Colin Van Noordt
email: colin.van-noordt@ext.ec.europa.eu

Gianluca Misuraca
email: gianluca.misuraca@ec.europa.eu

Keywords: Digital Transformation, e-Government, Artificial Intelligence, Chatbot

Advances in Artificial Intelligence technologies have revived the interest in Chatbots in both the private and the public sector. Chatbots could improve public service delivery by being able to answer frequently asked questions and conduct transactions, relieving staff from mundane tasks. However, previous e-Government research shows that the adoption of newer technologies does not always mean public services get improved. It is therefore of interest to research to which degree newer, advanced technologies such as Chatbots are able to improve, change and restructure public service delivery. This paper gives an exploratory insight using desktop research into three Chatbots currently used in the public administrations of Latvia, Vienna and Bonn. The findings suggest that minor organisational changes are accompanied with the introduction of Chatbot-technology in public administrations, but question whether Chatbots are able to transform traditional services to digital, integrated public service transactions.

Introduction

There has been a big interest in the possible gains of using Information and Communication Technologies (ICT) for the delivery of public services to citizens. Already during the 1990s, there was a strong belief that information technology services are able to create a new, better functioning government of the future [START_REF] Torres | E-government and the transformation of public administrations in EU countries: Beyond NPM or just a second wave of reforms?[END_REF]. Government operations would be able to become more efficient, of higher quality and also more accessible to the public.

The internet is always available 24 hours every day, so citizens would be able to avoid the slow and hierarchical structures of traditional government. They would not have to rely on the opening times of the government anymore since the Internet allows citizens to find information themselves online and is able to deliver services through the web [START_REF] Torres | E-government and the transformation of public administrations in EU countries: Beyond NPM or just a second wave of reforms?[END_REF][START_REF] Bekkers | The Myths of E-Government: Looking Beyond the Assumptions of a New and Better Government[END_REF]. Lately, another technology has captured the attention of the field. Coming from the realm of Artificial Intelligence, advances in Natural Language Processingtechnologies have revived the potential of Chatbots [START_REF] Androutsopoulou | Transforming the communication between citizens and government through ai-guided chatbots[END_REF]. Early Chatbots were limited in their functionalities as they were only able to respond to simple queries. Recent advances in Artificial Intelligence technologies, in particular the ability for machines to understand the context of languages better, made it possible for Chatbots to tackle more complex tasks and host more human-like conversations [START_REF] Androutsopoulou | Transforming the communication between citizens and government through ai-guided chatbots[END_REF].The optimism for this technology is great; it has already been predicted by Gartner that by 2020, the average person will have more conversations with Chatbots than with their own spouse [START_REF] Levy | Gartner Predicts a Virtual World of Exponential Change[END_REF].

In this paper, three cases of Chatbot used in European public administrations are described and briefly discussed on their transformative potential and integrated service delivery. As these Chatbots are frequently mentioned and have won numerous awards, they could be an indicator of how the future of Chatbots in the European public sector might look like in the upcoming years. The main aim of this research is therefore to answer ''Which organizational changes occur within public service delivery due to the introduction of Chatbot-technologies?". By analysing the transformative impact of three cases, a greater understanding could be achieved on the impact of Chatbots within the public sector. In order to answer the research question, this paper follows a multiple case study design to identify which kind of changes the Chatbot technology introduced. By analysing three well-known cases of Chatbots technologies, the findings could be more robust and generalizable rather than relying on one single case study [START_REF] Yin | Case study research and applications[END_REF]. The data collection had been done by document based desktop research. While this enables research from a distance, it does limit the correct interpretation of documents found online and restricts the researcher from gaining additional information not found on websites by for example conducting interviews [START_REF] Bryman | Social research methods[END_REF].

2

The promise of e-Government

An ICT-driven government is argued to be more responsive to citizen-needs, more democratic, transparent and efficient than a traditional government [START_REF] Bekkers | The Myths of E-Government: Looking Beyond the Assumptions of a New and Better Government[END_REF]. Early e-Government documents have showed that there was a great wish that technologies would enable a more joined up government apparatus, where different sectors of the government work together across organisational barriers to tackle public problems in an integrated approach, rather than different public organisations working isolated from each other [START_REF] Bekkers | The Myths of E-Government: Looking Beyond the Assumptions of a New and Better Government[END_REF][START_REF] Dunleavy | New public management is dead-long live digital-era governance[END_REF].Government-wide information structures would allow different departments to work together in a more quicker and efficient way as ICT would ease the communication across organisational barriers [START_REF] Lips | E-government is dead: Long live public administration 2.0[END_REF].

The ICT-reformed public services would then improve government-citizen relations, reducing democratic gaps and other disappointments experienced by citizens [START_REF] Norris | E-Government 2020: Plus ça change, plus c'est la même chose[END_REF]. For digital public service delivery, it was expected that there would be continuous progress from information provision online to one and two-way communication between citizens and the public organisation, transactional services and lastly cross-agency integrative e-services with more citizen engagement [START_REF] Bertot | Universal and contextualized public services: Digital public service innovation framework[END_REF]. However, many of the proclaimed benefits of e-government have not been realized [START_REF] Savoldelli | Understanding the e-government paradox: Learning from literature and practice on barriers to adoption[END_REF]. Despite many investments and projects to realize new innovative forms of governance and government service delivery, no substantial gains have been made in the e-government field. While there are many government services now available online, there is a significant mismatch within the supply and the demand for these online services [START_REF] Savoldelli | Understanding the e-government paradox: Learning from literature and practice on barriers to adoption[END_REF]. The techno-deterministic premise that ICT-introduction within the public sector would eventually lead to significant reforms within public organisations did not come by as expected [START_REF] Norris | E-Government 2020: Plus ça change, plus c'est la même chose[END_REF]. In fact, most government agencies did not change their organizational practices towards more citizen-oriented public services if they adopted ICT as there is still a lack of integration between different public organisations [START_REF] Bekkers | The Myths of E-Government: Looking Beyond the Assumptions of a New and Better Government[END_REF]. When public organisations actually do provide public services online, it is frequently only possible to gain information from the website rather than being able to conduct interactions or transactions with the public organisation [START_REF] Torres | E-government and the transformation of public administrations in EU countries: Beyond NPM or just a second wave of reforms?[END_REF][START_REF] Norris | Local e-government in the United States: Transformation or incremental change?[END_REF]. It has been argued that this strong focus on information provision exists because it is seen as ''lowhanging fruit''; implementing transactional digital services would require much more resources and effort [START_REF] Norris | Local e-government in the United States: Transformation or incremental change?[END_REF].

The promise of fully integrated public service delivery, without the need to go to multiple organisations, is usually not implemented [START_REF] Weerakkody | Transformational change and business process reengineering (BPR): Lessons from the British and Dutch public sector[END_REF]. This lack of integration among different public organisations was one of the challenges e-Government was supposed to solve, but rather, it is one of the greatest challenges which hinder the potential of e-Government [START_REF] Bekkers | The Myths of E-Government: Looking Beyond the Assumptions of a New and Better Government[END_REF]. IT-adoption in governments rather supports current organisational practices and power rather than changing the processes towards citizen's needs [START_REF] Kraemer | Information technology and administrative reform: will egovernment be different[END_REF].

The introduction of eGovernment-technologies has been argued to enable changes of different magnitudes within public administrations: at the workplace level, organisational level and inter-organisational level [START_REF] Nograšek | E-government and organisational transformation of government: Black box revisited[END_REF]. Firstly, technology allows for small, incremental changes by automating existing processes and thereby improving the efficiency of government operations. Secondly, ICT could allow more general organizational changes to support the introduction of newer technologies. These changes are small adaptions and internal changes, commonly referred to as first-order changes [START_REF] Misuraca | Shaping public sector innovation theory: an interpretative framework for ICT-enabled governance innovation[END_REF]. Technology introductions in the public sector frequently bring about these kinds of changes [START_REF] Nograšek | E-government and organisational transformation of government: Black box revisited[END_REF]. Thirdly, ICT could also enable transformative or even disruptive changes by enabling new mechanisms for public service delivery or policymaking, but limited empirical examples of these changes exist. Lastly, there could be more radical changes which change the governance systems or radically transform policymaking mechanisms [START_REF] Misuraca | Shaping public sector innovation theory: an interpretative framework for ICT-enabled governance innovation[END_REF]. These second-order changes are much more substantial as they radically alter traditional practices, but are more difficult to organize, especially in the public sector [START_REF] Nograšek | E-government and organisational transformation of government: Black box revisited[END_REF].

3

The revival of Chatbots

Chatbots, shorter for conversational agents, are computer programmes which are able to detect and understand language, through text or through speech, and have the ability to communicate back [START_REF] Androutsopoulou | Transforming the communication between citizens and government through ai-guided chatbots[END_REF]. Simply put, Chatbots are computer programs which are able to recognise the input from a user using pattern matching technologies, access information and reply with the information found in the knowledge database [START_REF] Zumstein | Chatbots--An Interactive Technology for Personalized Communication[END_REF]. Conversational agents are not really a new technology; the first Chatbot was already programmed in 1966 in order to discover if humans would be able to find out if they would be talking to a person or a machine [START_REF] Weizenbaum | ELIZA---a computer program for the study of natural language communication between man and machine[END_REF]. However, the potential for Chatbots is now taken much more seriously due to advances in AI-technologies and changing communication patterns. A lot of our daily communication occurs through messaging apps and we have grown quite comfortable with communicating with them; this makes the introduction of Chatbots quite frictionless [START_REF] Dale | The return of the chatbots[END_REF]. Currently, there are already numerous applications of Chatbots used by the private sector, with the most wellknown being the virtual assistants of our mobile phones: Siri, Alexa and Google. Chatbots are starting to appear into numerous other business sectors in order for people to obtain information or to complete interactions without the need for humans [START_REF] Androutsopoulou | Transforming the communication between citizens and government through ai-guided chatbots[END_REF].

Common usages of Chatbots are as customer service assistants, making reservations, paying bills and allowing customers to buy products or services online [START_REF] Przeganlinska | Policy Department for Economic, Scientific and Quality of Life Policies[END_REF].

The public sector has also been looking into the usage of Chatbots to improve public service delivery. The main proclaimed benefits of Chatbots are that they allow organisations to reduce their administrative burden and enhance communication with citizens [START_REF] Androutsopoulou | Transforming the communication between citizens and government through ai-guided chatbots[END_REF]. In addition, Chatbots would enable people to overcome information overload; rather than having to find information themselves, the Chatbot will help them to find what they need [START_REF] Brandtzaeg | Why people use chatbots[END_REF]. Early use cases of Chatbots within public organisations focus on answering citizen's questions or complaints through customer support, searching documents, routing citizens to the correct office, translations or drafting documents [START_REF] Mehr | Artificial intelligence for citizen services and government[END_REF]. Most Chatbots are well suited to help citizens navigate through websites with lots of information, answer simple questions or conduct transactions.

This removes the need for humans to answer the same kinds of questions over and over again, allowing human operators to spend more time on complex cases [START_REF] Lommatzsch | A Next Generation Chatbot-Framework for the Public Administration[END_REF][START_REF] Eggers | AI-augmented government: Using cognitive technologies to redesign public sector work[END_REF]. Others even see the potential of Chatbots to radically improve the citizen experience, improve citizen engagement and enabling new forms of decision-making with the help of citizen's interactions with Chatbots. Chatbots could be used to conduct surveys and gain feedback on public services in a more useful way as the Chatbot would be able to ask follow-up questions [START_REF] Bousquet | Five Ways Chatbots Could Transform Government Services[END_REF][START_REF] Desouza | Chatbots move public sector toward artificial intelligence, towards-artificial-intelligence/ 27. Ministry of Justice of the Republic of Latvia: Register of Enterprises opens the first public administration virtual assistant in Latvia -UNA[END_REF]. There is certainly a potential value for government organisations to embrace Chatbots, but based on the history of e-Government progress, there is a strong need to gather empirical evidence from its effects.

4

Current Chatbots in government service delivery

UNA in Latvia:

In 2018, the Register of Enterprises of Latvia introduced the Chatbot UNA to answer frequently asked questions regarding the process of enterprise registration. The name UNA has a symbolic meaning as it stands for the Future Support of Entrepreneurs in the Latvian language. This way, UNA acts as an indicator for the future of the Latvian public administration; Chatbots are available 24/7 and thus able to make communication between citizens and the state accessible and friendly [27]. UNA is available on both the website of the Register of Enterprises as well as on the Facebook page as part of the Facebook messenger application [START_REF]OECD: UNAthe first virtual assistant of public administration in Latvia[END_REF]. UNA is able to answer frequently asked about the registration of their businesses as well as the liquidation, merchants, companies and organizations. If citizens already have an application in progress, they are also able to ask about the progress of their documents. At the moment, UNA only works in the Latvian language [27].

The Chatbot has been developed because the organization had to respond to a lot of calls and emails, which were more or less the same each time. The high numbers of organizational resources spent to answering the same kinds of questions could easily be lessened by using Artificial Intelligence, especially Natural Language Processing techniques [29]. A Latvian company, Tilde, specializing in Artificial Intelligence technologies cooperated in the development of UNA. The usage of the conversational agent has been argued to be highly successful and has been nominated for numerous awards such as the OECD's Public Excellence, World Summit Awards and others [START_REF]UNA -The First Virtual Assistant of Public Administration in Lativa selected as the Best National Digital Solution for International World Summits Awards in the Category " ' Government & Citizen Engagement[END_REF][START_REF] Tilde | The virtual assistant UNA developed by Tilde helps win The International Quality Awards 2018![END_REF]. According to the first performance indicators, 44% of the questions asked on UNA are considered to be general of nature and easily taken care of by the Chatbot.

Other non-standard issues are still handled by the support staff, but now they have more time to focus on more complex tasks [START_REF]UNA -The First Virtual Assistant of Public Administration in Lativa selected as the Best National Digital Solution for International World Summits Awards in the Category " ' Government & Citizen Engagement[END_REF]. While there are plans for UMA to perform the registration of legal subjects and legal facts in the future, currently, the Chatbot is only available to provide information to commonly asked questions. Citizens are still required to collect and fill in numerous documents, sign and stamp, send the filled in documents to the Register and pay the fees using the traditional processes [START_REF]How to set up a company in Latvia[END_REF].

Another element worth considering is that UMA is not designed to assist citizens with the whole process of starting a business, but solely answers questions about the process of registering the Enterprise as this is the task of the Register of Enterprises of Latvia. Arguably, there are numerous other services which new business owners have to conduct such as applying for licenses, permits, getting a business bank account, buying property, paying taxes and others which UMA is not able to answer questions about. 2

WienBot in Vienna:

In 2017, the Chatbot WienBot was launched in Vienna. This conversational bot has been designed to provide answers to frequently asked questions people have. The City of Vienna discovered that there are thousands of searches every month on the municipal website in order to gain more information about the online services available in the city. WienBot improves this process by enabling citizens to find information "quickly, smart and on-the-go" [START_REF]Urban Innovation[END_REF].Rather than having to search for the correct page on the municipal website, citizens are able to ask the WienBot which will provide an immediate answer. The amount of information WienBot provides is very broad and diverse as the website of the municipality has many different online services [START_REF]Urban Innovation[END_REF]. At the moment, WienBot is able to provide answers to around 350 different topics and services of the city. WienBot works solely in the German language, but is also able to reply in the local dialect [START_REF] Wien | Redaktion: WienBot -der digitale Assistent der Stadt Wien[END_REF].

The WienBot has been developed in order to make the information about the different services the City of Vienna provides more easy and understandable. It follows the current trend that much more information about the municipal services is looked up on the smartphone. However, rather than having the citizen to look up the information themselves, the Chatbot will give a quick answer to any question someone might have [START_REF]Urban Innovation[END_REF].

Citizens will still be able to find additional information on the websites, but for quick information, WienBot should be sufficient. Especially information about the availability of public parking spaces in the city is mentioned as a well-desired functionality of WienBot [START_REF]Urban Innovation[END_REF]. The City of Vienna was responsible for the development of the application themselves. It won the World Summit Award in 2017 for the best Government & Citizen Engagement application [35].

Even though there are a large number of topics WienBot is able to answer, the Chatbot is solely aimed at information provision for already existing governmental information. It is not possible to transact any governmental services through the Chatbot. Instead, citizens will get a link with more information about where to go to in order to obtain certain government services [START_REF]Urban Innovation[END_REF]. While the WienBot is arguably very useful to gain information, there is no possibility to avoid going to the office by conducting transactions online through the Chatbot. It is unclear if there are future plans in order to incorporate the future of transactions through the applications. For example, if a person tells the WienBot that he has lost an item; it will provide him or her with a link to the relevant pages of the lost property office (Fundamt) rather than allowing citizens to use the services through the Chatbot [START_REF]Urban Innovation[END_REF].

The transformative potential for the WienBot is hereby severely reduced as citizens would still be required to go through the traditional public services in order to gain what they need, rather than being able to ask WienBot to conduct these transactions for them.

GovBot/ Botty Bonn in Bonn

In the City of Bonn, Germany, the GovBot [36] has been implemented in order to assist citizens with their administrative services. Citizens are able to ask for application forms, opening hours or are able to book an appointment through an interactive process with the Chatbot [START_REF]Stadt Bonn -Smartphone-Dienste der Stadtverwaltung Bonn [City of Bonn -Smartphone Service of the city administration Bonn[END_REF]. The City of Bonn didn't develop the Chatbot themselves but are using the GovBot developed by the software companies Publicplan and Materna. They developed a Chatbot which has been specifically designed for usage in the (German) public administration. Currently, the GovBot technology is used in the search engine of the administration of North-Rhein Westphalia, the City of Bonn and in the City of Krefeld [START_REF] Ehneß | Ein Chatbot für eGovernment [A Chatbot for eGovernment[END_REF]. Citizens are able to access the Chatbot on a specialized website.

GovBot is a Chatbot based on machine learning and an integrated knowledge base of administrative knowledge. The main aim of GovBot is to relieve the administrative staff within the public administration with labour-intensive and recurrent tasks of handling the same kind of citizen requests. Rather than having citizens ask the civil service themselves with questions, they are able to gain the same answers immediately through the use of the GovBot [START_REF]Stadt Bonn -Smartphone-Dienste der Stadtverwaltung Bonn [City of Bonn -Smartphone Service of the city administration Bonn[END_REF][START_REF]PublicPlan: GovBot -Dialogisches E-Government mit Chatbots | publicplan GmbH [GovBot -Dialogical e-Government with Chatbots | Publicplan GmbH[END_REF].

In addition, the GovBot is able to assist citizens with administrative processes by helping citizens fill in administrative forms. Citizens are then able to come prepared to their appointment with the documents filled in correctly, such as the application of a license plate [START_REF]PublicPlan: Typische Anwendungsfälle | publicplan GmbH [Typical applications | publicplan GmbH[END_REF]. Currently, the Chatbot is still in a testing phase and will be added with more information in the future [START_REF]Stadt Bonn -Smartphone-Dienste der Stadtverwaltung Bonn [City of Bonn -Smartphone Service of the city administration Bonn[END_REF].

Even though the Chatbot is still in a testing phase, the main aim of the GovBot is to facilitate better information provision to citizens about general affairs or current administrative procedures. As of now, it seemed not to be possible to conduct any transactions or government services through the Chatbot rather than scheduling an appointment at the office. There is much to praise about assisting citizens with difficult forms and the GovBot definitely could play a big role in this. However, there is no actual change on existing administrative processes with the introduction of the Chatbot; citizens still need to make an appointment at the civil service after filling in the forms and go through the standard procedure, rather than being able to finish the transaction through the GovBot.

Discussion and conclusions

This brief exploratory insight suggests that current Chatbots which have been implemented within the European public sector certainty provide a certain value for the citizens. All three Chatbots aim to improve the communication between citizens and the administrative bodies by providing easy answers to often asked questions. Citizens are able to find the information they are looking for in a quick and reliable way without the need to navigate the governmental websites themselves or contact the customer service, enabling staff to spend their time on other tasks.

Integrated information

No No No

Organisational changes

First order changes by having staff focus on more complex tasks

None identified None identified

Even with the introduction of advanced technologies, there is a significant focus on information provision towards citizens, rather than using them to provide better government services to citizens. Instead of using Chatbots in such a way that citizens don't even need to come to the administrative office, citizens are still required to follow the traditional procedures, although this time empowered by the knowledge provided by the Chatbot. It would be truly a change if citizens would be able to send the documents online as well or conduct the whole process through the Chatbot. There seems to be awareness that transactions provide more value towards citizens. The developers from UNA in Latvia aim to facilitate transactions in the future through the Chatbot, but at the moment this is not yet the case.

There are technologies in place to facilitate these transactions; most countries have some form of e-ID system in place already which citizens could use to identify themselves with. An online payment system or e-Signature system would make it possible for citizens to conduct their government transactions fully digital. However, this does require that the actual administrative procedures should be replaced, a task which is significantly more challenging to accomplish.

Just like the lack of transactions, the e-Government literature frequently mentions that the lack of an integrative approach with joined-up public services hinders the potential of e-Government services. All of the mentioned Chatbots seemed to be fully based on the knowledge from the developing organization and don't take into account the knowledge from other, relevant public organizations. This is unfortunate as citizens frequently have to contact different public institutions when they are in need of public services.

The aim of this paper was to explore whether the introduction of Chatbottechnology within the public sector woul be accompanied with transformational changes. However, based on these early findings, we suggest that only first-order occur: namely the automation of current activities and some (minor) organizational changes to facilitate or as an effect to its introduction. Civil servants would be able to devote more time towards more complex cases when many questions get answered by the Chatbots, but the actual nature of their work doesn't seem to change at all.

They are still conducting the same processes as before, even when some of these tasks could be done by different technologies too.

Our findings do not suggest that any second-order changes happened due to the introduction of Chatbots. Public service delivery has not been radically changed, nor was there any mentioning of changes in the governance system, citizen engagement or reforms of the policy-making processes due to the implementation of the Chatbot.

If these practices are left unchanged, more institutions might implement Chatbots in order to improve their information provision towards their citizens. While this goal is very noble and paved with good intentions, there is a serious chance that these Chatbots are going to reflect the current fragmented landscape of governance. Instead of the current practice that citizens have to find the information they need from 10 different government websites, they will have to ''talk'' with 10 different Chatbots because the knowledge bases of the Chatbots are not integrated. Each of the Chatbots will only be able to answer the citizens the questions they have that correspond to the activities of the organization, rather than giving citizens a full, integrative respond that will cover the whole journey they will take.

If there is no sufficient amount of data sharing between public organizations, citizens will still be required to provide the same kind of information multiple times. Filling in the same kind of information on a government form is, with or without a Chatbot, a tedious and annoying task for all. Just having a Chatbot is not going to make this procedure any more value adding. If the public sector truly wants to gain maximum benefits from emerging technologies, such as Chatbots, it will require massive public reform, a change in administrative culture and a strong reflection on the current organizational practices. Rather than having technology help understand citizens with the current administrative procedures, there should be questions raised if certain administrative procedures could be made easier or removed at all! There is much more research needed to make more valid conclusions as this paper so far briefly scanned a couple of Chatbot implementations within the public sector. The field is still rapidly evolving and the reflections given here might quickly become invalid if multiple public organisations realise the potential which digital transformation could provide them. Furthermore, the lack of interviews limits the scope of changes which might have been introduced with the Chatbot technology. Possibly, certain organisational changes did occur but were not mentioned online. This restricts the current conclusion but should be seen as an invitation to conduct further research on these cases. Artificial Intelligence technologies such as Chatbots are an intriguing set of new technologies, likely to leave a big impact on our society in the near-future. However, it is advisable to take the transformative discourse of these technologies with a pinch of salt. A true understanding of the impact these technologies will bring the public sector requires a clear and realistic view on how they get adopted and used in practice, by institutions and by citizens.

Disclaimer: The views expressed in this article are purely those of the authors and may not be regarded as stating the official position of the European Commission they are affiliated to.

Table 1 .

 1 Overview of Chatbots in government

	Chatbot	UNA	WienBot	GovBot
	Value
	Information provi-	Yes	Yes	Yes
	sion
	Transactional ser-	Planned	No	No
	vices

Public Sector Innovation & eGovernance. This is a joint-master degree from the KU Leuven, WWU Münster and the Tallinn Technological University.

Starting a business is considered a ''life event'', whereby numerous processes from different (public) organizations have to be followed by a citizen. See also the Quality of Public Administration Toolbox from the European Commission about why redesigning digital services based on these events has many benefits to citizens.

New Wine in Old Bottles: Chatbots in Government

Exploring the transformative impact of Chatbots in public service delivery


Morumotto (former SiQaCo project) is a tool that automatically fills a seismic data archive, corrects overlaps and requests data to fill the gaps. Data are regularly fetched from a pool of different sources. It also performs data quality control. This software is to be used by network operators.

The software was designed to be able to integrate new sources plugins (to be able to fit any kind of Seismic Network) and new data format/structure in the future.

  Geber, Constanza Pardo, Jean-Marie Saurel, Arnaud Lemarchand, Philippe Kowalski, Nicolas Leroy, El-Madani Aissaoui, Claudio Satriano 4èmes Rencontres Scientifiques et Techniques RESIF -Biarritz 12, 13 & 14 Novembre 2019 Interface Monitoring interface : daily gaps per component view ►Morumotto is running on a server, users can access it with a classical web browser ►Administration interface for configuration ►Different user levels : normal, advanced and admin • Normal user can monitor network gaps, overlaps and perform quality control (not affecting data) • Advanced user can create data requests, change update settings and statistics • Admin can configure, add & delete requests, statistics, users, etc. Web interface Administation panel : source plugin configuration QC interface : station location view ►(*) Release candidate version will be pushed to the RESIF GitHub ►Source plugins currently under development : Centaur, Q330, NAQS ►Quality Control currently under development ►Installation & testing in observatories & RESIF ►API & Documentation in progress Plugins ►New plugins can be easily integrated into the software ►FDSN Webservice plugin code : ►On the left : I/O (always the same), ►On the right : the part that needs to be modified to fetch data from a new source ►Then create a new class in source.py : Beta version available github.com/IPGP/morumotto ►Source code available on GitHub (*) : Command line interface All data processing & configuration can be done by command line. Few examples : ►Create a request manually : ►Execute stack manually ►Update data for a specific window of time Implementation ►Built with Django framework, which includes a template system for the frontend and an ORM for the backend and database management ►Supported RDBMS: MySQL, PostgreSQL ►Algorithms for request creation and stack management are in Python, data quality control and data processing are done using the Obspy library ►Frontend includes several Javascript libraries (Cal-heatmap, Datatables, Leaflet…) ►Multithreading and background tasks is done with Celery, which has a specific django plugin ►Stack monitoring is done with Flower, a Celery monitoring tool ►Source plugin scripts are written in Shell Daemon, crontab, configuration ►The software is deamonized, so that it runs on the startup of the system (using supervisor) ►It currently uses the crontab to execute according to a frequency defined by the user ►install.sh script provided, tested in Debian 9 and Ubuntu > 16.04 ►First use of the software comes with an initialisation wizard Software Yes, that's the full class definition Configuration Design ►Object oriented ►Modular: • New plugin integration is easy • New data & metadata formats can be added ►Works indifferently from data format & structure ►Currently supports miniSEED, dataless SEED and stationXML (FDSN) ►For evenly sampled generic timeseries including-but not only-seismic data MOdular aRchive bUilder from Multiple Origin Temporal Traces (& Other stuff) ►Functional specitication & design dossier shared with RESIF community in April & September 2018 ►Available on github : • github.com/IPGP/siqaco/blob/master/CDC_ValidationDonnees.pdf • github.com/IPGP/siqaco/blob/master/SiQaCo-Dossier-de-conception.pdf ►Development from Sep. 2018, involving RESIF, volcano observatories from IPGP community & Geoscope Development Perspectives Calendar ►Each source has a priority, data coming from higher priority source will be favoured ►Limitations in local or distant CPU and bandwidth can be modified to fit networks specificities ►Tasks are handled either manually or running automatically in a crontab Stack management License ►This program is free and open source, under the GNU General Public License version 3


Joana Reis

Nicola Manzella

Fernando Cagide

Jeanne Mialez-Perez

Eugenio Uriarte

Angelo Parini

Andrea Mattevi

Fernanda Borges

Claudia Binda

Tight-binding inhibition of human monoamine oxidase B by chromone analogs: a kinetic, crystallographic and biological analysis

about 150 words) Monoamine oxidase B (MAO-B) is a validated drug target for Parkinson's disease. Chromone derivatives were identified as novel potent and reversible MAO-B inhibitors and herewith we report on a crystallographic and biochemical analysis to investigate their inhibition mechanism. The crystal structures of human MAO-B in complex with three chromone analogs bearing different substituents on the exocyclic aromatic ring (determined at 1.6-1.8 Å resolution) showed that they all bind in the active site cavity of the protein with the chromone moiety located in front of the FAD cofactor. These inhibitors form two hydrogen bonds with Tyr435 and Cys172 and perfectly fit the hydrophobic flat active site of human MAO-B. This is reflected in their tight-binding mechanism of inhibition with K i values of 55, 17 and 31 nM for N-(3′,4′dimethylphenyl)-4-oxo-4H-chromene-3-carboxamide (1), N-(3'-chlorophenyl)-4-oxo-4H-chromene-3-carboxamide (2) and N-(3'-fluorophenyl)-4-oxo-4H-chromene-3carboxamide (3), respectively. These compounds were also 1000-fold more effective in reducing the cellular levels of reactive oxygen species (ROS) in comparison to Ldeprenyl.

Introduction

Monoamine oxidases (MAOs, EC 1.4.3.4) are broadly distributed enzymes that contain a flavin adenine dinucleotide (FAD) cofactor covalently bound to a cysteine residue.

They are expressed in several living organisms and two isoforms are present in mammals, namely MAO-A and MAO-B, which are responsible for the major neurotransmitter metabolism in both the central nervous system (CNS) and peripheral tissues. [START_REF] Edmondson | Molecular and Mechanistic Properties of the Membrane-Bound Mitochondrial Monoamine Oxidases[END_REF][START_REF] Rona | Monoamine Oxidases: The Biochemistry of the Proteins As Targets in Medicinal Chemistry and Drug Discovery[END_REF] Both isoenzymes are bound to the outer mitochondrial membrane and catalyze the oxidative deamination of aromatic amine substrates (Figure 1). MAO-A and MAO-B display different substrate specificity that depends on the tissue distribution of these enzymes. Serotonin and catecholamines are preferentially metabolized by MAO-A, whereas dopamine and phenethylamine are mainly metabolized by MAO-B. In light of their key role in neurotransmitter metabolism in the brain, these enzymes are validated drug targets for neurological diseases. MAO inhibitors are currently used in the clinical practice for the treatment of depression and Parkinson's disease. Most of them, such as tranylcypromine and rasagiline, irreversibly inactivate the enzyme by forming a covalent adduct with the flavin cofactor. More recently, safinamide, a MAO-B selective and reversible inhibitor, was newly approved in Europe and United States as Xadago® as adjunct therapy for Parkinson's disease. [START_REF] Blair | Safinamide: A Review in Parkinson's Disease[END_REF] Additionally, the expression of MAO-B in neuronal tissues increases 4-fold with aging, resulting in an intensification of dopamine metabolism and higher production of hydrogen peroxide. [START_REF] De La Fuente-Fernández | Levodopa-induced changes in synaptic dopamine levels increase with progression of Parkinson's disease: implications for dyskinesias[END_REF] Indeed, in the clinical treatment of Parkinson's disease MAO-B inhibition is thought to be beneficial both in restoring physiological levels of dopamine and in exerting a neuroprotective effect by preventing the production of excessive hydrogen peroxide (Figure 1). [START_REF] De La Fuente-Fernández | Levodopa-induced changes in synaptic dopamine levels increase with progression of Parkinson's disease: implications for dyskinesias[END_REF] This aspect is emerging as a relevant issue also in the context of MAO activity and reactive oxygen species (ROS) in non-neuronal tissues including heart, [START_REF] Maggiorani | Oxidative Stress, and Altered Mitochondrial Dynamics in Cardiac Ageing[END_REF] prostate, [START_REF] Wu | MAOA-Dependent Activation of Shh-IL6-RANKL Signaling Network Promotes Prostate Cancer Metastasis by Engaging Tumor-Stromal Cell Interactions[END_REF] and adipose macrophages. Chromones (4H-benzopyran-4-one), a class of heterocyclic compounds largely distributed in nature, have attracted attention in the field of drug discovery due to their interesting biological activities (Figure 2). [START_REF] Gaspar | A Valid Scaffold in Medicinal Chemistry[END_REF][START_REF] Reis | Chromone as a Privileged Scaffold in Drug Discovery: Recent Advances[END_REF] Moreover, several studies have shown that chromone is a privileged structure for the rational discovery and development of new MAO-B inhibitors. [START_REF] Gaspar | Chromone 3-phenylcarboxamides as potent and selective MAO-B inhibitors[END_REF][START_REF] Gaspar | a Privileged Scaffold for the Development of Monoamine Oxidase Inhibitors[END_REF][START_REF] Cagide | Discovery of two new classes of potent monoamine oxidase-B inhibitors by tricky chemistry[END_REF][START_REF] Reis | Discovery of New Chemical Entities for Old Targets: Insights on the Lead Optimization of Chromone-Based Monoamine Oxidase B (MAO-B) Inhibitors[END_REF] Inhibition studies performed with microsomal recombinant human MAO-B showed that a series of chromone analogs selectively and reversibly bind to this enzyme with IC 50 values in sub-nanomolar range. [START_REF] Reis | Discovery of New Chemical Entities for Old Targets: Insights on the Lead Optimization of Chromone-Based Monoamine Oxidase B (MAO-B) Inhibitors[END_REF] Preliminary kinetic studies demonstrated that compound 1 acted as a competitive MAO-B inhibitor while compound 2 displayed a non-competitive profile. [START_REF] Reis | Discovery of New Chemical Entities for Old Targets: Insights on the Lead Optimization of Chromone-Based Monoamine Oxidase B (MAO-B) Inhibitors[END_REF] Despite several computational docking studies to predict chromone binding to MAOs, [START_REF] Gaspar | Chromone 3-phenylcarboxamides as potent and selective MAO-B inhibitors[END_REF][START_REF] Reis | Discovery of New Chemical Entities for Old Targets: Insights on the Lead Optimization of Chromone-Based Monoamine Oxidase B (MAO-B) Inhibitors[END_REF][START_REF] Giulio | Monoamine Oxidase Inhibitors: Ten Years of Docking Studies[END_REF] no experimental structural information was available so far. In order to clarify the MAO-B inhibition mechanism of this class of compounds, we undertook a crystallographic analysis combined with a more in-depth kinetic characterization using purified recombinant human MAO-B. Moreover, in light of the recent findings highlighting the role of MAOderived ROS in several cellular processes, we have also evaluated the biological activity of these compounds in reducing the cellular ROS levels in HEK-293 cells.

Results and discussion

Chemistry

Compounds 1 and 2 (Figure 2) were previously synthesized in moderate yields through the in situ generation of an acyl chloride intermediate, using phosphoryl chloride (POCl 3 ) in N,N-dimethylformamide (DMF), and the subsequent addition of the appropriate arylamine, as reported by Reis et al. [START_REF] Reis | Discovery of New Chemical Entities for Old Targets: Insights on the Lead Optimization of Chromone-Based Monoamine Oxidase B (MAO-B) Inhibitors[END_REF] Likewise, compound 3 was synthesized from chromone 3-carboxylic acid in an amidation reaction using POCl 3 (Scheme 1). Scheme 1 -Strategy followed for the synthesis of chromone-3-phenylcarboxamide derivative 3, from chromone 3-carboxylic acid. Reagents and conditions: POCl 3 , DMF, 3-fluoroaniline, r.t., 5h.

Structures of human MAO-B in complex with chromone inhibitors

The crystal structures of human MAO-B in complex with inhibitors 1, 2 and 3 were solved at 1.8, 1.6 and 1.7 Å resolution, respectively. Crystallographic statistics are reported in Table 1. The unbiased electron density map was of extraordinary good quality, which allowed to unambiguously model the inhibitor molecules in the enzyme active site. For all three structures, no relevant differences in inhibitor binding and active site conformation were found between the two molecules present in the asymmetric unit (rmsd for the C  atoms are 0.28, 0.23 and 0.22 Å between monomers A and B for the structures in complex with compounds 1, 2 and 3, respectively). Hereafter we will refer to subunit A of each structure for the following discussion. All chromone analogs clearly bind in the hydrophobic active site of human MAO-B 1 (Figure 3a). In particular, the 1,4-benzopyrone moiety occupies the substrate-binding site in front of the flavin ring, whereas the amide linker and the differently-substituted exocyclic ring moieties extend into the entrance space whose access is negotiated by loop 99-105 (Figures 3a, 3b, 3c, 3d). The pyrone carbonyl group points towards the upper part of the active site whereas the oxygen atom of the ring is located in the more hydrophilic part of the cavity and establishes a weak hydrogen bond with Tyr435 (about 3.4 Å in all structures). This binding mode is in agreement with previous docking studies [START_REF] Gaspar | a Privileged Scaffold for the Development of Monoamine Oxidase Inhibitors[END_REF][START_REF] Giulio | Monoamine Oxidase Inhibitors: Ten Years of Docking Studies[END_REF] except for a hydrogen bond predicted to exist between the pyrone carbonyl group and Tyr326 that instead is not observed.

The amide linker of all three inhibitors lies nearby the gating residue Ile199 that is in the open conformation similarly to other human MAO-B structures in complex with bulky inhibitors. [START_REF] Edmondson | Molecular and Mechanistic Properties of the Membrane-Bound Mitochondrial Monoamine Oxidases[END_REF][START_REF] Binda | Structures of Human Monoamine Oxidase B Complexes with Selective Noncovalent Inhibitors: Safinamide and Coumarin Analogs[END_REF] The carbonyl group of the linker is H-bonded to Cys172 with distances ranging from 3.2 to 3.4 Å (Figures 3b,3c,3d), which was also predicted by previous docking studies and which is fully consistent with the longer hydrogen bonds that are known to involve sulphur atoms. [START_REF] Mazmanian | Preferred Hydrogen-Bonding Partners of Cysteine: Implications for Regulating Cys Functions[END_REF] Interestingly, careful inspection of the electron density showed that the side chain of Cys172 exists in a double conformation (Figures 3b,3c,3d) and by using the program PHENIX [START_REF] Echols | Automating crystallographic structure solution and refinement of protein-ligand complexes[END_REF] we refined the occupancy values for the two rotamers of Cys172 in each of the three structures. This resulted to be 0.5 in the case of the complex with inhibitor 1, whereas for the structures with 2 and 3

the conformation H-bonded with the inhibitor was preferred with respect to the other one (occupancy is about 0.7 versus 0.3 of the other rotamer; Figures 3c and3d).

Interestingly, the cysteine rotamer that is H-bonded with the chromone linker moiety is the conformation normally present in other human MAO-B structures (Figure 4).

Nevertheless, Cys172 double conformation was previously observed in the structures of human MAO-B in complex with the slow substrate p-NO 2 -benzylamine [START_REF] Li | Functional Role of the "Aromatic Cage" in Human Monoamine Oxidase B: Structures and Catalytic Properties of Tyr435 Mutant Proteins[END_REF] and in complex with a rasagiline analog bearing an OH substituent on the aminoindan ring. [START_REF] Li | Functional Role of the "Aromatic Cage" in Human Monoamine Oxidase B: Structures and Catalytic Properties of Tyr435 Mutant Proteins[END_REF][START_REF] Binda | Crystal Structures of Monoamine Oxidase B in Complex with Four Inhibitors of the N-Propargylaminoindan Class[END_REF] In both cases, one of the two Cys172 rotamers forms a hydrogen bond with the oxygen atom of the ligand, similarly to that formed with the carbonyl group of the chromone inhibitors. These observations suggest that the involvement of Cys172 in hydrogen bond formation as well as its flexibility in adopting a double conformation, though rarely found in other MAO-B crystal structures, [START_REF] Edmondson | Molecular and Mechanistic Properties of the Membrane-Bound Mitochondrial Monoamine Oxidases[END_REF] is not exclusive to the complexes with chromones and might be relevant to stabilize binding of some inhibitors.

The differently-substituted aromatic ring linked to the chromone unit through the amide spacer represents the varying moiety of the inhibitors under study (Figure 2). In all structures the substituted exocyclic ring binds in the entrance space of MAO-B active site with the halogen atom of 2 and 3 occupying the same position as one of the two methyl groups of compound 1 (Figure 4). No differences in the active site architecture were found between 2 and 3, indicating that the halogen atomic radius does not affect inhibitor binding. Interestingly, structure superposition with MAO-B in complex with safinamide (Figure 4) and coumarin analogs [START_REF] Binda | Structures of Human Monoamine Oxidase B Complexes with Selective Noncovalent Inhibitors: Safinamide and Coumarin Analogs[END_REF] showed that their halogen substituents perfectly overlaps with those of 2 and 3 (Figure 4) suggesting that this part of the active site may represent a subsite favorable for halogen binding. A direct comparison between coumarin and chromone inhibitors is not applicable as different drug design strategies were undertaken, with the spacer-phenyl chain added to the aromatic part of coumarin rather than to the pyrone ring as in the case of chromone analogs. This comparative analysis also highlighted another aspect related to loop 99-105 whose flexibility allows the admission of ligands into the enzyme active site (Figure 3a). [START_REF] Edmondson | Molecular and Mechanistic Properties of the Membrane-Bound Mitochondrial Monoamine Oxidases[END_REF] In particular, Pro102 and Phe103 were previously found to adopt different conformations depending on the size of the bound inhibitor, as observed also in the structures in complex with chromone analogs described in this study (Figure 4). Nevertheless, in the case of compounds 2 and 3, we observed a shift of about 1.5 Å in the position of Pro104-Pro105 towards the inhibitor, which was not found in any other MAO-B structure including that in complex with 1. This conformation is probably due to the fact that the halogenated chromone analogs do not extend as long as safinamide and lack the second substituent on the aromatic ring as it happens in 1 (Figure 4). However, as this conformational difference is moderate and does not correlate with binding affinity of chromone analogs, this aspect is not worth further discussion.

An important feature in the conformation of all chromone analogs bound to human MAO-B active site is the intramolecular hydrogen bond between the carbonyl group of the pyrone ring and the nitrogen atom of the spacer (about 2.7 Å in all three structures; not shown in Figures 3b,3c,3d). This interaction was found also in the crystallographic structure of chromone compounds [START_REF] Cagide | Discovery of two new classes of potent monoamine oxidase-B inhibitors by tricky chemistry[END_REF] and it is conserved when bound to the enzyme active site. The human MAO-B active site cavity, with its flat and elongated shape, [START_REF] Edmondson | Molecular and Mechanistic Properties of the Membrane-Bound Mitochondrial Monoamine Oxidases[END_REF] provides an ideal niche for this class of molecules, which may be related to their inhibition mechanism as described below.

Inhibition studies on chromone analogs

As the crystallographic analysis demonstrated that all chromone inhibitors bind in the MAO-B active site in a highly conserved way, we carried out a more in-depth biochemical characterization to clarify their inhibition mechanism. For this task, we used a fluorimetric horseradish peroxidase-Amplex Red-coupled assay [START_REF] Zhou | A One-Step Fluorometric Method for the Continuous Measurement of Monoamine Oxidase Activity[END_REF] The effect of the inhibitors on the enzyme was determined using both the nonlinear regression on GraphPad Prism software 5.0 (left panels) and the double reciprocal plot analysis (right panels). Apparent k cat (y axis) were calculated as v 0 /[MAO-B].

It is well-known that this mixed or non-competitive behaviour might occur when ligands bind to the enzyme active site in a way that is defined as "tight-binding inhibition". [START_REF] Zhou | A One-Step Fluorometric Method for the Continuous Measurement of Monoamine Oxidase Activity[END_REF] In this case the standard steady-state kinetic model used to describe the mechanism of inhibition is no longer valid and the resulting double-reciprocal plots appear similar to the classical pattern for non-competitive inhibitors. To test whether this may hold for the chromone analogs, we applied a protocol by plotting the IC 50 values for each chromone analog determined at different substrate concentrations, [START_REF] Copeland | Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis[END_REF] using the same fluorimetric assay employed for the classical steady-state experiments.

In the case of tight-binding inhibitors, higher concentrations of substrate are needed in order to compete with the inhibitor in binding to the enzyme active site. With both 1

(not shown) and 2 (Figure 6), a linear correlation between IC 50 values and substrate concentration was observed, in full agreement with tight-binding competitive inhibition. 21 After having assessed that chromone analogs, independently from the type of substituent present on the aromatic ring, feature a tight-binding inhibition mechanism, the next step was to determine an accurate value of inhibition constants. This was accomplished by following published protocols, [START_REF] Copeland | Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis[END_REF][START_REF] Cha | Tight-binding inhibitors-II: Non-steady state nature of inhibition of milk xanthine oxidase by allopurinol and alloxanthine and of human erythrocytic adenosine deaminase by coformycin[END_REF] which use the Morrison equation (Eq. 1), to describe the fractional velocity of an enzymatic reaction as a function of inhibitor concentration, at fixed concentrations of enzyme and substrate:

where v i /v 0 represents the enzyme activity, whereas [E] is the total enzyme concentration, [I] is the total inhibitor concentration, and K i app is the apparent inhibition constant. The real inhibition constant (K i ) is obtained using a different equation in accordance with the inhibitor type. In the case of a competitive tight-binding inhibitor, this is achieved through Eq. 2:

(

Accordingly, we used the fluorimetric Amplex Red assay to obtain the data that were plotted in a dose-response curve described by the Morrison equation for both chromone 1 (Figure 7a) and chromone 2 (Figure 7b). By using Eq. 2, the tight-binding K i values for the inhibitors were determined to be 55 ± 9 nM and 17 ± 2 nM for 1 and 2, respectively. Based on these results, we extended the kinetic analysis to other chromone analogs to probe both the type of halogen substituent on the exocyclic phenyl ring and the importance of the carbonyl group on the amide spacer (forming the hydrogen bond with Cys172) in enhancing binding affinity. For the former task, inhibitor 3 bearing a fluorine group (Figure 2) and its bromine analog N-(3-bromophenyl)-4-oxo-4Hchromene-3-carboxamide [START_REF] Reis | Discovery of New Chemical Entities for Old Targets: Insights on the Lead Optimization of Chromone-Based Monoamine Oxidase B (MAO-B) Inhibitors[END_REF] were tested for their binding mechanism using the procedures described above for 1 and 2. Both 3 and the bromine analog showed a tightbinding profile with the nonlinear regression curves and double-reciprocal plots displaying a mixed and a non-competitive type of inhibition, respectively (data not shown). Accordingly, the tight-binding inhibition K i values were determined by doseresponse curve analysis (Figures 8a and 8b To investigate the role of the amide spacer and its interaction with Cys172, a chromone analog bearing a chlorine group in meta position but lacking the carbonyl group in the linker moiety (chromone 4, Figure 2), was synthesized and used for both kinetic and crystallographic analysis. Even though we could still observe a tight-binding inhibition profile, chromone 4 acted as a competitive inhibitor with a K i of 2927 ± 488 nM, i.e.

about 100-fold higher than that of the other chromone analogs. This suggests that, although the chromone scaffold is likely to trigger the tight-binding inhibition mechanism of these compounds, the hydrogen bond between the amide carbonyl group and Cys172 observed in the crystal structures (Figures 3b,3c,3d) is relevant for binding affinity. To confirm this hypothesis we attempted to determine the structure of human MAO-B in complex with chromone 4. Although X-ray data could be collected at 2.0 Å resolution, the electron density map for the inhibitor was of very bad quality, which hampered modelling of the molecule in the enzyme active site. However, we cannot rule out the hypothesis that the lower rigidity of chromone 4 determined by the absence of the planar amide spacer may give a significant contribution to destabilize inhibitor binding in the active site. Indeed, previous studies showed that analogs bearing a spacer with the carbonyl group but with either a sulphur or an oxygen atom in place of the NH group (i.e. lacking the full planar amide conformation and therefore featuring a lower rigidity) displayed higher IC 50 values. [START_REF] Reis | Discovery of New Chemical Entities for Old Targets: Insights on the Lead Optimization of Chromone-Based Monoamine Oxidase B (MAO-B) Inhibitors[END_REF] Nevertheless, those analogs are expected to lack the intramolecular hydrogen bond that instead in 4 is likely to exist and confer a good level of rigidity to the molecule. Therefore, the hydrogen bond between chromone analogs and Cys172 may significantly contribute to the high binding affinity of 1, 2 and 3 inhibitors.

Biological evaluation of ROS production assay

On the basis of their potent effect in inhibiting recombinant human MAO-B, we next carried out a biological evaluation of chromones 1 and 2 on ROS production in HEK-293 cells transfected with human MAO-B and stimulated with the substrate tyramine.

As shown in Figure 9

Conclusion

A series of chromone 3-phenylcarboxamide analogs were previously identified as novel potent and reversible inhibitors of human MAO-B (with essentially no activity on MAO-A). [START_REF] Reis | Discovery of New Chemical Entities for Old Targets: Insights on the Lead Optimization of Chromone-Based Monoamine Oxidase B (MAO-B) Inhibitors[END_REF] As their exact mechanism of enzyme inhibition was not clarified and no experimental structural information was available, we carried out a crystallographic and biochemical analysis of selected chromone analogs (Figure 2). Our studies showed that chromone inhibitors bind in the enzyme active site cavity with the 1,4-benzopyrone moiety ring in front of the flavin cofactor and they establish two hydrogen bonds with Tyr435 and Cys172 (Figures 3b,3c,3d). The position of the substituted exocyclic ring is also well conserved for all three chromone analogs, with the halogen atoms of 2 and 3 overlapping with one of the two methyl groups of 1 and with the fluorine atom of safinamide (Figure 4). The planar conformation of these inhibitors, enhanced by an intramolecular hydrogen bond between the pyrone carbonyl group and the amide spacer, perfectly fits the flat hydrophobic active site cavity of human MAO-B. These results are in agreement with most of previous docking predictions, [START_REF] Gaspar | a Privileged Scaffold for the Development of Monoamine Oxidase Inhibitors[END_REF][START_REF] Giulio | Monoamine Oxidase Inhibitors: Ten Years of Docking Studies[END_REF] and were interpreted in light of the tight-binding inhibition mode of these chromone analogs. As a matter of fact, the steady-state inhibition studies resulting in either mixed or non-competitive inhibition profiles (confirmed also with the purified recombinant enzyme) were in contrast with what observed in the crystal structures. Accordingly, a dose-response curve approach 21 was adopted, which clearly revealed that chromone analogs are tightbinding inhibitors with K i values of 55, 17 and 31 nM for 1, 2 and 3, respectively.

Although it is known that the chromone moiety itself displays a high affinity for MAO-B active site, we postulate that the hydrogen bond provided by the spacer carbonyl to Cys172 and the perfect match of the whole molecule of these analogs with the enzyme cavity significantly enhance their inhibitory activity. Remarkably, this was also observed in the cellular context with a 1000-fold stronger reduction of ROS levels in treated HEK-293 cells for both 1 and 2 compared to the well-known covalent MAO-B inhibitor L-deprenyl. In conclusion, the chromone core represents a valuable scaffold for the development of novel potent and reversible inhibitors of human MAO-B.

Experimental section

Reagents. Chromone-3-carboxylic acid, 3-formylchromone, sodium sulphate, dimethylformamide (DMF), phosphorus oxychloride (POCl 3 ), aniline derivatives were purchased from Sigma-Aldrich. All other reagents and solvents were pro analysis grade and were acquired from Carlo Erba Reagents and Scharlab and were used without additional purification. All reagents for protein purification and crystallization and for enzymatic assays were purchased from Sigma-Aldrich, except for detergents that were from Anatrace (USA). Enzymatic assays. Human recombinant MAO-A and MAO-B were expressed in Pichia pastoris and purified as described. [START_REF] Li | High-Level Expression of Human Liver Monoamine Oxidase A in Pichia pastoris: Comparison with the Enzyme Expressed in Saccharomyces cerevisiae[END_REF][START_REF] Newton-Vinson | High-Level Expression of Human Liver Monoamine Oxidase B in Pichia pastoris[END_REF] Purified protein samples were stored in 50 mM potassium phosphate buffer pH 7.5, 0.8% (w/v) β-octylglucoside, 20% glycerol.

General

Enzyme concentration was determined by measuring the absorbance UV/Vis spectrum using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). MAO-A and MAO-B activity was determined by a fluorescence-based method with benzylamine and kynuramine as substrates, respectively, which detects the increase in H 2 O 2 (side-product of the MAO reaction) with time using the horseradish peroxidase-Amplex Red coupled assay. [START_REF] Zhou | A One-Step Fluorometric Method for the Continuous Measurement of Monoamine Oxidase Activity[END_REF] All measurements were done in in 50 mM Hepes buffer, pH 7.5, containing 0.25% (w/v) reduced Triton X-100 at 25 °C using either a CLARIOstar plate reader (BMG Labtech) or a Cary Eclipse fluorimeter (Agilent) depending on the type of experiment.

drop vapour diffusion method following published protocols. [START_REF] Binda | Crystal Structures of Monoamine Oxidase B in Complex with Four Inhibitors of the N-Propargylaminoindan Class[END_REF] X-ray diffraction data were collected at the beamlines of the Swiss Light Source in Villigen (Switzerland) and European Synchrotron Radiation Facility in Grenoble (France). For data collection, crystals were transferred into a mother liquor solution containing 18% (v/v) glycerol and flash-cooled in a stream of gaseous nitrogen at 100 K. Data processing and scaling (Table 1) were performed using XDS 26 and the CCP4 package. [START_REF]The CCP4 Suite: Programs for protein Crystallography[END_REF] The coordinates of the MAO B-safinamide complex, [START_REF] Binda | Structures of Human Monoamine Oxidase B Complexes with Selective Noncovalent Inhibitors: Safinamide and Coumarin Analogs[END_REF] deprived of all water and inhibitor atoms, were used as initial model. The program Coot [START_REF] Emsley | Features and development of Coot[END_REF] was used for electron density inspection and model building, whereas crystallographic refinement was performed with the program REFMAC5. [START_REF] Murshudov | REFMAC5 for the refinement of macromolecular crystal structures[END_REF] Figures were generated by the program CCP4mg. [START_REF] Mcnicholas | Presenting your structures: the CCP4mg molecular-graphics software[END_REF] Biological evaluation of ROS production.

HEK-293 cells were grown in DMEM containing 10% heat-inactivated FBS under 5% CO2 at 37°C. Cells were transfected with the plasmid pcDNA3.1-MAO-B (rat cDNA) using Lipofectamine (Thermo Fisher scientific). Tyramine and L-deprenyl were from Sigma-Aldrich. Intracellular ROS were measured using the DCFDA probe assay (Thermo Fisher Scientific). Briefly, cells were loaded for 45 minutes with DCFDA probe (5 µM), washed with HBSS and stimulated with tyramine (500 M) in HBSS for 15 min, in the presence of the indicated inhibitors. Fluorescence was measured using Varioskan microplate reader (Thermo Fisher Scientific).

PDB ID Codes

Authors will release the atomic coordinates and experimental data upon article publication.

Table 1. Data collection and refinement statistics for the crystal structures of human MAO B in complex with chromone inhibitors. d R cryst =|F obs -F calc |/|F obs | where F obs and F calc are the observed and calculated structure factor amplitudes, respectively. R cryst and R free were calculated using the working and test sets, respectively.

Figure 1 .

 1 Figure 1. Scheme of the FAD-dependent oxidative reaction catalyzed by MAOs. Dopamine is showed as substrate that is preferentially metabolized by MAO-B in the CNS. Besides the aldehyde product (dopanal) and ammonia derived from substrate oxidation, hydrogen peroxide is also generated upon cofactor re-oxidation by molecular oxygen.

7

 7

Figure 2 .

 2 Figure 2. Chemical structures of MAO-B inhibitors based on the chromone scaffold.

Compound 4 Scheme 2 -

 42 Scheme 2 -Synthetic strategy followed for the synthesis of chromone derivative 4 from 3formylchromone. Reagents and conditions: 3-chloroaniline, Na(AcO) 3 BH, r.t., 15 min.

Figure 3 .

 3 Figure 3. Crystal structure of human MAO-B in complex with chromone inhibitors. (a) Overall ribbon diagram (ice blue) of human MAO-B structure oriented with the membrane-bound C-terminal helix pointing at the bottom of the figure. The loop formed by residues 99-105 that admits to the enzyme active site is highlighted in bold style. The FAD cofactor is in stick representation with carbon, nitrogen, oxygen and phosphorus atoms colored in yellow, blue, red and magenta, respectively. Inhibitor 1 is showed as reference of chromone analogs binding mode with atom color code as FAD except for carbons that are in dark green. (b) Zoomed view of human MAO-B active site in complex with inhibitor 1. The orientation of the molecule is rotated approximately 45° around an axis perpendicular to the figure plane with respect

Figure 4 .

 4 Figure 4. Structural superposition between structures of human MAO-B in complex with inhibitor 1,inhibitor 2 and safinamide[START_REF] Binda | Structures of Human Monoamine Oxidase B Complexes with Selective Noncovalent Inhibitors: Safinamide and Coumarin Analogs[END_REF] (PDB code 2V5Z) depicted with carbon atoms in gray, white and black, respectively (inhibitor 3, whose fluorine-substituted aromatic ring perfectly overlaps with that of 2, was omitted for clarity). Color code for FAD and the other atoms is as in Figure3.

Figure 5 -

 5 Figure 5 -Kinetic studies on the mechanism of human MAO-B inhibition by chromones 1 (a) and 2 (b).

Figure 6 .

 6 Figure 6. Effects of substrate (S) concentration on the IC 50 values for chromone 2. A similar pattern was obtained for chromone 1 (not shown).

Figure 7 .

 7 Figure 7. Plot of enzyme velocity as a function of inhibitor (I) concentration for chromone 1 (a) and chromone 2 (b). The solid curve drawn through the data points represents the best fit to the Morrison equation.

Figure 8 .

 8 Figure 8. Plot of enzyme velocity as a function of inhibitor (I) concentration for N-(3-bromophenyl)-4oxo-4H-chromene-3-carboxamide (a), chromone 3 (b) and chromone 4 (c). The solid curve drawn through the data points represents the best fit to the Morrison equation.

  , incubation of MAO-B-transfected cells with tyramine for 15 min induced ROS production, measured with DCFDA fluorescence. The specific involvement of MAO-B in ROS generation was supported by the complete prevention of DFCDA production by the MAO-B inhibitor L-deprenyl. As observed with Ldeprenyl, pre-incubation of the cells with compounds 1 or 2 dose-dependently inhibited ROS production following tyramine addition (Figure 9). Remarkably, both 1 and 2 were 1000 fold more potent than L-deprenyl for inhibition of ROS production by MAO B in HEK-293 cells.

Figure 9 .

 9 Figure 9. Cellular inhibition of ROS production by inhibitors 1 and 2. HEK-293 cells transfected with MAO-B were incubated with tyramine for 15 min and intracellular ROS generation was measured with the fluorescent probe DCFDA. Pre-incubation with L-deprenyl (Dep, 1 M) or compounds 1 or 2 was performed 10 min before addition of tyramine. Results are the mean +/-s.e.m. of 4 experiments. ***p<0.001 for Ct vs tyramine; *p<0.01, **p<0.001 for tyramine vs tyramine+inhibitors, Anova post-Tukey's statistical analysis.

c

  As in previous human MAO-B structures, one molecule of the Zwittergent 3-12 detergent (used in crystallization experiments) is partly visible in the electron density of each of the two protein monomers present in the asymmetric unit (15 and 11 atoms out of 22 in monomer A and B, respectively).

  Assignments were also made from DEPT (Distortionless Enhancement by Polarization under inert atmosphere at room temperature. After 15 minutes, the mixture was diluted with CH 2 Cl 2 (40 mL) and washed with saturated NaHCO 3 solution (2 x 10 mL). The organic phase was dried, filtered and evaporated. The residue was purified by flash

	procedures. Thin-layer chromatography (TLC) was carried out on precoated chromatography (CH 2 Cl 2 ). The compound was obtained in 60% yield and recrystallized
	silica gel 60 F254 (Merck) with layer thickness of 0.2 mm. For analytical control the from CH 2 Cl 2 . 1 H NMR (400 MHz, CDCl 3 ) δ 8.23 (1H, ddd, J = 8.0, 1.7, 0.4 Hz, H5)),
	following systems were used: ethyl acetate/methanol and chloroform/methanol in 7.91 (1H, t, J = 1.0 Hz, H2), 7.67 (1H, ddd, J = 8.6, 7.1, 1.7 Hz, H7), 7.48 -7.35 (2H,
	several proportions. The spots were visualized under UV detection (254 and 366 nm). m, H6, H8), 7.06 (1H, dd, J = 8.0, 8.0 Hz, H5'), 6.68 (1H, ddd, J = 8.0, 2.1, 0.9 Hz,
	Flash chromatography was performed using silica gel 60 0.2-0.5 or 0.040-0.063 mm H4'), 6.63 (1H, dd, J = 2.1, 2.1 Hz, H2'), 6.52 (1H, ddd, J = 8.2, 2.0, 0.9 Hz, H6'), 4.24
	(Carlo Erba Reagents). Following the workup, the organic phases were dried over (2H, d, J = 1.0 Hz, CH 2 ). 13 C NMR (101 MHz, CDCl 3 ) δ 177.9 C4, 156.6 C8a, 153.1 C2,
	Na 2 SO 4 . Solutions were decolorized with activated charcoal, when necessary. The 148.6 C1', 135.1 C3', 133.8 C7, 130.3 C5, 125.7 C6, 125.3 C4', 123.8 C4a, 120.8 C3,
	recrystallization solvents were ethyl acetate, dichloromethane or ethyl ether/n-hexane. 118.2 C5', 118.0 C8, 113.1 C2', 111.8 C6', 40.4 CH 2 . ESI/MS m/z (%): 285 (M + + 1,
	Solvents were evaporated in a Buchi Rotavapor. The purity of the final products (>97% 20), 159 (96), 131 (100).
	purity) was verified by high-performance liquid chromatography (HPLC) equipped with
	a UV detector. Chromatograms were obtained in an HPLC/DAD system, a Jasco
	instrument (pumps model 880-PU and solvent mixing model 880-30, Tokyo, Japan),
	equipped with a commercially prepacked Nucleosil RP-18 analytical column (250 mm x
	4.6 mm, 5 m, Macherey-Nagel, Duren, Germany), and UV detection (Jasco model
	875-UV) at the maximum wavelength of 254 nm. The mobile phase consisted of a
	methanol/water or acetonitrile/water (gradient mode, room temperature) at a flow rate of
	1 mL/min. The chromatographic data was processed in a Compaq computer, fitted with
	CSW 1.7 software (DataApex, Czech Republic). NMR data were acquired, at room
	temperature, on a Brüker AMX 400 spectrometer operating at 400.15 MHz for 1 H and
	100.62 MHz for 13 C. Chemical shifts were expressed in δ (ppm) values relative to

tetramethylsilane (TMS) as internal reference; coupling constants (J) were given in Hz.

  R sym =|I i -<I>|/I i , where I i is the intensity of i th observation and <I> is the mean intensity of the reflection. Values in parentheses are for reflections in the highest resolution shell.

					3
	Resolution (Å)	1.8	1.6	1.7
	R sym a,b (%)	16.4 (97.1)	11.4 (99.4)	10.5 (66.2)
	CC 1/2 (%)	99.4 (53.5)	99.6 (45.9)	99.5 (66.2)
	Completeness b (%)	100.0 (100.0)	99.9 (100.0)	99.4 (98.9)
	Unique reflections	117,061	164,973	137,691
	Redundancy	6.8 (6.4)	4.5 (4.3)	4.5 (4.4)
	I/ b		10.2 (1.5)	8.6 (1.3)	10.1 (1.9)
	N of non-hydrogen atoms
	protein/FAD/inhibitor	7944/2x53/2x22	7944/2x53/2x21	7944/2x53/2x21
	detergent c /glycerol/water	26/2x7/846	26/2x7/1051	26/2x7/981
	Average B value for
	protein/inhibitor atoms (Å 2 )	17.9/20.2	17.9/19.4	16.4/17.2
	R cryst	b,d (%)	16.8 (27.1)	16.5 (27.9)	16.7 (24.8)
	R free b,d (%)	19.9 (29.3)	19.1 (27.3)	19.2 (29.1)
	Rms bond length (Å)	0.011	0.009	0.010
	Rms bond angles ()	1.48	1.42	1.48

a b

ABBREVIATIONS

MAO-A, monoamine oxidase A; MAO-B, monoamine oxidase B; FAD, flavin adenine dinucleotide; CNS, central nervous system; MD, molecular dynamics; ROS, reactive oxygen species; DMF, N,N-dimethylformamide.

Funding Sources

This work was supported by Fondazione Cariplo (grant n. 2014-0672 to C.B.), Foundation for Science and Technology (FCT), FEDER/COMPETE2020 (UID/QUI/00081/2015 and POCI-01-0145-FEDER-006980).

Steady-state kinetic analysis. The chromone analogs inhibition mechanism towards human MAO-B was determined by measuring the initial rates of substrate oxidation in the presence of varying concentrations of inhibitor. Data were fit to the appropriate form of the Michaelis-Menten equation and analyzed using GraphPad Prism software 5.0.

The mode of inhibition was determined using global fit analysis of the enzyme velocity versus substrate concentration curves in the presence and absence of the inhibitor to equations for competitive, mixed, non-competitive, and uncompetitive inhibition.

Visual inspection of the fitting curves and evaluation of the r 2 value were used to select the best fitting for determination of the inhibition constants. The results were also analyzed as double reciprocal plots (1/k cat vs. 1/[S]).

Tight-binding inhibition assays and K i determination.

Further inhibition studies were carried out using the same fluorescence-based assay described above following protocols developed for tight-binding inhibition [START_REF] Li | Functional Role of the "Aromatic Cage" in Human Monoamine Oxidase B: Structures and Catalytic Properties of Tyr435 Mutant Proteins[END_REF] . In particular, the tight-binding