Advances in antiviral drug discovery and development - Future Medicine

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process that spans many scientific disciplines, including biology, chemistry, antiviral metabolism and clinical observation.” Shailendra K Saxena†, Niraj Mishra & Rakhi Saxena

Editorial

“Antiviral discovery is a complex, multistage

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Advances in antiviral drug discovery and development: Part II: Advancements in antiviral drug development

Author for correspondence: Centre for Cellular & Molecular Biology, Uppal Road, Hyderabad 500 007 (AP), India n Tel.: +91 40 2719 2630 n Fax: +91 40 2716 0591 n [email protected]

†

Prelude to the saga

In spite of advances in scientific, technological and cultural development, microbes still remain a powerful threat and continue to evade treatment, causing disastrous infectious diseases worldwide. Within the last three decades, several viruses have emerged/re-emerged and created potential public health problems  [101] . By contrast, the development of science and technology has helped us to understand genetic, molecular, structural as well as functional diversity of viruses, the intricacy of viral multiplication and association of host-cell machinery with infection. The information obtained provided road for development of many antivirals, as well as novel therapeutic strategies/targets against viruses. Figure 1 demon­strates the classification of antiviral therapeutics with examples of each group. Antiviral development is a process in which modeling and experiments unite and, therefore, involves various stages as well as various techniques (Figure 2) . Antiviral development approaches can be broadly divided into three levels: physiological, mechanistic and genetic. The physiological approaches attempt to identify antiviral targets through studies in whole organisms since infections only manifest at the level of the organism. This is the most popular method for developing therapeutics. The mechanistic approaches attempt to identify antiviral targets by comparing the intracellular pathways that regulate biological responses in cell culture, model animals and patients. However, variation in host response and complexity of interactions during infection often limit the applicability of this approach for therapeutics. The genetic approaches involve the identification of potential antiviral targets 10.2217/FVL.09.1 © 2009 Future Medicine Ltd

by comparing the differential expression of genes and proteins in normal and abnormal physiological conditions. This is the most recent, technically challenging approach, and it could produce large numbers of potential antiviral targets [1] . Developmental stages of antivirals: stepping up the stairs

The identification of an antiviral target requires a significant understanding of structural, mole­ cular and biological properties. Furthermore, the development of the antiviral candidate requires in vivo screenings in order to understand its pharmacokinetic properties, meta­ bolism and toxicity. Thus, the development of antiviral therapy is a tedious and time-consuming process and traditionally involves the following stages: n Target identification and screening of antivirals n Lead generation and optimization n Preclinical and clinical studies n Final registration of the antiviral (Figure 3) [2,102] Target identification & screening

“Antiviral development approaches can be broadly divided into three levels: physiological, mechanistic and genetic.”

During development of the antiviral, the viral components are the first choice of target. However, owing to the small size of viral genome, variable mutation frequencies and resistance/cross-resistance to antivirals, it is not always favorable. Host factors are the next components to target the virus. This stage of antiviral development requires further understanding of the biochemical, cellular and structural mechanisms of the antiviral to specifically inhibit viral replication in order to select appropriate dose ranges for early Future Virol. (2009) 4(3), 209–215

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Antiviral therapeutics

Virucides

Antivirals

Fusion inhibitors

DNA pol inhibitors

RT inhibitors

Cyclosporine Maraviroc Enfuviritide Docosanol

Idoxuridine Vidarabine Phosphonoacetic acid Trifluridine Aciclovir Foscarnet Ganciclovir Penciclovir Cidofovir Famciclovir Valaciclovir Valganciclovir

Rimatadine Zidovudine Didanosine Zalcitabine Nevirapine Lamivudine Delavirdine Efavirenz Adefovir Abacavir Tenofovir Emtricitabine Entecavir Etravirine Telbivudine

Immunomodulators

Integrase inhibitors

Protease inhibitors

Signaling inhibitors

Raltegravir

Saquinavir Indinavir Amprenavir Nelfinavir Ritonavir Tipranavir Atazanavir Darunavir Zanamivir Oseltamivir

Resveratrol Ribavirin

Figure 1. Classification of antiviral therapeutics. Virusides, immunomodulators and antivirals are the three classes of antiviral therapeutics. Antivirals may be classified into fusion inhibitors, DNA pol inhibitors, RT inhibitors, integrase inhibitors, protease inhibitors and signaling inhibitors. Examples are mentioned below each class. pol: Polymerase; RT: Reverse transcriptase.

clinical trials  [3] . Thus, the target identification and screening of antivirals is costly, time consuming and exhaustive. Several methods, such as microarray, siRNA, hairpin ribozymes and aptamers, have recently evolved, however, owing to their limiations, further investigation is required. Lead generation & optimization

The lead generation could be demonstrated by the screening of chemical libraries, substratebased screening and biostructural approaches. During screening of chemical libraries, all available chemicals are assayed for antiviral activity. The substrate-based approach has been applied to targets, such as HIV-protease inhibitors and influenza virus neuraminidase inhibitors. In the biostructural approach, high-resolution structural data on target biomolecules are used to design lead compounds. The process of lead optimization involves the hypothesis–synthesis–activity cycle [4] . Money, 210

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time and manpower are constraints at this level as well. Molecular imaging and computational screening may prove to be handy in the future; however, there is a need for further development of these technologies. Preclinical & clinical studies

Preclinical studies are performed to observe absorption, metabolism, toxicity, secondary characteristics, rate of excretion and in  vitro and in vivo effect of antivirals. This pharmaco­ logical profiling is important to evaluate the efficiency, efficacy and safety of antivirals over longer periods of time. A compound that successfully emerges from preclinical testing becomes a candidate antiviral and, at this stage, the inventor must file an Investigational New Drug application that is forwarded to a suitable agency. The agency reviews the application to ensure that patients covered in the clinical trials will not face unreasonable risks. Clinical trials involve the following three phases: future science group

Advances in antiviral drug discovery & development

Phase  1 trials involve 20 –100 healthy v­olunteers to examine the safety of antivirals in humans;

n

Phase  2 trials involve 100–500  patients to examine whether antiviral works by the proposed mechanism and actually produces measurable results;

n

Phase 3 trials involve testing antivirals thousands of patients for safety, specificity, efficacy and overall b­enefit–risk profile.

n

This is the rate-limiting step of antiviral develop­ment since translation of preclinical information to clinical output is a constraint and most antivirals fail at this stage. This makes antiviral development costly and it is considered to be very risky [102] . The high failure rate of antivirals that have undergone extensive validation before entering clinical trials may be owing to the lack of provision of cell-culture and animal models that are truly predictive of infectious conditions that are comparable to humans. Molecular

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imaging, animal-model development and characterization of secondary properties of antiviral molecules may help us to overcome the pitfalls. Final registration of the antiviral

Following preclinical tests and clinical trials, and prior to marketing, the approval and final registration of the antiviral by government authorities must be obtained for protection of intellectual property rights and proper manufacturing, marketing and distribution rights. The antiviral is then ready for public use [102] . Techniques: strategem

The antiviral-development cycle is a bidirectional f low of information from clinics to the research laboratories and from research laboratories to the clinics (F igu r e  2) . New techniques/approaches are emerging daily to ensure antiviral discovery, which include the approaches of genomics, epigenomics, pharmacogenomics, proteomics, glycomics,

Patients

PubMed

Clinic

Genes

Proteins

SAR analysis

• ELISA • CT scan/MRI • PET • Tissue array • Microarray

• PCR • Sequencing • Biomarker analysis • Genetic association

• Molecular biology • PAGE/2D/MALDI-MS • Spectroscopy • CD/ORD/ITC • x-ray/NMR/QSAR • Chromatography

• In silico analysis • FRET, FRAP • Combinational chemistry • In vitro and in vivo analysis by cell culture and animal testing • SAR • Synthetic chemistry • Microarray • 2D/MALDI • NMR/MS

HTS

Virus

Antivirals

Chemical assimilation

Chemical library

Biological testing Future Virol. © Future Science Group (2009)

Figure 2. Techniques used in various steps of antiviral development. Antiviral research begins at the clinic/patient and, during various steps, many techniques are used. Patient ana­lysis involves ELISA, molecular imaging, tissue array and microarray and the genetic ana­lysis of host factors and viruses involve PCR, sequencing, biomarker ana­lysis and polymorphic association with infection. Host-factor and viral protein ana­lyses involve molecular biology and PAGE/2D/MALDI/spectroscopy/x-ray/NMR/chromatography. Screening and development of antivirals involve combinational/synthetic chemistry, FRET/FRAP/bioinformatics/animal and cell cultures/SAR/NMR/MS. FRAP: Fluorescence recovery after photobleaching; FRET: Förster resonance energy transfer; HTS: High-throughput screening; MALDI: Matrix-assisted laser desorption ionization; MS: Mass spectrometry; NMR: Nuclear magnetic resonance; PAGE: Polyacrylamide gel electrophoresis; QSAR: Quantitative structure–activity relationship; SAR: Structure–activity relationship.

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Genomic analysis

Clinical samples

New antiviral

Proteonomics analysis Patient information Approval/registration of antiviral

Genetic association Forward genetics

Cell model

Modulcation by KO/transgenic/ overexpression screening

Experimental information

Phase III

Forward/reverse genetics

Animal model Viral infection

Phase II Target identification and screening Literature information

Phase I

Target information and probable antiviral Clinical trials

In silico analysis

Synthesis Hypothesis

Antiviral compunds

Preclinical trials on animals

Activity Problem

Hypothesis

Lead generation and optimization Future Virol. © Future Science Group (2009)

Figure 3. Stages of antiviral development. Viral infection that poses a threat to human health leads to the development of an antiviral, which involves target identification against viruses, screening of antiviral compounds, generation of lead and its optimization, preclinical and clinical trials and, finally, approval and registration of the compound as a new antiviral. KO: Knockout.

interactomics, metabonomics and metabonolomics. These technologies may be classified into two groups: conventional and current. Conventional techniques: customary practices

Conventional techniques are those that have long been used for antiviral development; for example, cell culture, biomarker analysis, HPLC and ELISA. 212

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Cell culture

The random screening of antivirals involves the evaluation of antiviral activity and determination of cytotoxicity to the referred molecules [102] . This is a tedious, costly and time-consuming process. Cell cultures can be used to study the effect of antiviral on viral replication and host cells, but it is not comparable with results obtained from living systems owing to the absence of various autocrine and other conditioning factors. Therefore, future science group

Advances in antiviral drug discovery & development

knowledge of these autocrine and other conditioning factors is essential in order to make the cell culture comparable to the living system.

biochips, quantitative structure–activity relationship (QSAR), Förster resonance energy transfer (FRET) and surface plasmon resonance.

Biomarkers

Computational screening

Biomarkers are molecular, biological or physical characteristics of an individual that indicate a specific, underlying physiologic state. Biomarkers can be used to identify risk for infection, make a diagnosis, assess severity, identify the organs involved and, ultimately, guide treatment against infection. Moreover, biomarkers can help antiviral-approval a­gencies to decide on the safety, specificity and efficacy of antivirals; for example, biological biomarkers for HIV/AIDS include viral load (the number of free virus particles in the blood) and the count of CD4 + cells of the immune system [5] . The expression of biomarkers are time bound, unstable and varied from individual to individual. Problems related to validity, reliability, accuracy and precision need to be clarified. Legal, ethical and cost considerations are also important [6] .

Antiviral discovery is a complex, multistage process that spans many scientific disciplines, including biology, chemistry, antiviral metabolism and clinical observation. All aspects of the field have witnessed an explosion in the volume of electronic data. At present, computer-aided drug designing (CADD) is used, which has helped in expediting the process of antiviral development [11,12] . CADD involves determining the 3D structure of the viral molecules using x-ray/nuclear magnetic resonance, computer simulation and thermo­ dynamic computation [13] . Using this approach, it has become much easier to select active molecules (hits and selection of leads) and transform them into suitable antivirals by improving their physico­chemical, pharmaceutical, absorption, metabolism, excretion, toxicity and pharmacokinetic properties [14] . CADD is still under development; however, the rapid advances in this field demonstrate promise for the efficient, selective identification of new antivirals. In silico prediction of toxicity and cellular-interacting molecules may also help to improve CADD.

HPLC

In antiviral development, HPLC could be used in the screening of viral inhibitors, detection of the proteolytic activity and determination of primaryinhibitory activity. This technique has proved to be very useful in testing the ch­romogenic compounds, however, it is very tedious and time consuming. It has been successfully used for detecting the proteolytic activity of SARS-coronavirus (SARS-CoV) 3CL proinhibitors [7] and oseltamivir [8] . It is an automated, rapid and highly sensitivive technique that is applicable to diverse samples. The precise quantification of samples can be carried out without loss of sample, and development of universal detectors will make it more efficient and cost effective [9] . ELISA is a technique that is used to detect the presence of an antibody or an antigen in a sample. ELISA can be used to screen for inhibitors of viral functions that can be used as antivirals. This method has been used for screening inhibitors against SARS-CoV 3CL proinhibitors [10] . ELISA is a sensitive technique in which background interference and false positives and negatives are problems that still need to be addressed. Current technology: modish approach

Biochips array

Current screening technologies are those that have evolved in recent times, for example, computational screening, molecular imaging,

Many arrays, such as DNA microarrays, miRNA arrays and tissue arrays, have been developed. These are important tools used to study host–viral

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“...the development of the antiviral candidate requires in vivo screenings in order to understand its pharmacokinetic properties, metabolism and toxicity,”

Molecular imaging

Molecular imaging is a powerful technique for characterizing biological processes in vivo using imaging probes. Recent developments in this field have allowed for early detection and characterization of infection and evaluation of therapy [15,16] . Failure of antivirals in clinical trials is a problem, and this can be attributed to the unavailability of useful models for evaluation of antivirals. Molecular imaging may effectively be used to monitor the effects of antivirals in vivo. Therefore, it may enhance the quality of selection of the lead candidate and may reduce late-stage failures by shifting abrasion to earlier, less expensive stages of the development process. Molecular imaging is still evolving and specific resolution, signal amplification, signal-to-noise ratio, toxicity, in vivo pharmacodynamic and cross-reactivity caused by imaging probes and assay molecules are considered to be large limitations. Development of molecular beacons and nuclear imaging may enable high s­ensitivity and specificity with less toxicity [17] .

ELISA

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interaction and global gene expressions [18] . The ana­lysis of the global response to infection is an important means for finding new treatments in relatively fewer steps and, thus, pave the path for the development of new antiviral. The biggest advantage with this technique is that a large number of samples can be analyzed in a short time for high-throughput validation of targets, evaluation of efficacy, distribution and toxicity. Key factors that will eventually determine the future success of the array are experimental and computational standardization, reproducibility of data, cost-limited access, quality of sample preparation and background [19] .

“The overall focus of antiviral development should be to translate the expanding knowledge in molecular and genomic research into patient care.”

Quantitative structure–activity relationship

Quantitative structure–activity relationship is the process by which chemical structure is quantitatively correlated with biological activity or chemical reactivity, along with CADD. Antiviral discovery often involves the use of QSAR to identify chemical structures that could have strong inhibitory effects on specific targets and possess low toxicity as well as high specificity (e.g., for study of HIV-1 integrase inhibition) [20] . With QSAR, it is easy to calculate descriptors for molecules and functions can be performed accurately and quickly. The main disadvantage of the technique is that a superimposable conformation for analysis must be selected [21] . Fluorescence-based approaches

A number of fluorescence-based assays have been developed for understanding the mechanism of inhibitors in vivo. Fluorescent protein substrates, internally quenched substrates and near-infrared fluorescence probes are used for this purpose [22] . FRET and fluorescence lifetime imaging micro­ scopy are examples of this approach. FRETbased assays have been used to identify many potential inhibitors of SARS‑CoV [23] . Surface plasmon resonance

This technique is used to analyze the interaction between antivirals and target proteins. This method is mainly used to screen compounds for receptor binding and to study their binding kinetics. The technique involves immobilization of the target protein on the surface of a sensor chip. If a compound binds to this chip, it results in a change in the refractive index at the surface of the chip; however, poor refractive index resolution remains a problem with this technique. SARs‑CoV 3CL proinhibitors have recently been discovered using this technique [24] . 214

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Combinatorial libraries

Combinatorial libraries of coactamer along with the proteomics approach is used to discover, screen and validate the targets and, has been proven to be a potential source of antiviral peptides. This integrated, sequential antiviral discovery process for selection of peptides with antiviral activity may include two-hybrid selection, viral reverse genetic assay, protein chip mass spectrometry and assay for inhibition of viral infection. This is a tedious process and requires support from computational as well as system biology, both of which are still in the development phase [25] . Future perspective: provisions beforehand

Every stage of antiviral discovery is a tedious, time-consuming and costly process. Many techniques have been developed to minimize the problem, however, they require further refinement and the development of novel techniques, such as fragment-based lead discovery, phenotypic screening of antivirals, siRNA, ribozymes, aptamers and antibody for the betterment of antiviral development. Development of improved in silico, in vitro and in vivo models for predicting antiviral-induced injury and toxicity is imperative. The development of molecular imaging, computational screening and system biology to determine antiviral targets, screening of antiviral, lead generation, optimization, understanding infection and the effect of antivirals in vivo is considered to be imperative. There is also a need to develop a collaborative network between academics and pharmaco­logical companies for the rapid, cost-effective antiviral development, and for applying successful strategies to guarantee antiviral reimbursement in order to safely deliver an antiviral. The overall focus of antiviral development should be to translate the expanding knowledge in molecular and genomic research into patient care. Attempts to support and encourage the imaging clinicians, researchers, structural biologists, molecular biologists and chemists to undertake interdisciplinary, cooperative and integrated projects must be emphasized. Conclusion: convergent inference

Owing to the combined application of molecular biology, structural biology, cell biology, chemistry and bioinformatics, the development of several antivirals against treating life-threatening or debilitating infections, such as HIV, hepatitis B virus, herpes virus and influenza virus, have been observed. This has resulted from technical advancement in respective fields and their continuation may lead to future improvements future science group

Advances in antiviral drug discovery & development

of current therapies and development of new therapeutics. All these technologies have their limitations. The combinational approach in technology development may be better suited to the purpose. At present, current antiviral therapies are restricted to a limited number of viral diseases and emerging viruses are potential threats to humans. Therefore, the development of efficient and cost-effective antivirals against all viruses still remains a challenge. Bibliography 1.

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Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes empl=oyment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

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Shailendra K Saxena, PhD, DCAP, FAEB, FCMS Centre for Cellular & Molecular Biology, Uppal Road, Hyderabad 500 007 (AP), India Tel.: +91 40 2719 2630; Fax: +91 40 2716 0591; [email protected] Niraj Mishra Centre for Cellular & Molecular Biology, Uppal Road, Hyderabad 500 007 (AP), India Tel.: +91 40 2719 2505 ; Fax: +91 40 2716 0591; Rakhi Saxena Centre for Cellular & Molecular Biology, Uppal Road, Hyderabad 500 007 (AP), India Tel.: +91 40 2719 2505 ; Fax: +91 40 2716 0591;

severe acute respiratory syndrome coronavirus 3CL protease inhibitors: virtual screening,

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