1. LIFE CYCLE OF MEASLES

The measles virus (MeV) is a member of the Morbillivirus genus within the Paramyxoviridae family. Its virions are spherical to pleomorphic particles, with diameters ranging from 120 to 300 nm. The MeV genome is a single-stranded negative-sense RNA molecule. It consists of approximately 16,000 nucleotides and is enclosed in a lipid-containing shell derived from the host cell membrane during the budding process. The genome contains six genes that encode such structural proteins as nucleocapsid (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin (H), and large protein (L). The P gene encodes two accessory non-structural proteins identified as V and C1. The transmembrane glycoproteins H and F are exposed on the surface of virus. Binding of protein H to host cell receptors triggers conformational changes in protein F, driving the fusion of the viral envelope with the plasma membrane. This process releases viral ribonucleoprotein (RNP) complexes into the cytoplasm of target cells. The viruses replicate and transcribe their genomes entirely within the cytoplasm [5]. Viral RNA-dependent RNA polymerase (RdRp) uses the viral RNA genome as a template for both transcription and replication [6]. Primary transcription begins at the 3’ end of the viral genome. Viral genes are transcribed 3’-to-5’ along the template using a stop-start mechanism. Newly synthesized viral mRNAs use the host cell’s translation factors to produce viral proteins. Positive-sense RNA viruses use their genome as a template for synthesizing a negative-sense (–RNA) complimentary strand, which is then packaged into new virions. Coordinated interactions between viral components (assembly of the protein M, RNP complex, and glycoproteins in certain areas of the plasma membrane of infected cells) and between viral and cellular factors lead to the formation of fully infectious virus particles. During these processes, the virus particles are collected and released from infected cells. In addition, expression of the H and F proteins on the surface of MeV-infected cells triggers fusion with neighboring cells, creating multinucleated giant cells known as syncytia. Infectious progeny viruses remain associated with the host cell membrane, and direct cell-to-cell transmission via infectious synapses is a key mechanism for viral spread within a host [5].

2. PATHOGENESIS OF MEASLES

Cells of the upper respiratory epithelium are the primary targets of the measles virus. Following infection, MeV within the respiratory tracts is first captured by dendritic cells and macrophages, and then transferred to thymocytes, T and B cells, and hematopoietic stem cells [:lit_7, 8;]. MeV utilizes infected mononuclear cells to spread to non-lymphoid organs, where the virus replicates in endothelial and epithelial cells. The measles virus starts spreading throughout the body. The process commonly takes 7–14 days and is clinically asymptomatic. During the prodromal phase of measles, high fever accompanied by cough, coryza, or conjunctivitis occurs due to virus replication in epithelial cells [9]. Being a typical measles rash, maculopapular exanthema is a hallmark of the immune response to the measles virus and is present in nearly all immunocompetent patients. Infected keratinocytes can result in keratoconjunctivitis and blindness [10]. The measles-like rash, swelling, and redness are primarily caused by the immune system destroying infected lymphoid and myeloid cells.

2.1 Measles virus-induced suppression of immune responses

Viral proteins V, C, and P can antagonize the host’s innate immune response to an infection. To do this, they suppress immune signaling pathways and effector functions and block the induction and production of interferons. All the processes allow to completely evade the host’s innate immune response and obtain exceptionally high virulence of MeV in vivo [11]. Measles also suppressed the adaptive immune response through various mechanisms possibly leading to increased susceptibility to opportunistic infections. MeV induces cell death in both infected and uninfected lymphocytes [12]. Immunological abnormalities last for at least several weeks, months or even years. Thus, measles can cause delayed mortality for 2–3 years following a measles infection [2].

Pneumonia is one of the leading causes of measles-related morbidity and mortality [13]. Measles can cause permanent blindness, particularly in children with vitamin A deficiency or deafness, and intellectual disabilities associated with encephalitis [13, 14;].

Subacute sclerosing panencephalitis (SSPE) is a neurodegenerative disease caused by a measles virus infection in the central nervous system. It can manifest 5–10 years post-infection being one of severe complications of measles. SSPE causes progressive personality changes, myoclonic seizures, and motor dysfunctions, resulting in coma and death within 1–3 years of symptom onset [15].

3. MEASLES CONTROL MEASURES

3.1. Vaccination

Measles is a vaccine-preventable disease. Measles immunization in immunocompetent individuals induces strong long-term immunity against the virus.

Live attenuated measles vaccines were developed in the late 1950s and early 1960s in different countries of the world. The most successful ones are parts of both mono- and combined (associated) forms today. All modern vaccines contain a live attenuated strain of measles virus. Most measles vaccine strains are derived from the prototypical Edmonston strain (Edmonston B, Moraten, Schwartz, and Edmonston-Zagreb strains), while others were developed from independent wild-type isolates. The Leningrad-16 strain was developed at Pasteur Research Institute of Epidemiology and Microbiology and has been a cornerstone of measles preventive vaccination in the USSR and Eastern Europe since 1967. Today, it is included in all Russian-made mono- and associated vaccines.

The development of an effective measles vaccine enabled inclusion of measles in the WHO’s Expanded Programme on Immunization in 1974. Its aim was to ensure active immunization of children 1 year of age globally to treat the 6 most common and dangerous childhood infections (polio, diphtheria, tuberculosis, tetanus, whooping cough and measles) that resulted in a significant decline in measles incidence and mortality in the following years. Integrating a second (revaccination) vaccine to routine immunization further increased protection against the disease. The second dose of the measles vaccine is specifically intended to provide protection to individuals who did not develop immunity from the first dose (those with immunosuppression, intercurrent diseases), as well as to children vaccinated at the age of 9 months following the valid national immunization schedule as, for example, in many countries of the WHO African region. Vaccination before 1 year of age can result in weakening immunity over time as the immune system of young children fails to develop long-term, intense post-vaccination immunity.

WHO adopted the Global Measles Elimination Program in the early 2000s following the successful efforts toward measles elimination. It targeted measles elimination in 5 of the 6 WHO regions by 2020.

However, though the global measles vaccination program has been successful in general, measles outbreaks are still surging globally, affecting both developing and industrialized nations [16, 17].

Thus, in 2018–2019, a particularly high incidence of measles was recorded in the WHO European Region (82,599 and 104,420 cases, respectively) [18]. In 2019, Ukraine (57,282 cases), Kazakhstan (13,326 cases), and Georgia (3,920 cases) reported a major measles resurgence in the European Region. In 2019, India (76,588 cases) and the Philippines (46,689) had higher levels of measles infections in Southeast Asia and Western Pacific region respectively. In Africa, large-scale measles outbreaks have been reported in Madagascar (151,032 cases), Nigeria (27,195 cases), and a number of other countries [19].

Anti-epidemic measures against COVID-19 significantly reduced measles transmission. The lockdown effectively reduced the transmission of not only SARS-CoV-2, but also the measles virus. The COVID-19 pandemic, however, caused a significant drop in measles vaccination coverage, primarily driven by a surge in medical exemptions and the diversion of healthcare resources toward pandemic response. It resulted in a significant surge in measles cases, observed in different WHO regions in 2023–2024, which confirms the insufficiency of preventive measures in several areas. 175 cases of measles were detected across the European region, including 51 cases in Turkey, 38 cases in Azerbaijan, 16 cases in France, 15 cases in Ukraine, 14 cases in Poland, and 10 cases in Tajikistan. In Russia, 1 case of measles was confirmed in the laboratory in 2021; 117 cases in 2022, and 3,378 cases of measles were reported in the first nine months of 2023.

Current causes of the spread of measles include as follows:

• active migration processes and measles cases imported from epidemiologically disadvantaged countries;
• during imports of measles, people from risk groups are infected such as medical, educational and social workers, students, migrant workers, and tourists, with further spread of measles due to a decreased population immunity;
• failure to recognize measles by medical workers due to prolonged absence of measles outbreaks in 2000– 2010;
• vaccine refusal among specific populations (nomadic and religious communities) and lack of commitment to vaccination, increased activity and number of anti-vaccinators.

Thus, there exist under-vaccinated communities that allow the highly contagious virus to spread. In such conditions, antiviral chemotherapeutic agents can become an additional measure to inhibit virus replication, to reduce disease severity in a patient and to lower population-level transmission.

3.2. Specific measles therapy

Uncomplicated measles is commonly managed with supportive care, including antipyretics and antitussives, hydration, and environmental control (moisturizing) [20]. Proper nutrition and vitamin A supplementation are highly effective in protecting against more severe measles-related symptoms [21].

As measles is considered highly vaccine-controllable, little attention has traditionally been paid to the development of specific antiviral chemotherapy drugs. As a result, there are currently no antiviral therapy to treat measles. Nevertheless, there are certain improvements in this area.

The measles virus life cycle occurs entirely in the cytoplasm and involves key structural components. These are the attachment protein, the fusion protein, and viral RNA polymerase conducting transcription and replication of the viral genome. Currently, the components have promising inhibitors that have failed to reach clinical trials. In addition, no target has been identified for many compounds with a demonstrated antiviral activity. Moreover, substances targeting not viral, but cellular mechanisms are used to combat the measles virus and enhance the immune response. Let’s take a closer look at each of the listed groups of compounds.

3.2.1. Receptor binding inhibitors

The first stage of the viral life cycle is the binding of a viral particle to a specific receptor on the host cell surface. Similar to many other viruses, the measles virus uses glycosaminoglycans (GAGS) as the main entry factors. These are negatively charged linear sulfated polysaccharides including chondroitin sulfate (HC) and heparan sulfate (HC). They represent complex natural glycans on cell surfaces and in the extracellular matrix. Viral glycoproteins use GAG as initial attachment of viral particles to host cells, which further improves the probability of affinity binding to virus-specific cellular receptors and penetration of the virus as a whole [22].

Two polyphenolic tannins, chebulagic acid and punicalagin (fig. 1), inhibit the reproduction of various enveloped viruses including the measles virus [23].

It is assumed that tannins bind to viral glycoproteins on the surface of these viruses and infected cells, blocking virus attachment, penetration and cell-to-cell spread. In this case, both tannins can target multiple infection stages, including attachment to cell surfaces, inhibit membrane fusion, and act as potent inhibitors of virus-mediated fusion of infected cells with each other.

3.2.2. Fusion protein inhibitors

The measles virus is an enveloped virus, and its entry into a host cell is entirely dependent on the successful fusion of its viral envelope with the host cell membrane. The measles virus fusion machinery is composed of two viral surface proteins forming hetero-oligomeric complexes. These complexes consist of tetrameric adsorption glycoprotein (H) and trimeric fusion glycoprotein (F). When H binds to its specific host cell receptor, a sequential cascade of conformational changes is initiated (first in H, and then in F). Finally, these structural rearrangements lead to viral-cell membrane merging and formation of fusion pores. The pores are used by the viral genome to penetrate the host cytosol. Membrane fusion is critical for both viral entry and cell-to-cell spread, leading to the formation of syncytia (multinucleated cells), which is the hallmark of morbilliviruses and many other paramyxovirus infections [23, 24].

The viral fusion protein F is associated with the envelope and organized as trimmers consisting of the head, which binds the protein to the cellular receptor, the stem, which is necessary for the subsequent membrane fusion process, and the transmembrane domain, which anchors the protein in the viral envelope.

Two highly conserved sequences of heptad repeat (HR) regions are critical components of membrane fusion, one of which is located next to the fusion peptide and near the N-terminus of the protein (HRN domain, or HRA), and the other is located next to the transmembrane domain and near the C-terminus (HRC or HRB). After activation and fusion peptide insertion, the protein F undergoes a conformational transition from the pre-hairpin intermediate to a highly stable six-helix bundle (6-HB) core structure.

In protein H-mediated activation, trimmers F, which are initially found in a metastable and inactive prefusion state, undergo a series of spontaneous and irreversible conformation changes. The structural rearrangements represent a sequence of several stages: 1) opening of the stem domain formed by three compact HRB domains; 2) subsequent rearrangement of the eleven segments of the HRA region of the three monomers into an expanded spiral conformation, which allows the released fusion peptide to be moved and embedded into the cell membrane, forming the so-called “pre-staple” intermediate; and 3) rotation of the three HRB peptides around the base of the globular head to attach to the helix, thereby forming the final 6HB core structure typical of the postfusion conformation of the protein.

These refolding stages are thought to be related to membrane fusion, as the complete assembly of 6HB means that the TM transmembrane domain (embedded in the viral envelope) and the fusion peptide (anchored in the membrane of the host cell) are close, which, in turn, causes membrane curvature, mixing of the outer lipid monolayers of both cellular and viral membranes and their subsequent spontaneous fusion. It should be noted that the energy released during refolding of F from the metastable prefusion state to the thermodynamically highly stable postfusion conformation exceeds the free energy of the intermediate lipid fusion structure, which allows the formation of a fusion pore [24]. Due to the fact that the membrane fusion process is specific to the virus and is provided by virus-specific proteins that have no cellular analogues, these proteins have long been an attractive target for the development of specific inhibitors. Research has identified several promising groups of compounds with high antiviral activity and favorable pharmacological properties.

Thus, compounds ZF and Zf and a structurally similar small hydrophobic fusion peptide inhibitor (carbobenzoxy [:lit_Z;]-D-Phe-L-Phe-Gly, FIP) (fig. 2) have been shown to block virus production and syncytium formation when Vero cells, CV-1 and HeLa were infected with measles [25].

It is known that FIP blocks fusion pore formation by inhibiting mixing of outer lipid layers of the viral envelope and the cell membrane. It occurs due to prevention of the essential conformational change of protein F from prefusion to postfusion. It has also been shown that the amino acid residue at position 94 affects the fusogenic activity of protein F and also makes it resistant to FIP [26, 27]. Using a model based on the postfusion structure of F, a small conservative hydrophobic cavity containing this residue was described, which includes the HRC region at the C-terminus of F2 [28]. The structure of this cavity served as the basis for further rational identification of medicines. The lead candidates such as OX-1 and AM-4 (fig. 3) demonstrated 50% inhibitory concentrations (IC50) of 50 μm and 260 nM, respectively, against MeV Edmonston strain and specifically inhibited virus penetration and syncytium formation [26].

The V94A mutation developed resistance to OX-1, as it was previously done with FIP. AM-4 subsequently underwent optimization, which allowed to develop AS-48, a first-generation lead (fig. 4). It had lower cell toxicity, increased stability, and potent inhibitory activity against wild-type MeV isolates.

Thus, research from 2004–2006 identified low molecular weight compounds that inhibited MeV penetration by targeting the fusion protein (F) [26, 29, 30]. The mechanism of action for the inhibitors has been meticulously analyzed in theory using drug-resistant mutants [31, 32]. Ten mutations were identified within the HRB region of the MeV fusion protein when FIP-resistant mutants of the virus were generated (fig. 5). These mutations were located in a small junction region between the head and the stalk of prefusion conformation F [33]. It must be noted that FIP-resistant MeV mutants commonly exhibit cross-resistance to AS-48.

Resistant escape MeV mutants were isolated by cultivation in the presence of OX-1 and AM-4 and by subsequent plaque purification [32]. The major resistance mutations were N462S, N462D, and N462K found mainly in the HRB region. N462S, N462D, N462K, and A367T function in a region comprised of HRB, HRB linker, and the globular head (domain I) acting as a “conformational switch”.

The changes to more hydrophilic amino acid residues within the region decreased the activation threshold making transition to the postfusion F trimer easier. AS-48 was assumed to interact with an intermediate structure along the fusion conformational pathway and to interfere with the rearrangements bringing HRB in proximity to HRA that were needed to form the postfusion 6HB. Binding of AS-48 to the intermediate would increase the energy barrier from the intermediate to the postfusion structure. Escape mutations to FIP (I452T, L454W, D458G, D458N, D458G/V459A, N462H, N462K, G464E, G464R, and I483R) could also lower fusion activation energy. It seems that FIP employs a mechanism of action similar to that of AS-48 and aims to inhibit protein F rearrangements by binding to it in the prefusion conformation.

It is important that resistance mutations could commonly be found in the HRB region, but never occurred in the HRA region. FIP escape mutations were found exclusively in HRB (I452T, L454W, D458G, D458N, D458G/V459A, N462H, N462K, G464E, G464R, and I483R) and, similar to AS-48, did not appear in proximity to the F peptide (V94M) or the cysteine-rich region (A362T). Removal of inhibitor selection enabled rapid reversion of FIP-resistant viruses to the original phenotype.

3.2.3 Viral polymerase inhibitors

As other members belonging to the family Paramyxoviridae, the measles virus proimerase is composed of nucleoprotein (N), phosphoprotein (P), and large protein (L). The enzymatic activity is localized in the L subunit. The RdRp complex acts as a transcriptase and replicase to produce a negative-sense genome and a positive-sense antigenome. The binding of the viral L protein to the N-RNA matrix of the nucleocapsid is mediated by L-P and P-N-RNA interaction. Apart from viral components, cellular factors are essential for viral RNA synthesis [34, 35]. After infection, the viral genome serves as a template to produce viral mRNAs. To initiate the replication cycle, infectious viral particles must contain protein P in addition to RNP and protein L. The switch of RdRp from transcription mode to replication mode allows to synthesize full-length antigenomes. They subsequently serve as templates for the synthesis of the negative-sense genomic RNA molecules, which are then packaged into offspring virions. As eukaryotic cells possess no enzymatic complexes capable of RNA synthesis using an RNA template, the measles virus polymerase seems to be a very attractive target for developing targeted inhibitors [36].

In 2007, a cellular system was developed to screen large-scale compound libraries for anti-measles activity. It was used to screen a chemical library of 34,000 diverse molecules [37]. As a result, 4 lead compounds were identified. The most potent ones inhibited virus replication in the submicromolar concentration range (IC50 = 0.24 microns), with a selectivity index being over 2000. The compound was active against different measles viruses, including viruses resistant to the previously described AS-48 fusion inhibitor. It shows that the targets of these two compounds are different. In a series of experiments, it was found out that compound 16677 binds reversibly to the polymerase complex, which allows to identify it as the first-class inhibitor of the measles virus RNA-dependent RNA polymerase.

Synthesis of a library of 16 677 analogues through the modification of pyrazole and pyrimidine rings and analysis of the structure-activity relationship allowed to identify compound 15f (fig. 6) with IC 50 = 5 nM and a selectivity index of 85,000 (10.1021/jm701239a). Subsequent optimization of 16 677 chemical structure made it possible to obtain compound AS-136A (fig. 6), [36, 38] with more favorable pharmacological characteristics.

Further similar studies allowed to obtain compound ERDRP-0519 (fig. 7) [39]. It is active against Morbilliviruses in general, including measles virus isolates with different genotypes (IC50 from 0.07 to 0.3 μM) and the related canine distemper virus. Just like precursors 16677, 15f and AS-136A, the compound is an allosteric inhibitor of the polymerase L subunit. It is designed to target specifically morbilliviruses and lacks efficacy against other members of the Paramyxoviridae family such a respiratory syncytial virus.

The inhibitor was active against the virus when administered orally. In the corresponding experiments, 100% achieved protection with 100% mortality rate in the control group; a significant decrease was observed in the viral titer in the blood. It is also important that strains resistant to this compound were less pathogenic to animals, i. e. resistance to the inhibitor was achieved through virulence reduction.

ERRP-0519 uniquely blocks all phosphodiester bond formation both during de novo initiation of RNA synthesis at the promoter and during RNA elongation. It makes the compound different from all other known polymerase inhibitors with negative-sense viruses. ERDRP-0519 acts as a measles virus polymerase inhibitor by simultaneously binding the polyribonucleotidyl transferase (PRNTase)-like domain and a flexible intrusion loop of the viral L protein. This locks the polymerase in a pre-initiation conformation, sterically preventing the accommodation of viral RNA [40]. Few reports suggested potential benefits from intravenous or aerosol administration of ribavirin in severe cases [41, 42]. Favipiravir (6-fluoro-3-hydroxypyrazine-2-carboxamide) that has shown activity against influenza and COVID-19 is also demonstrating activity against measles virus strains [43]. Favipiravir can be recognized by the polymerase complex as a substrate that can mimic both adenine and guanine producing a lethal mutagenic effect on virus replication. Other nucleoside analogues modified at the sugar residue or the nitrogenous base also show high activity against MeV through mechanisms associated with chain termination or lethal mutagenesis during the synthesis of daughter RNA strands by viral RNA polymerases [44, 45]. A derivative of aristeromycin is also included in the group (:media_8). Being a nucleoside analog, it exhibits a specific activity against the measles virus but not against many other viruses, both related (paramyxoviruses, picornaviruses and reoviruses) and unrelated (herpes and adenoviruses).

3.2.4 Inhibitors with unidentified target and inhibitors of indirect action

Effective compounds against the measles virus have also been identified. Their target, however, has not been reliably established. Thus, the plant-derived naphthoquinone droseron (fig. 9) inhibits the measles virus at the viral entry stage, when viral particles initiate fusion of the virus with the cell membrane, and the viral ribonucleoprotein complex (RNP) is absorbed by the cell [46]. Droseron, therefore, can interact with viral structures to inhibit receptor recognition and/or membrane fusion. The specific viral target structure for droseron is currently unknown.

(22R,23R)—2a,3α,22,23-tetrahydroxy-β-homo-7-oxa-stigmastan-6-one, the most active substance from the library, had CC50=427 μM, EC50=8 μM, and SI=53. Its mechanism of the action has not been determined yet.

Significant inhibitory activity is found in triterpene compounds of the brassinosteroid class (fig. 10) [48]. The most active substance from the library, (22R,23R)—2a,3α,22,23- tetrahydroxy-β-homo-7-oxa-stigmastan-6-one, had CC50=427 μM, EC50=8 μM, and SI=53. Their target of action has not been determined yet.

Compound 09167 (fig. 11) [49], which produces an indirect action, is active against all myxoviruses such as influenza viruses of different subtypes, measles virus, and respiratory syncytial virus. Meanwhile, IC50 values range from dozens of nanomoles, and the selectivity indices range from 200 to 1,500, depending on the target virus. Its mechanism triggers the innate immune response, directly stimulating the expression of interferon-stimulated genes such as ISG15, RIG-I и IFIT1 bypassing the actual induction of interferon. Thus, 09167 is an effective inhibitor, which is active against a broad range of myxoviruses.

Vitamin A is a clinically accepted treatment for measles. While Vitamin A is clinically proven to reduce morbidity and mortality in children with measles, the exact biological pathway remains incompletely understood. Its effectiveness is probably linked to its role in producing a beneficial effect on epithelial cells and supporting the host immune response [50].

Thus, it can be summed up that some critical points of preventive vaccination reduce effectiveness of this method of controlling measles. Meanwhile, numerous studies have identified various low-molecular-weight compounds that effectively inhibit measles virus reproduction. Thus, reasonable combination of methods of measles prevention and therapy can significantly improve outcomes.