrishnan T.A. Steitz A.E. Yonath

9 (Courtesy A.Yonath) Myoglobin 16 kDa

Ribosome 2.5 MDa 10 Key methodological developments in X‐ray structural biology

• Isomorphous replacement (heavy atoms) • Molecular replacement • Crystallization, data collection & detectors • Early use of anomalous scattering • Multiwavelength Anomalous diffraction (MAD) • Single wavelength anomalous diffraction (SAD) • X-RAY SOURCES

11 European and National Facilities

12 (Courtesy G. Evans) 13 X‐ray data collection strategies

(Courtesy G. Evans) 14 From crystal to structure: a linear process?

15 3D protein structures within the Protein Data Bank

applications

discovery

16 «Unique protein folds» within the PDB

NEW FOLDS

17 FROM BASIC TO APPLIED SCIENCE

STRUCTURAL BIOLOGY IN DRUG DISCOVERY

18 Mean life expectancy for man has risen from 25 to about 80 years in the UK thanks to the eradication of infectious diseases The explosion of new infectious diseases • About 40 newly diagnosed infectious diseases during the last 30 years, including HIV, VCJD, Ebola, SARS, West Nile, MERS, and several documented human infections with avian influenza viruses (potentially pandemic). • Never in the history so many new Infectious Diseases have emerged in such a short period of time Trends in Infectious diseases Infectious diseases are rooted in

• increased global population • overcrowded cities • increased travel trends • intensive food production • sexual practices • poverty • global warming • insufficient public health measures Recently emerged infectious agents originate or are transferred from animals The reservoir for new infectious agents is unlimited SILVER SEARCH STRATEGY

Small‐molecule Inhibitor Leads Versus emerging and neglected RNA viruses

EU HEALTH‐2010/14 Collaborative Project 24 Norovirus threat to global health

Noroviruses (NV) are the leading cause of foodborne disease outbreaks worldwide, causing significant morbidity and mortality among children. NV is highly infective. No vaccine is known and effective treatments are not available, largely because studies on human norovirus suffer from the lack of a permissive cell-culture system.

25 The viral infection cycle

26 Druggable targets in HNV genome (ca. 7.4 kb)

RNA‐dependent RNA polymerase (RdRp) is considered one of the most interesting targets for chemotherapeutic intervention, since it is essential for the replication of viral RNA.

We used an in silico docking procedure to identify small synthetic compounds predicted to bind with high affinity to the selected site, and then validated these experimentally.

27 Our crystal structures and MD analyses of murine and human NV RdRp allowed us to start a search for binders at a specific region

of the enzyme, selecting two in silico hits 28 NF023 SURAMIN 29 Crystal structure of hNV RdRp bound to NF023 30 31 Molecule Fragments

primary binding site

secondary binding site IC50 (nM) Cpd MNVRdRp hNVRdRp hNVRdRp (mut)

Suramin 70±3 24.6±0.8 140±6

hNV RdRp inhibition shows a mixed NF023 200±2 71.5±0.9 280±6 mechanism relative to both GTP and the nucleic acid substrates, implying that the inhibitor is able to bind to the Naf2 200±40 1300±200 1600±200 free enzyme as well as to the enzyme‐ substrate complex. Naf3 190±30 1400±300 1400±200

PPNDS 200±40 350±80 500±5033 CC 25 nM 12.5 nM 6.3 nM

Yellow Fever Virus Infection

3.1 nM 1.5 nM 0.7 nM VC

E074419‐BEE‐EC patent A novel therapeutic use of an active ingredient belonging to the class of Ivermectins 34 PROTEIN STRUCTURE IN VACCINE RESEARCH

35 36 Rappuoli (2007) Nat. Biotech. 25, 1361 Conventional vaccine development

Killed vaccines

Cultivate Live attenuated vaccines Microorganism Subunit vaccines

Test Test immunogenicity Convalescent Antigen sera selection Purify components Identify Clone genes components 5-15 Years

Immunogenicity testing in Vaccine VACCINE DEVELOPMENT animal models

Courtesy R. Rappuoli – Novartis Vaccines37 New technologies for vaccine development

Reverse Vaccinology

Immunogenicity testing VACCINE Vaccine in animal models DEVELOPMENT

1-2 years Express recombinant proteins Antigen cloning

In silico antigen candidates

Computer Prediction Start From the Whole Genomic Sequence

Courtesy R. Rappuoli – Novartis Vaccines38 Vaccine Development

Structural Biology Vaccinology

Structure‐based rational design •Stabilize antigen conformations

Reverse •Facilitate laboratory production •Generate chimeras for cross‐protection

39 Structural vaccinology to develop vaccines against variant microbes.

42 Burkholderia pseudomallei

• Soil‐borne Gram‐negative bacterium • Endemic in Tropical and sub‐Tropical regions of world • Several routes of infection (lungs, wounds, rain season, … ) • Melioidosis  Pneumonia, septicemia, abscesses and chronic disease • Poor diagnosis and treatment: antibiotic resistance problems • Class 3 pathogen • Best option –Vaccine development

43 Acute and chronic disease

Acute disease –rapid onset Chronic disease –low level peristent infection Recurrent disease

Latent disease – asymptomatic until activated

‐ There are numerous reports of chronic melioidosis that have been initially misdiagnosed as TB (Vidyalakshmi K et al., Int J Tuberc Lung Dis 2008).

44 Acute and chronic disease

C57BL/6 mice (n=6) were challenged i.n. with 1000 or 100 CFU Bps 576. Mice monitored for survival.

Courtesy R.W. Titball 45c Our Structural Vaccinology Pipeline for epitope discovery and testing commencing from 3D structural information

46 RED: Acute phase antigens, priority 47 ORANGE: Chronic phase antigens BPSL2063 BPSL2287

BPSL0280

OppA

BPSL3319: FliC BPSL2522

N-PilO2 BPSL2765 48 BPSL2765 (PalBp)

RV approach to antigen identification • Peptidoglycan‐associated lipoprotein • 100% conserved in 28 B. pseudomallei genomes analyzed • Immunogenic in both mice and melioidosis > 1200 protein microarray patients

• 10‐fold higher levels of anti‐PalBp antibodies in individuals, recovered from one episode of the disease Probing with immune sera from • Offers only 50% protection in mice immunization melioidosis patients experiments (acute  chronic phase) • Good Structural Vaccinology (SV) target: • 3D atomic level information  antigen tailoring Identification of 49 seroreactive proteins

Aims: Felgner et al., (2009) PNAS, 106, 13499‐13504 1. To solve the 3D structure of PalBpas a SV target 2. Carry out 3D‐structure based in silico epitope predictions and validate them experimentally 3. Synthesize consensus epitopes as peptides 4. Assess immunological activities of identified epitopes 49 1.To solve the 3D structure of PalBp as a SV target

• 170 residues • Cloned and heterologously expressed as a His‐tag fusion protein in E. coli cells (35mg/L culture) • > 500 crystallization conditions screened • His‐tag cleavage essential for diffraction‐quality crystals • 2.3Å structure: canonical Pal alpha‐beta sandwich fold

50 From crystal to antigen structure

51 BPSL2765 crystal structure

PalBp Data collection Space group C2 Cell dimensions a, b, c (Å) 102.2, 75.1, 72 a b, g (°) 90, 133.7, 90 Resolution (Å) 40‐2.3

Rmerge 0.050 (0.271) I / sI 13 (3.5) Completeness (%) 96.8 (95.7) Redundancy 2.2 (2.2)

Refinement Resolution (Å) 40‐2.3 No. unique reflections 16804

Rgen / Rfree 20.4/24.9 No. atoms Protein 2853 Acetate ions 42 Water 62 B‐factors (Å2) Protein 37.5 Acetate ions 34.8 Water 35.4 R.m.s. deviations Bond lengths (Å) 0.002 Bond angles (°) 0.513 Ramachandran Plot (%) Favoured Regions 98.6 Allowed Regions 100 52 2. Carry out 3D-structure based in silico epitope predictions and validate them experimentally

Two complementary in silico methods applied to the crystal structure of PalBp

1. MLCE: Matrix of Local Coupling Energies • (Scarabelli et al., (2010) Biophys J. 98, 1966‐1975) • Based on idea that antibody recognition depends on epitope structure, dynamics and stability • Identifies antigenic epitopes (red) from structures derived from MD simulations • Identifies substructures with minimal interaction networks with structure • 2 patches identified

2. EDP: Electrostatic Desolvation Profiles • (Fiorucci et al.(2010), Biophys J. 98, 1921‐1930) • Identifies protein‐protein interaction interfaces in general (blue) • Regions with low desolvation penalties  interfaces • X. Daura group (Universitat Autònoma de Barcelona) • 6 patches identified

• All MLCE residues were also found by EDP

• Two epitopes representing the consensus of both methods were identified

53 54 Scarabelli et al. Biophys. J. (2010) 98, 1966. 55 Fiorucci & Zacharias. Biophys. J. (2010) 98, 1921. Computationally predicted BPSL2765 epitopes

MLCE and EDP

56 Experimental Epitope Mapping

• Immunocapturing approach using

recombinant PalBp and pAbs • Identified diverse peptides with a common 19‐ residue sequence (C). • 3 residues (YLK) overlap with in silico predictions (purple) • Mario Ferrer‐Navarro (UAB, Spain)

57 In silico predictions, in vitro mapping and Peptide synthesis

58 BPSL2765 - OmpA

EDP prediction Epitope 1 Consensus region

MLCE prediction Epitope 2 Epitope 3 (mapped) 59 3. Peptide Synthesis

Based on in silico and in vitro epitope mapping, 3 peptides designed and synthesized (Alessandro Gori; Prof R. Longhi group CNR‐ICRM):

EPITOPE1: (yellow) consensus of EDP and MLCE EPITOPE 2: (green) consensus of EDP, MLCE and houses in vitro YLK tripeptide EPITOPE 3: extended from YLK consensus tripeptide to include a helix‐loop region that resists cleavage during in vitro mapping a conformationally‐ selected unit recognised by pAbs

60 BPSL2765 - OmpA

Artistic representation of HSA conjugated to Epitope 3 peptides.

C-PEG-PEG-SYSVQDQYQALLQQHAQYLK free

Biotyl-C-PEG-PEG-SYSVQDQYQALLQQHAQYLK Biotyl-conjugated

HSA-C-PEG-PEG-SYSVQDQYQALLQQHAQYLK HSA-conjugated 61 Assess immunological activities of identified epitopes

• Seroreactivity of EPITOPES 1‐3 tested vs recombinant antigen against Sera from healthy, BPS infected, and recovered melioidosis patient groups

RESULTS • EPITOPE 3 recognised to same extent as recombinant protein suggesting conformation maintained as free peptide (confirmed by CD)

•EPITOPES 1 and 2 not antigenic

62 EPITOPE 3 Antibodies: Further Immunological Tests…..

Antibody‐dependent agglutination •Important pathogen defense mechanism

• Anti‐EPITOPE 3 Abs specifically agglutinate BPS and to a greater extent in comparison with Anti‐

PalBp Abs

63 EPITOPE 3: Further Immunological Tests…..

•Anti‐EPITOPE 3 antibodies raised and tested in opsonization killing experiments using human neutrophils •Anti‐EPITOPE3 antibodies increase phagocytosis by human neutrophils by 50% via oxidative burst function indicated by the production of superoxide by 95% neutrophils

•Anti‐BPSL2765 do not have this effect

•Phagocytosis, oxidative burst and bacterial killing are [Ab]‐dependent

64 BPSL2765 - OmpA

Conclusions Structure-based antigen engineering allowed us to generate an epitope that induces antibodies with elevated bactericidal activities relative to the full- length, recombinant antigen

MICE PASSIVE IMMUNIZATION.

Is underway in Gregory Bancroft’s lab, using Anti-Epitope 3 antibodies

65 GTA partner involvement: UMIL, UAB, ICRM-CNR New collaborations: U. Khon Kaen and ICRM (Longhi Lab)

Paper Reports: •Crystal structure of OppA at 2.1Å •Computational and experimental epitope mapping •Synthesis of 3 computational and 3 experimental epitope peptides • Epitope recognition by immune sera from melioidosis patients (recovered, healthy positive or negative) •All COMP peptides immunoreactive : COMP1-3 are real epitopes reactivity of COMP3 was significantly diverse between the three groups : tool for diagnosis?

Conclusions Our combined effort defines a new approach that may be used in Structural vaccinology field. Understanding epitope location could help develop epitope-driven subunit vaccines and diagnostic tools, e.g. as for COMP-3. 66 67 EPITOPE TARGETING

PROTOTYPING THE CONCEPT FOR IMMUNODIAGNOSTICS

Peri, C., et al.(2013). ACS Chem Biol 8, 68397-404. Future ideas

MULTIPLE EPITOPE PRESENTATION

Rather than focusing on single epitopes as elicitors of neutralizing antibodies…

Analyze the immune response in term of the collective activities of antibodes directed to several epitopes

epitope1 epitope2 epitope3

SCAFFOLD

Synthesis of one «chimera» construct, based on our most promising epitopes, from different antigens

Gori A., et al. (2013)Amino Acids, 69epub 70 There are several unanswered questions in vaccine development, e.g. what is the molecular nature of immunodominant epitopes? Or, why only some antigens are strongly protective?

To solve these problems we need basic science, and likely explain such open questions from molecular structures, dynamics and first principles.

71 Dipartimento di Bioscienze – Universita’ di Milano

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